Evaluation of long-term immune response in cattle to botulism using a recombinant E. coli bacterin formulated with Montanide™ ISA 50 and aluminum hydroxide adjuvants.

Botulism is a severe disease caused by potent botulinum neurotoxins (BoNTs) produced by Clostridium botulinum. This disease is associated with high-lethality outbreaks in cattle, which have been linked to the ingestion of preformed BoNT serotypes C and D, emphasizing the need for effective vaccines. The potency of current commercial toxoids (formaldehyde-inactivated BoNTs) is assured through tests in guinea pigs according to government regulatory guidelines, but their short-term immunity raises concerns. Recombinant vaccines containing the receptor-binding domain have demonstrated potential for eliciting robust protective immunity. Previous studies have demonstrated the safety and effectiveness of recombinant E. coli bacterin, eliciting high titers of neutralizing antibodies against C. botulinum and C. perfringens in target animal species. In this study, neutralizing antibody titers in cattle and the long-term immune response against BoNT/C and D were used to assess the efficacy of the oil-based adjuvant compared with that of the aluminum hydroxide adjuvant in cattle. The vaccine formulation containing Montanide™ ISA 50 yielded significantly higher titers of neutralizing antibody against BoNT/C and D (8.64 IU/mL and 9.6 IU/mL, respectively) and induced an immune response that lasted longer than the response induced by aluminum, extending between 30 and 60 days. This approach represents a straightforward, cost-effective strategy for recombinant E. coli bacterin, enhancing both the magnitude and duration of the immune response to botulism.

Combined Immunodeficiency Caused by a Novel De Novo Gain-of-Function RAC2 Mutation.

Ras-related C3 botulinum toxin substrate 2 (RAC2) is a GTPase exclusively expressed in hematopoietic cells that acts as a pivotal regulator of several aspects of cell behavior via various cellular processes. RAC2 undergoes a tightly regulated GTP-binding/GTP-hydrolysis cycle, enabling it to function as a molecular switch. Mutations in RAC2 have been identified in 18 patients with different forms of primary immunodeficiency, ranging from phagocyte defects caused by dominant negative mutations to common variable immunodeficiency resulting from autosomal recessive loss-of-function mutations, or severe combined immunodeficiency due to dominant activating gain-of-function mutations. Here, we describe an 11-year-old girl with combined immunodeficiency presenting with recurrent respiratory infections and bronchiectasis. Immunological investigations revealed low T-cell receptor excision circle/K-deleting recombination excision circles numbers, lymphopenia, and low serum immunoglobulin G. Targeted next-generation sequencing identified a novel heterozygous mutation in RAC2, c.86C > G (p.P29R), located in the highly conserved Switch I domain. The mutation resulted in enhanced reactive oxygen species production, elevated F-actin content, and increased RAC2 protein expression in neutrophils, as well as increased cytokine production and a dysregulated phenotype in T lymphocytes. Furthermore, the dominant activating RAC2 mutation led to accelerated apoptosis with augmented intracellular active caspase 3, impaired actin polarization in lymphocytes and neutrophils, and diminished RAC2 polarization in neutrophils. We present a novel RAC2 gain-of-function mutation with implications for immunodeficiency and linked to functional dysregulation, including abnormal apoptosis and cell polarization arising from altered RAC2 expression. Thus, our findings broaden the spectrum of known RAC2 mutations and their underlying mechanisms.

Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.

Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.

New Engineered-Botulinum Toxins Inhibit the Release of Pain-Related Mediators.

Targeted delivery of potent inhibitor of cytokine/pain-mediator into inflammatory or pain-sensing cells is a promising avenue for treating chronic pain, a world-wide major healthcare burden. An unmet need exists for a specific and effective delivery strategy. Herein, we describe a new approach using sortase to site-specifically ligate a non-toxic botulinum neurotoxin D (BoNT/D) core-therapeutic (synaptobrevin-cleaving protease and translocation domains) to cell-specific targeting ligands. An engineered core-therapeutic was efficiently ligated to IL-1ß ligand within minutes. The resultant conjugate specifically entered into cultured murine primary macrophages, cleaved synaptobrevin 3 and inhibited LPS/IFN-γ evoked IL-6 release. Likewise, a CGRP receptor antagonist ligand delivered BoNT/D protease into sensory neurons and inhibited K+-evoked substance P release. As cytokines and neuropeptides are major regulators of inflammation and pain, blocking their release by novel engineered inhibitors highlights their therapeutic potential. Our report describes a new and widely-applicable strategy for the production of targeted bio-therapeutics for numerous chronic diseases.

