BACKGROUND
clostridium botulinum is a gram-positive, spore-forming anaerobe commonly found in soil, and is responsible for production of botulinum neurotoxin , a potent lethal toxin  <cit> .
seven bont serotypes  have been identified  <cit> : serotypes a, b, e, and f are primarily responsible for human botulism; serotypes c and d are mostly limited to intoxication of animals  <cit> ; and host specificity for serotype g has not been fully determined  <cit> .
three clinical manifestations are related to naturally acquired botulism: foodborne, infant, and wound botulism  <cit> .
however, recently, three other forms have been described  <cit> : undefined or adult intestinal, inadvertent injection-related and inhalational botulism.
all forms of bont intoxication present with the same clinical syndrome of symmetrical and descending flaccid paralysis of motor and autonomic nerves, which can cause respiratory arrest and death  <cit> .
since natural exposure to c. botulinum spores is rare, the necessity to create a vaccine is primarily for high-risk individuals.
moreover, an intentional release to contaminate food and water supplies or for use as an aerosolized weapon  <cit>  warrants the development of vaccines to protect high-risk groups.
currently, there are no certified vaccines for preventing human botulism.
in the united states, an investigational pentavalent botulinum toxoid that includes serotypes a through e is used to vaccinate high risk individuals  <cit> .
although toxoid-based vaccines can be conveniently produced, these have a number of limitations, including isolation of active toxins, loss of essential neutralizing epitopes by formalin treatment, and multiple immunizations required to sustain elevated ab titers  <cit> .
to overcome these limitations, recent efforts have shifted to adapting recombinant bont heavy  chain to develop subunit vaccines  <cit> , as well as employing dna vaccine approaches  <cit> .
such approaches eliminate the reliance on handling active intact bonts.
bonts are originally produced as  <dig> kda chains that are post-translationally nicked into a c-terminal h chain  and n-terminal light  chain   <cit> .
functionally, the toxins contain three domains  <cit> : a receptor-binding domain, a translocation domain, and an enzymatic domain.
part of the receptor-binding domain is located in the carboxyl-terminal segment of the h chain, a region referred as the fragment c or hc.
the amino-terminal segment of the h chain is responsible for the translocation and internalization of the l chain into the cell  <cit> .
the l chain acts as a zinc-dependent endoprotease, which selectively attacks three crucial proteins, disabling the docking and fusion of acetylcholine-containing synaptic vesicles to the plasma membrane  <cit> .
a number of studies  have reported that recombinant  <dig> kda hc domain possesses the ability to stimulate protective abs capable of neutralizing native bont.
the hc is an effective immunogen and simultaneously capable of binding and penetrating epithelial barriers in the gut and airway, suggesting that this molecule could be used as a mucosal vaccine against botulism  <cit> .
the crystal structure of bont shows that the hc fragment is composed of two subdomains: n-terminal developing jelly roll motif and a c-terminal forming β-trefoil domain  <cit> .
the β-trefoil motif, especially the final approximately  <dig> residues, significantly diverges among serotypes with sequence similarity as low as 15%  <cit> , but maintains a ganglioside binding motif that is conserved  <cit> .
since there is a lack of significant cross-reactivity among the serotypes, it is speculated that the protective epitopes are located within this fragment of hc.
in our previous study, vaccination with the hc β-trefoil domain, referred to as hcβtre/a, rather than the intact hc, was shown to protect against bont/a  <cit> .
a major impediment in forwarding mucosal vaccines into humans is the absence of mucosal adjuvants suitable for human use.
significant efforts have focused on live, attenuated vaccine approaches to circumvent the requirement for exogenous adjuvants.
one such vaccine vector has been a modified adenovirus widely tested for a number of experimental vaccines  <cit> .
a major benefit of adenoviral vectors is their affinity for the mucosal epithelium, which makes them optimal for mucosal vaccination  <cit> , although this may be problematic for use in humans because of pre-existing abs  <cit> .
initial attachment of adenovirus to the host cell surface via coxsackie-adenovirus receptor is mediated by the knob domain of adenovirus fiber  <cit> .
to circumvent use of a live vector, the adenovirus  <dig> fiber  protein was selected and tested for its abilities to deliver bont/b hcβtre vaccine.
previously, immunization with the fusion protein, hcβtre/a-ad2f, increased onset of plasma and mucosal ab responses and conferred protection against bont/a  <cit> .
in light of these findings, we queried whether this approach could be adapted to generate a vaccine for bont/b.
thus, hcβtre/b was genetically fused to ad2f, and immunization with hcβtre/b-ad2f was found to be more immunogenic than hcβtre alone, further suggesting the utility of ad2f as a targeting moiety to improve a mucosal vaccine's immunogenicity.
RESULTS
comparison of ab responses by hcβtre-or hcβtre-ad2f-vaccinated mice
groups of mice were immunized by the intranasal  route with either hcβtre or hcβtre-ad2f to assess the ability of each to evoke a mucosal and systemic immune response .
in parallel experiments, mice were given the same vaccines, but via the i.m.
route .
all mice were boosted on days  <dig> and  <dig>  serum and fecal samples were collected at weekly intervals beginning on day  <dig> until day  <dig> post-primary immunization and then evaluated for anti-hcβtre ab titers by standard elisa methods.
significant s-iga ab responses were detected in fecal extracts from mice dosed nasally with hcβtre-ad2f, either in the presence or absence of ct adjuvant, as opposed to mice dosed nasally with hcβtre, which produced weak hcβtre-specific s-iga abs .
the intramuscular  hcβtre-ad2f-immunized mice also exhibited elevated fecal hcβtre-specific s-iga ab titers, but these tended to wane at the later time points .
in the presence of an adjuvant, i.n.
and i.m.
immunization  with hcβtre-ad2f and hcβtre induced elevated serum igg abs.
although mice i.n.
immunized with hcβtre-ad2f alone or i.m.
or i.n.
with hcβtre alone exhibited only weak serum igg responses , mice i.m.
immunized with hcβtre-ad2f without ct showed modest induction of serum igg ab responses .
to assess whether s-iga anti-hcβtre abs were induced in nasal secretions, nasal wash samples collected on day  <dig> post-primary immunization showed significantly elevated s-iga abs in those mice immunized i.n.
with hcβtre-ad2f plus ct when compared with mice given adjuvanted hcβtre .
mice dosed with the vaccines, but without adjuvant, showed no s-iga abs in their nasal washes .
sera from the immunized mice were also evaluated for the induced igg subclass responses from the ct co-immunized mice .
both groups of i.n.-immunized mice showed elevations in their igg <dig>  igg2a, and igg2b anti-hcβtre responses, but only the hcβtre-ad2f plus ct-immunized mice showed significant elevations in igg <dig> responses  .
in contrast, the i.m.
hcβtre-ad2f plus ct-immunized mice showed significant elevations  in both igg <dig> and igg2a anti-hcβtre responses compared to the i.m.
hcβtre plus ct-immunized mice, and both groups showed equivalent igg2b responses .
these results clearly show that hcβtre evokes both mucosal and systemic immune ab responses influenced by route of immunization, and the ab titers are significantly improved when hcβtre is genetically fused to ad2f.
distribution of immune b cells following i.n.
or i.m.
immunization
to learn the source of the immune abs obtained upon immunization with hcβtre-based vaccines, ag-specific b cell responses from the various mucosal and systemic lymphoid tissues were measured using an ab elispot assay.
ag-specific ab-forming cells  were minimally detected in lymphoid tissues of mice vaccinated without ct .
elevated hcβtre-specific iga afcs were detected in nasal-associated lymphoreticular tissue   and nasal passages   from mice i.n.
immunized with hcβtre-ad2f plus ct when compared to mice i.n.
immunized with adjuvanted hcβtre .
the numbers of ag-specific iga afcs in head and neck lns , submaxillary glands , spleens, peyer's patches , and intestinal lamina propria  were similar to mice immunized with either hcβtre-ad2f or hcβtre .