C3 Transferase Gene Therapy for Continuous RhoA Inhibition.

The identification of RhoA inhibition as a therapeutic target in neurodegenerative diseases and traumatic central nervous system (CNS) injuries has introduced a need to develop tools that effectively modulate intracellular RhoA-dependent signaling. In neurons, the bacterial exoenzyme C3 transferase irreversibly inactivates RhoA GTPase signaling to promote neuritogenesis and axon regeneration following an injury. Thus, we have adopted a gene therapy approach for the targeted inhibition of RhoA activity in the CNS by expressing C3 transferase. Herein we describe the construction of adeno-associated viral vectors for the expression of cell-permeable-C3 transferase and their functional characterization in vitro.

Rho-inhibiting C2IN-C3 fusion toxin inhibits chemotactic recruitment of human monocytes ex vivo and in mice in vivo.

Bacterial protein toxins became valuable molecular tools for the targeted modulation of cell functions in experimental pharmacology and attractive therapeutics because of their potent and specific mode of action in human cells. C2IN-C3lim, a recombinant fusion toxin (~50 kDa) of the Rho-inhibiting C3lim from Clostridium (C.) limosum and a non-toxic portion of the C. botulinum C2 toxin (C2IN), is selectively internalized into the cytosol of monocytic cells where C3lim specifically ADP-ribosylates Rho A and -B, thereby inhibiting Rho-mediated signaling. Thus, we hypothesized that these unique features make C2IN-C3lim an attractive molecule for the targeted pharmacological down-regulation of Rho-mediated functions in monocytes. The analysis of the actin structure and the Rho ADP-ribosylation status implied that C2IN-C3lim entered the cytosol of primary human monocytes from healthy donors ex vivo within 1 h. Moreover, it inhibited the fMLP-induced chemotaxis of human monocytes in a Boyden chamber model ex vivo. Similarly, in a 3-dimensional ex vivo model of extravasation, single cell analysis revealed that C2IN-C3lim-treated cells were not able to move. In a clinically relevant mouse model of blunt chest trauma, the local application of C2IN-C3lim into the lungs after thorax trauma prevented the trauma-induced recruitment of monocytes into the lungs in vivo. Thus, C2IN-C3lim might be an attractive lead compound for novel pharmacological strategies to avoid the cellular damage response caused by monocytes in damaged tissue after trauma and during systemic inflammation. The results suggest that the pathophysiological role of clostridial C3 toxins might be a down-modulation of the innate immune system.

Rational design of botulinum neurotoxin A1 mutants with improved oxidative stability.

Botulinum neurotoxins (BoNTs) are the most potent toxic proteins to mankind known but applied in low doses trigger a localized muscle paralysis that is beneficial for the therapy of several neurological disorders and aesthetic treatment. The paralytic effect is generated by the enzymatic activity of the light chain (LC) that cleaves specifically one of the SNARE proteins responsible for neurotransmitter exocytosis. The activity of the LC in a BoNT-containing therapeutic can be compromised by denaturing agents present during manufacturing and/or in the cell. Stabilization of the LC by reducing vulnerability towards denaturants would thus be advantageous for the development of BoNT-based therapeutics. In this work, we focused on increasing the stability of LC of BoNT/A1 (LC/A1) towards oxidative stress. We tackled this task by rational design of mutations at cysteine and methionine LC/A1 sites. Designed mutants showed improved oxidative stability in vitro and equipotency to wildtype toxin in vivo. Our results suggest that suitable modification of the catalytic domain can lead to more stable BoNTs without impairing their therapeutic efficacy.

Spatiotemporally Controlled Release of Rho-Inhibiting C3 Toxin from a Protein-DNA Hybrid Hydrogel for Targeted Inhibition of Osteoclast Formation and Activity.