only minimal ag-specific iga afcs were detected in mice i.m.
immunized with our hcβtre-based vaccines .
analysis of ag-specific igg afc responses in i.n.-dosed mice exhibited elevated afcs in hnlns, smg, spleen, and pps  of mice that received adjuvanted hcβtre-ad2f when compared with mice given hcβtre .
the presence of hcβtre-specific igg afcs was detected in nalt and nps in both groups of i.n.-vaccinated mice, but these were not statistically significant .
mice i.m.
immunized with hcβtre-ad2f plus ct showed increased levels of ag-specific igg afcs in the peripheral lns , pps , and hnlns  in contrast to mice that received hcβtre plus ct .
minimal iga afc responses were detected in any of the lymphoid tissues examined .
from these studies, we showed that nasal immunization with adjuvanted hcβtre-ad2f enhances iga afc responses in np and nalt and igg afc responses in hnlns, smg, spleen, and pps when compared with adjuvanted hcβtre.
in addition, these data show that i.m.
immunization with hcβtre-ad2f in presence of ct enhances the number of igg afcs in plns, hnlns, and pps when compared to mice immunized with hcβtre plus ct.
hcβtre-based vaccines elicit a mixed th cell response
since immunization with our hcβtre-based vaccines induced elevated ag-specific ab responses in both mucosal and systemic tissues, it was important to examine the nature of th cells that support these hcβtre-specific b cell responses.
splenic, hnln, and mesenteric ln  lymphocytes from mice dosed with adjuvanted hcβtre and hcβtre-ad2f vaccines were restimulated with hcβtre and subsequently evaluated for the production of ifn-γ, il- <dig>  il- <dig>  il- <dig>  and il- <dig> by cytokine elispot method.
analysis of th <dig> and th <dig> cytokine production by ag-stimulated t cells revealed a varied th cell phenotype.
for i.n.-immunized mice, splenic il- <dig> and il- <dig> cytokine-forming cells  were induced to similar levels for both vaccine groups, but increased ifn-γ and il- <dig> cfcs were observed in hcβtre-immunized mice and il- <dig> in hcβtre-ad2f-dosed mice .
in the hnlns, the ifn-γ, il- <dig>  il- <dig>  and il- <dig> were particularly enhanced in hcβtre-vaccinated mice .
in the mlns, the levels of il- <dig>  il- <dig>  and il- <dig> cfcs were significantly greater than in hcβtre-ad2f-dosed mice .
for i.m.-immunized mice, splenic ifn-γ, il- <dig>  il- <dig>  and il- <dig> cfcs , hnln il- <dig>  il- <dig>  il- <dig>  and il- <dig> cfcs , and mln ifn-γ,il- <dig>  and il- <dig> cfcs  were significantly elevated in the hcβtre-ad2f-immunized group when compared to hcβtre-immunized mice.
these results showed that nasal immunization with adjuvanted hcβtre-ad2f elicited an enhanced th <dig> cell bias as opposed to mice immunized with hcβtre plus ct that elicited a mixed th cell response.
i.m.
immunization with either vaccine combined with adjuvant showed mixed th cell responses.
for either immunization route, it was evident that the igg <dig> ab responses were supported by elevations in th <dig> cell cytokines and igg2a ab responses supported by ifn-γ.
mouse neutralization assay
to evaluate the protective ab responses, serum samples from the various i.n.
immunization groups were tested for their ability to neutralize native bont/b.
three sources of sera were assessed:  sera from unimmunized mice;  sera from mice vaccinated with hcβtre plus ct; and  sera from mice vaccinated with hcβtre-ad2f plus ct. mice were immunized four times on days  <dig>   <dig>   <dig>  and  <dig>  and serum samples were collected on day  <dig> post-primary immunization, at which time endpoint titers were determined for hc/b.
mice given either hcβtre or hcβtre-ad2f showed elevated serum igg responses against hc/b, while naive sera showed no reactivity .