In osteoporosis, bone structure can be improved by the introduction of therapeutic molecules inhibiting bone resorption by osteoclasts. Here, biocompatible hydrogels represent an excellent option for the delivery of pharmacologically active molecules to the bone tissue because of their biodegradability, injectability, and manifold functionalization capacity. The present study reports the preparation of a multifunctional hybrid hydrogel from chemically modified human serum albumin and rationally designed DNA building blocks. The hybrid hydrogel combines advantageous characteristics, including rapid gelation through DNA hybridization under physiological conditions and a self-healing and injectable nature with the possibility of specific loading and spatiotemporally controlled release of active proteins, making it an advanced biomaterial for the local treatment of bone diseases, for example, osteoporosis. The hydrogels are loaded with a recombinant Rho-inhibiting C3 toxin, C2IN-C3lim-G205C. This toxin selectively targets osteoclasts and inhibits Rho-signaling and, thereby, actin-dependent processes in these cells. Application of C2IN-C3lim-G205C toxin-loaded hydrogels effectively reduces osteoclast formation and resorption activity in vitro, as demonstrated by tartrate-resistant acid phosphatase staining and the pit resorption assay. Simultaneously, osteoblast activity, viability, and proliferation are unaffected, thus making C2IN-C3lim-G205C toxin-loaded hybrid hydrogels an attractive pharmacological system for spatial and selective modulation of osteoclast functions to reduce bone resorption.

C3 transferase gene therapy for continuous conditional RhoA inhibition.

Regrowth inhibitory molecules prevent axon regeneration in the adult mammalian central nervous system (CNS). RhoA, a small GTPase in the Rho family, is a key intracellular switch that mediates the effects of these extracellular regrowth inhibitors. The bacterial enzyme C3-ADP ribosyltransferase (C3) selectively and irreversibly inhibits the activation of RhoA and stimulates axon outgrowth and regeneration. However, effective intracellular delivery of the C3 protein in vivo is limited by poor cell permeability and a short duration of action. To address this, we have developed a gene therapy approach using viral vectors to introduce the C3 gene into neurons or neuronal progenitors. Our vectors deliver C3 in a cell-autonomous (endogenous) or a cell-nonautonomous (secretable/permeable) fashion and promote in vitro process outgrowth on inhibitory chondroitin sulfate proteoglycan substrate. Further conditional control of our vectors was achieved via the addition of a Tet-On system, which allows for transcriptional control with doxycycline administration. These vectors will be crucial tools for promoting continued axonal regeneration after CNS injuries or neurodegenerative diseases.

Survival of corticostriatal neurons by Rho/Rho-kinase signaling pathway.

Developing cortical neurons undergo a number of sequential developmental events including neuronal survival/apoptosis, and the molecular mechanism underlying each characteristic process has been studied in detail. However, the survival pathway of cortical neurons at mature stages remains largely uninvestigated. We herein focused on mature corticostriatal neurons because of their important roles in various higher brain functions and the spectrum of neurological and neuropsychiatric disorders. The small GTPase Rho is known to control diverse and essential cellular functions through some effector molecules, including Rho-kinase, during neural development. In the present study, we investigated the role of Rho signaling through Rho-kinase in the survival of corticostriatal neurons. We performed the conditional expression of Clostridium botulinum C3 ADP-ribosyltransferase (C3 transferase) or dominant-negative form for Rho-kinase (Rho-K DN), a well-known inhibitor of Rho or Rho-kinase, respectively, in corticostriatal neurons using a dual viral vector approach combining a neuron-specific retrograde gene transfer lentiviral vector and an adeno-associated virus vector. C3 transferase markedly decreased the number of corticostriatal neurons, which was attributed to caspase-3-dependent enhanced apoptosis. In addition, Rho-K DN produced phenotypic defects similar to those caused by C3 transferase. These results indicate that the Rho/Rho-kinase signaling pathway plays a crucial role in the survival of corticostriatal neurons.

Botulinum neurotoxin type C protease induces apoptosis in differentiated human neuroblastoma cells.

Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C. We show that following retinoic acid treatment, human neuroblastoma cells, SiMa and SH-SY5Y, acquire a neuronal phenotype evidenced by axonal growth and expression of neuronal markers. Botulinum neurotoxin type C which cleaves neuron-specific SNAP25 and syntaxin1 caused apoptotic death only in differentiated neuroblastoma cells. Direct comparison of translation-inhibiting enzymes and the type C botulinum protease revealed one order higher cytotoxic potency of the latter suggesting a novel neuroblastoma-targeting pathway. Our mechanistic insights revealed that loss of ubiquitous SNAP23 due to differentiation coupled to SNAP25 cleavage due to botulinum activity may underlie the apoptotic death of human neuroblastoma cells.

Structural analysis of Clostridium botulinum neurotoxin type D as a platform for the development of targeted secretion inhibitors.

The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the 'LHn fold' across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.

Botulinum neurotoxin: Progress in negating its neurotoxicity; and in extending its therapeutic utility via molecular engineering. MiniReview.

While the poisonous effects of botulinum neurotoxin (BoNT) have been recognized since antiquity, the overall actions and mechanisms of effects of BoNT have been elucidated primarily over the past several decades. The general utility of BoNT is described in the paper, but the focus is mainly on the approaches towards negating the toxic effects of BoNT, and on the projection of an engineered BoNT molecule serving as a Trojan Horse to deliver a therapeutic load for treatment of a host of medical disorders. The BoNT molecule is configured with a binding domain, a zinc-dependent protease with specificity primarily for vesicular proteins, and a translocation domain for delivery of the metalloprotease into the cytoplasm. The anti-toxin approaches for BoNT include the use of vaccines, antibodies, block of BoNT binding or translocation, inhibition of metalloprotease activity, impeded translocation of the protease/catalytic domain, and inhibition of the downstream Src signaling pathway. Projections of BoNT as a therapeutic include its targeting to non-cholinergic nerves, also targeting to non-neuronal cells for treatment of hypersecretory disorders (e.g., cystic fibrosis), and treatment of hormonal disorders (e.g., acromegaly). Still in the exploratory phase, there is the expectation of major advances in BoNT neuroprotective strategies and burgeoning utility of engineered BoNTs as therapeutics.

Characterization of SNARE proteins in human pituitary adenomas: targeted secretion inhibitors as a new strategy for the treatment of acromegaly?

CONTEXT: Targeted secretion inhibitors (TSIs), a new class of recombinant biotherapeutic proteins engineered from botulinum toxin, represent a novel approach for treating diseases with excess secretion. They inhibit hormone secretion from targeted cell types through cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor-activating protein receptor) proteins. qGHRH-LH(N)/D is a TSI targeting pituitary somatotroph through binding to the GHRH-receptor and cleavage of the vesicle-associated membrane protein (VAMP) family of SNARE proteins. OBJECTIVE: Our objective was to study SNARE protein expression in pituitary adenomas and to inhibit GH secretion from somatotropinomas using qGHRH-LH(N)/D. DESIGN: We analyzed human pituitary adenoma analysis for SNARE expression and response to qGHRH-LH(N)/D treatment. SETTING: The study was conducted in University Hospitals. PATIENTS: We used pituitary adenoma samples from 25 acromegaly and 47 nonfunctioning pituitary adenoma patients. OUTCOME: Vesicle-SNARE (VAMP1-3), target-SNARE (syntaxin1, SNAP-23, and SNAP-25), and GHRH-receptor detection with RT-qPCR, immunocytochemistry, and immunoblotting. Assessment of TSI catalytic activity on VAMPs and release of GH from adenoma cells. RESULTS: SNARE proteins were variably expressed in pituitary samples. In vitro evidence using recombinant GFP-VAMP2&3 or pituitary adenoma lysates suggested sufficient catalytic activity of qGHRH-LH(N)/D to degrade VAMPs, but was unable to inhibit GH secretion in somatotropinoma cell cultures. CONCLUSIONS: SNARE proteins are present in human pituitary somatotroph adenomas that can be targeted by TSIs to inhibit GH secretion. qGHRH-LH(N)/D was unable to inhibit GH secretion from human somatotroph adenoma cells. Further studies are required to understand how the SNARE proteins drive GH secretion in human somatotrophs to allow the development of novel TSIs with a potential therapeutic benefit.

Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain.

Botulinum neurotoxins translocate their enzymatic domain across vesicular membranes. The molecular triggers of this process are unknown. Here, we tested the possibility that this is elicited by protonation of conserved surface carboxylates. Glutamate-48, glutamate-653 and aspartate-877 were identified as possible candidates and changed into amide. This triple mutant showed increased neurotoxicity due to faster cytosolic delivery of the enzymatic domain; membrane translocation could take place at less acidic pH. Thus, neutralisation of specific negative surface charges facilitates membrane contact permitting a faster initiation of the toxin membrane insertion.

GHRH receptor-targeted botulinum neurotoxin selectively inhibits pulsatile GH secretion in male rats.