sera samples were incubated with  <dig>  ld <dig> of the bont/b at room temperature for  <dig> min before i.p.
injection into naïve balb/c mice.
all mice given naive sera succumbed to intoxication by  <dig> h .
mice given bont/b plus pooled sera from mice nasally immunized with either adjuvanted hcβtre or hcβtre-ad2f survived.
these studies demonstrated that immunization with hcβtre-ad2f or hcβtre could elicit neutralizing abs that protect against systemic challenge.
a pooled sera from nasally vaccinated or naive mice incubated for  <dig> h with  <dig>
ld <dig> of bont/b in pbs- <dig> % gelatin and then injected into naive mice.
b source of sera used.
c endpoint titer for hc/b in pooled sera .
d survival of bont/b-challenged mice after  <dig> h.
e p-value determined by kaplan-meier method.
discussion
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  <cit> .
although natural exposure via the inhalational route is unlikely  <cit> , illicit aerosol releases have occurred  <cit> ; 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  <cit> , few studies have evaluated bont/b-based vaccines  <cit> .
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  <cit> , including the limited ability to stimulate mucosal abs when given parenterally  <cit>  and the required yearly boosts to maintain protective ab levels  <cit> .
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  <cit> .
current efforts to develop bont vaccines have focused primarily on immunizing with recombinant hc polypeptides capable of evoking protective immunity against bont challenge  <cit> .
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  <cit>  and suggested by others  <cit> , 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  <cit> .
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 .
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  <cit> .
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  <cit> .
in fact, recombinant adenovirus bearing the hc transgene has been shown to effectively stimulate neutralizing abs to bont/c  <cit> .
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  <cit> , thus, preventing the development of a protective immune response  <cit> .
admittedly, studies have shown that i.n.
immunization seems to avoid the stimulation of systemic neutralizing abs  <cit> .
however, another concern is the possible retrograde uptake of replication-deficient adenovirus vectors into the olfactory bulb  <cit> , 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  <cit> .
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  <cit> , 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 .
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 .
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 .
notably, i.m.
immunization with hcβtre-ad2f in the absence of ct showed elevated igg ab titers that remained elevated for at least  <dig> 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  <cit> , the hcβtre's immunogenicity was markedly improved by the addition of ad2f .
however, only a low level of mucosal abs after day  <dig> was observed , 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 , such evidence suggests that transient iga ab responses were induced during immunization.
few studies have evaluated whether th <dig> or th <dig> cells are beneficial for optimal ab production for the hc-based vaccines.
a recent study by kobayashi et al.
<cit>  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 .
notable enhancements in th2-type cells were detected in the spleens and mlns of i.n.
hcβtre-ad2f plus ct-immunized mice , and likewise for i.m.
hcβtre-ad2f plus ct-immunized mice  suggesting these responses supported the observed, elevated igg <dig> ab responses .
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  <cit> .
nasal administration of hcβtre-based vaccines induced significant systemic and mucosal abs.
to assess their protective efficacy, a mouse neutralization assay, was performed .
our data showed that passively administered sera from animals immunized with hcβtre or with hcβtre-ad2f protected 100% mice for  <dig> hr against  <dig>  ld <dig>  suggesting neutralizing abs are induced.
CONCLUSIONS
as previously shown for hcβtre/a  <cit> , the hcβtre/b is also very immunogenic capable of eliciting elevated serum igg and mucosal iga abs, especially when genetically fused to the adhesin, ad2f.
these enhanced ab responses are supported by mostly by th <dig> cell responses when hcβtre-ad2f is given nasally, and a mixed th1/th <dig> cell response is obtained subsequent i.m.
immunization.
the induced abs were able to neutralize bont/b as evident in the mouse neutralization assay.
thus, these studies demonstrate that the hcβtre immunogen can be used as a bont vaccine.

 clostridium botulinum is a gram-positive, spore-forming anaerobe commonly found in soil, and is responsible for production of botulinum neurotoxin, a potent lethal toxin. seven bont serotypes have been identified: serotypes a, b, e, and f are primarily responsible for human botulism; serotypes c and d are mostly limited to intoxication of animals; and host specificity for serotype g has not been fully determined. three clinical manifestations are related to naturally acquired botulism: foodborne, infant, and wound botulism. however, recently, three other forms have been described: undefined or adult intestinal, inadvertent injection-related and inhalational botulism. all forms of bont intoxication present with the same clinical syndrome of symmetrical and descending flaccid paralysis of motor and autonomic nerves, which can cause respiratory arrest and death. since natural exposure to c. botulinum spores is rare, the necessity to create a vaccine is primarily for high-risk individuals. moreover, an intentional release to contaminate food and water supplies or for use as an aerosolized weapon warrants the development of vaccines to protect high-risk groups. currently, there are no certified vaccines for preventing human botulism. in the united states, an investigational pentavalent botulinum toxoid that includes serotypes a through e is used to vaccinate high risk individuals. although toxoid-based vaccines can be conveniently produced, these have a number of limitations, including isolation of active toxins, loss of essential neutralizing epitopes by formalin treatment, and multiple immunizations required to sustain elevated ab titers. to overcome these limitations, recent efforts have shifted to adapting recombinant bont heavy chain to develop subunit vaccines, as well as employing dna vaccine approaches. such approaches eliminate the reliance on handling active intact bonts. bonts are originally produced as kda chains that are post-translationally nicked into a c-terminal h chain and n-terminal light chain. functionally, the toxins contain three domains: a receptor-binding domain, a translocation domain, and an enzymatic domain. part of the receptor-binding domain is located in the carboxyl-terminal segment of the h chain, a region referred as the fragment c or hc. the amino-terminal segment of the h chain is responsible for the translocation and internalization of the l chain into the cell. the l chain acts as a zinc-dependent endoprotease, which selectively attacks three crucial proteins, disabling the docking and fusion of acetylcholine-containing synaptic vesicles to the plasma membrane. a number of studies have reported that recombinant kda hc domain possesses the ability to stimulate protective abs capable of neutralizing native bont. the hc is an effective immunogen and simultaneously capable of binding and penetrating epithelial barriers in the gut and airway, suggesting that this molecule could be used as a mucosal vaccine against botulism. the crystal structure of bont shows that the hc fragment is composed of two subdomains: n-terminal developing jelly roll motif and a c-terminal forming β-trefoil domain. the β-trefoil motif, especially the final approximately residues, significantly diverges among serotypes with sequence similarity as low as 15%, but maintains a ganglioside binding motif that is conserved. since there is a lack of significant cross-reactivity among the serotypes, it is speculated that the protective epitopes are located within this fragment of hc. in our previous study, vaccination with the hc β-trefoil domain, referred to as hcβtre/a, rather than the intact hc, was shown to protect against bont/a. a major impediment in forwarding mucosal vaccines into humans is the absence of mucosal adjuvants suitable for human use. significant efforts have focused on live, attenuated vaccine approaches to circumvent the requirement for exogenous adjuvants. one such vaccine vector has been a modified adenovirus widely tested for a number of experimental vaccines. a major benefit of adenoviral vectors is their affinity for the mucosal epithelium, which makes them optimal for mucosal vaccination, although this may be problematic for use in humans because of pre-existing abs. initial attachment of adenovirus to the host cell surface via coxsackie-adenovirus receptor is mediated by the knob domain of adenovirus fiber. to circumvent use of a live vector, the adenovirus fiber protein was selected and tested for its abilities to deliver bont/b hcβtre vaccine. previously, immunization with the fusion protein, hcβtre/a-ad2f, increased onset of plasma and mucosal ab responses and conferred protection against bont/a. in light of these findings, we queried whether this approach could be adapted to generate a vaccine for bont/b. thus, hcβtre/b was genetically fused to ad2f, and immunization with hcβtre/b-ad2f was found to be more immunogenic than hcβtre alone, further suggesting the utility of ad2f as a targeting moiety to improve a mucosal vaccine's immunogenicity.  