Botulinum neurotoxin is a potent inhibitor of acetylcholine secretion and acts by cleaving members of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor family, which are critical to exocytotic vesicular secretion. However, the potential of botulinum neurotoxin for treating secretory disease is limited both by its neural selectivity and the necessity for direct injection into the relevant target tissue. To circumvent these limitations, a technology platform called targeted secretion inhibitors (TSIs) is being developed. TSIs are derived from botulinum neurotoxin but are retargeted to specific cell types to inhibit aberrant secretion. A TSI called qGHRH-LHN/D, with a GHRH receptor targeting domain and designed to specifically inhibit pituitary somatotroph GH release through cleavage of the N-ethylmaleimide-sensitive factor-attachment protein receptor protein, vesicle-associated membrane protein (VAMP), has recently been described. Here we show this TSI activates GHRH receptors in primary cultured rat pituicytes is internalized into these cells, depletes VAMP-3, and inhibits phorbol-12-myristate-13-acetate-induced GH secretion. In vivo studies show that this TSI, but not one with an inactive catalytic unit, produces a dose-dependent inhibition of pulsatile GH secretion, thus confirming its mechanism of action through VAMP cleavage. Selectivity of action has been shown by the lack of effect of this TSI in vivo on secretion from thyrotrophs, corticotrophs, and gonadotrophs. In the absence of suitable in vivo models, these data provide proof of concept for the use of somatotroph-targeted TSIs in the treatment of acromegaly and moreover raise the potential that TSIs could be used to target other diseases characterized by hypersecretion.

Molecular components required for resting and stimulated endocytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons.

Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCV-releasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.

C3 exoenzyme lacks effects on peripheral axon regeneration in vivo.

Peripheral nerve injury triggers the activation of the small GTPase RhoA in spinal motor and peripheral sensory neurons. C3 transferase, an exoenzyme produced by Clostridium botulinum that inactivates RhoA by ADP-ribosylation, has been successfully applied in central nervous system (CNS) lesion models to facilitate regeneration functionally and morphologically. Until now it has not been demonstrated if C3bot exerts positive effects on peripheral axon regeneration as well. In organotypic spinal cord preparations, C3bot reduced axonal growth of motoneurons, while no effect on sensory axon outgrowth from dorsal root ganglia (DRG) explants was observed. Enzymatically inactive C3E174Q was ineffective in both culture models. Spinal cord slices exhibited a significant increase in microglia/macrophages after treatment with C3bot suggesting an inflammatory component in the inhibition of axon growth. C3bot or C3E174Q were then applied into conduits implanted after transection of the sciatic nerve in rats. Functional evaluation by electrophysiology, nociception, and walking track tests did not show any significant difference between groups with active or mutant C3E174Q . Transmission electron microscopy of the regenerated nerves revealed no significant differences in the number of myelinated and unmyelinated axons 6 weeks after surgery. Compared to the CNS, the functional significance of RhoA may be limited during nerve regeneration in a growth-promoting environment.

Recombinant streptavidin-C3bot for delivery of proteins into macrophages.

We demonstrated previously that monocytes and macrophages are target cells for the Rho-modifying Clostridium botulinum C3 ADP-ribosyltransferase. Here, we report the construction, expression and characterization of a recombinant streptavidin-C3 fusion protein which allows for delivery of biotin-labelled molecules into the cytosol of macrophages via enzymatically inactive C3bot1E174Q. The enzyme domain of diphtheria toxin was used as cargo to demonstrate proof of principle. This transport system could represent an attractive tool for experimental monocyte/macrophage pharmacology.

Oral vaccination with an adenovirus-vectored vaccine protects against botulism.

We have previously shown that an adenovirus vectored vaccine delivered intramuscularly or intranasally was effective in protection against botulism in a mouse model. The adenoviral vector encodes a human codon-optimized heavy chain C-fragment (H(C)50) of botulinum neurotoxin type C (BoNT/C). Here, we evaluate the same vaccine candidate as an oral vaccine against BoNT/C in a mouse model. To elicit protective immunity, the mice were orally vaccinated with a single dose of 1×10(4) to 1×10(7)plaque forming units (pfu) of the adenoviral vector. The immune sera, collected six weeks after oral vaccination with 2×10(7)pfu adenovirus, have shown an ability to neutralize the biological activity of BoNT/C in vitro. Additionally, animals receiving a single dose of 2×10(6)pfu adenovirus or greater were completely protected against challenge with 100×MLD(50) of BoNT/C. The data demonstrated the feasibility to develop an adenovirus-based oral vaccine against botulism.