comparison of ab responses by hcβtre-or hcβtre-ad2f-vaccinated mice groups of mice were immunized by the intranasal route with either hcβtre or hcβtre-ad2f to assess the ability of each to evoke a mucosal and systemic immune response. in parallel experiments, mice were given the same vaccines, but via the i.m. route. all mice were boosted on days and serum and fecal samples were collected at weekly intervals beginning on day until day post-primary immunization and then evaluated for anti-hcβtre ab titers by standard elisa methods. significant s-iga ab responses were detected in fecal extracts from mice dosed nasally with hcβtre-ad2f, either in the presence or absence of ct adjuvant, as opposed to mice dosed nasally with hcβtre, which produced weak hcβtre-specific s-iga abs. the intramuscular hcβtre-ad2f-immunized mice also exhibited elevated fecal hcβtre-specific s-iga ab titers, but these tended to wane at the later time points. in the presence of an adjuvant, i.n. and i.m. immunization with hcβtre-ad2f and hcβtre induced elevated serum igg abs. although mice i.n. immunized with hcβtre-ad2f alone or i.m. or i.n. with hcβtre alone exhibited only weak serum igg responses, mice i.m. immunized with hcβtre-ad2f without ct showed modest induction of serum igg ab responses. to assess whether s-iga anti-hcβtre abs were induced in nasal secretions, nasal wash samples collected on day post-primary immunization showed significantly elevated s-iga abs in those mice immunized i.n. with hcβtre-ad2f plus ct when compared with mice given adjuvanted hcβtre. mice dosed with the vaccines, but without adjuvant, showed no s-iga abs in their nasal washes. sera from the immunized mice were also evaluated for the induced igg subclass responses from the ct co-immunized mice. both groups of i.n.-immunized mice showed elevations in their igg igg2a, and igg2b anti-hcβtre responses, but only the hcβtre-ad2f plus ct-immunized mice showed significant elevations in igg responses. in contrast, the i.m. hcβtre-ad2f plus ct-immunized mice showed significant elevations in both igg and igg2a anti-hcβtre responses compared to the i.m. hcβtre plus ct-immunized mice, and both groups showed equivalent igg2b responses. these results clearly show that hcβtre evokes both mucosal and systemic immune ab responses influenced by route of immunization, and the ab titers are significantly improved when hcβtre is genetically fused to ad2f. distribution of immune b cells following i.n. or i.m. immunization to learn the source of the immune abs obtained upon immunization with hcβtre-based vaccines, ag-specific b cell responses from the various mucosal and systemic lymphoid tissues were measured using an ab elispot assay. ag-specific ab-forming cells were minimally detected in lymphoid tissues of mice vaccinated without ct. elevated hcβtre-specific iga afcs were detected in nasal-associated lymphoreticular tissue and nasal passages from mice i.n. immunized with hcβtre-ad2f plus ct when compared to mice i.n. immunized with adjuvanted hcβtre. the numbers of ag-specific iga afcs in head and neck lns, submaxillary glands, spleens, peyer's patches, and intestinal lamina propria were similar to mice immunized with either hcβtre-ad2f or hcβtre. only minimal ag-specific iga afcs were detected in mice i.m. immunized with our hcβtre-based vaccines. analysis of ag-specific igg afc responses in i.n.-dosed mice exhibited elevated afcs in hnlns, smg, spleen, and pps of mice that received adjuvanted hcβtre-ad2f when compared with mice given hcβtre. the presence of hcβtre-specific igg afcs was detected in nalt and nps in both groups of i.n.-vaccinated mice, but these were not statistically significant. mice i.m. immunized with hcβtre-ad2f plus ct showed increased levels of ag-specific igg afcs in the peripheral lns, pps, and hnlns in contrast to mice that received hcβtre plus ct. minimal iga afc responses were detected in any of the lymphoid tissues examined. from these studies, we showed that nasal immunization with adjuvanted hcβtre-ad2f enhances iga afc responses in np and nalt and igg afc responses in hnlns, smg, spleen, and pps when compared with adjuvanted hcβtre. in addition, these data show that i.m. immunization with hcβtre-ad2f in presence of ct enhances the number of igg afcs in plns, hnlns, and pps when compared to mice immunized with hcβtre plus ct. hcβtre-based vaccines elicit a mixed th cell response since immunization with our hcβtre-based vaccines induced elevated ag-specific ab responses in both mucosal and systemic tissues, it was important to examine the nature of th cells that support these hcβtre-specific b cell responses. splenic, hnln, and mesenteric ln lymphocytes from mice dosed with adjuvanted hcβtre and hcβtre-ad2f vaccines were restimulated with hcβtre and subsequently evaluated for the production of ifn-γ, il- il- il- and il- by cytokine elispot method. analysis of th and th cytokine production by ag-stimulated t cells revealed a varied th cell phenotype. for i.n.-immunized mice, splenic il- and il- cytokine-forming cells were induced to similar levels for both vaccine groups, but increased ifn-γ and il- cfcs were observed in hcβtre-immunized mice and il- in hcβtre-ad2f-dosed mice. in the hnlns, the ifn-γ, il- il- and il- were particularly enhanced in hcβtre-vaccinated mice. in the mlns, the levels of il- il- and il- cfcs were significantly greater than in hcβtre-ad2f-dosed mice. for i.m.-immunized mice, splenic ifn-γ, il- il- and il- cfcs, hnln il- il- il- and il- cfcs, and mln ifn-γ,il- and il- cfcs were significantly elevated in the hcβtre-ad2f-immunized group when compared to hcβtre-immunized mice. these results showed that nasal immunization with adjuvanted hcβtre-ad2f elicited an enhanced th cell bias as opposed to mice immunized with hcβtre plus ct that elicited a mixed th cell response. i.m. immunization with either vaccine combined with adjuvant showed mixed th cell responses. for either immunization route, it was evident that the igg ab responses were supported by elevations in th cell cytokines and igg2a ab responses supported by ifn-γ. mouse neutralization assay to evaluate the protective ab responses, serum samples from the various i.n. immunization groups were tested for their ability to neutralize native bont/b. three sources of sera were assessed: sera from unimmunized mice; sera from mice vaccinated with hcβtre plus ct; and sera from mice vaccinated with hcβtre-ad2f plus ct. mice were immunized four times on days and and serum samples were collected on day post-primary immunization, at which time endpoint titers were determined for hc/b. mice given either hcβtre or hcβtre-ad2f showed elevated serum igg responses against hc/b, while naive sera showed no reactivity. sera samples were incubated with ld of the bont/b at room temperature for min before i.p. injection into naïve balb/c mice. all mice given naive sera succumbed to intoxication by h. mice given bont/b plus pooled sera from mice nasally immunized with either adjuvanted hcβtre or hcβtre-ad2f survived. these studies demonstrated that immunization with hcβtre-ad2f or hcβtre could elicit neutralizing abs that protect against systemic challenge. a pooled sera from nasally vaccinated or naive mice incubated for h with ld of bont/b in pbs- % gelatin and then injected into naive mice. b source of sera used. c endpoint titer for hc/b in pooled sera. d survival of bont/b-challenged mice after h. e p-value determined by kaplan-meier method. discussion 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. although natural exposure via the inhalational route is unlikely, illicit aerosol releases have occurred; 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, few studies have evaluated bont/b-based vaccines. 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, including the limited ability to stimulate mucosal abs when given parenterally and the required yearly boosts to maintain protective ab levels. 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. current efforts to develop bont vaccines have focused primarily on immunizing with recombinant hc polypeptides capable of evoking protective immunity against bont challenge. 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 and suggested by others, 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. 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. 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. 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. in fact, recombinant adenovirus bearing the hc transgene has been shown to effectively stimulate neutralizing abs to bont/c. 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, thus, preventing the development of a protective immune response. admittedly, studies have shown that i.n. immunization seems to avoid the stimulation of systemic neutralizing abs. however, another concern is the possible retrograde uptake of replication-deficient adenovirus vectors into the olfactory bulb, 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. 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, 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. 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. 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. notably, i.m. immunization with hcβtre-ad2f in the absence of ct showed elevated igg ab titers that remained elevated for at least 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, the hcβtre's immunogenicity was markedly improved by the addition of ad2f. however, only a low level of mucosal abs after day was observed, 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, such evidence suggests that transient iga ab responses were induced during immunization. few studies have evaluated whether th or th cells are beneficial for optimal ab production for the hc-based vaccines. a recent study by kobayashi et al. 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. notable enhancements in th2-type cells were detected in the spleens and mlns of i.n. hcβtre-ad2f plus ct-immunized mice, and likewise for i.m. hcβtre-ad2f plus ct-immunized mice suggesting these responses supported the observed, elevated igg ab responses. 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. nasal administration of hcβtre-based vaccines induced significant systemic and mucosal abs. to assess their protective efficacy, a mouse neutralization assay, was performed. our data showed that passively administered sera from animals immunized with hcβtre or with hcβtre-ad2f protected 100% mice for hr against ld suggesting neutralizing abs are induced.  as previously shown for hcβtre/a, the hcβtre/b is also very immunogenic capable of eliciting elevated serum igg and mucosal iga abs, especially when genetically fused to the adhesin, ad2f. these enhanced ab responses are supported by mostly by th cell responses when hcβtre-ad2f is given nasally, and a mixed th1/th cell response is obtained subsequent i.m. immunization. the induced abs were able to neutralize bont/b as evident in the mouse neutralization assay. thus, these studies demonstrate that the hcβtre immunogen can be used as a bont vaccine. 
these bont/b-specific abs induced by nasal immunization were generally supported in large part by th cells, as opposed to hcβtre-immunized mice that showed more mixed th and th cells. these results demonstrate that hcβtre-based immunogens are highly immunogenic, especially when genetically fused to ad2f, and ad2f can be exploited as a vaccine delivery platform to the mucosa. in combination with the mucosal adjuvant, cholera toxin, enhanced mucosal iga and serum igg ab titers were induced by nasal hcβtre-ad2f relative to hcβtre alone; however, similar ab titers were obtained upon intramuscular immunization. immunization with recombinant carboxyl-terminal domain of the heavy chain of botulinum neurotoxin stimulates protective immunity against native bont challenge. most studies developing a botulism vaccine have focused on the whole hc; however, since the principal protective epitopes are located within β-trefoil domain, we hypothesize that immunization with the hcβtre domain is sufficient to confer protective immunity. using a mouse neutralization assay, sera from animals immunized with hcβtre and hcβtre-ad2f protected mice against ld 
 immunization with recombinant carboxyl-terminal domain of the heavy chain of botulinum neurotoxin stimulates protective immunity against native bont challenge. most studies developing a botulism vaccine have focused on the whole hc; however, since the principal protective epitopes are located within β-trefoil domain, we hypothesize that immunization with the hcβtre domain is sufficient to confer protective immunity. in addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus fiber protein may enhance such uptake during vaccination.  the hcβtre serotype b immunogen was genetically fused to ad2f, and its immunogenicity was tested in mice. in combination with the mucosal adjuvant, cholera toxin, enhanced mucosal iga and serum igg ab titers were induced by nasal hcβtre-ad2f relative to hcβtre alone; however, similar ab titers were obtained upon intramuscular immunization. these bont/b-specific abs induced by nasal immunization were generally supported in large part by th cells, as opposed to hcβtre-immunized mice that showed more mixed th and th cells. using a mouse neutralization assay, sera from animals immunized with hcβtre and hcβtre-ad2f protected mice against ld  these results demonstrate that hcβtre-based immunogens are highly immunogenic, especially when genetically fused to ad2f, and ad2f can be exploited as a vaccine delivery platform to the mucosa. 