Discrimination of the Activity of Low-Affinity Wild-Type and High-Affinity Mutant Recombinant BoNT/B by a SIMA Cell-Based Reporter Release Assay.

Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.

Effects of membrane properties on the binding activities of the HN and HC heavy-chain domains of botulinum neurotoxin A.

Binding behaviors of the HN and the HC domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A HN and HC regions were prepared (HN519-845 and HC967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of HN519-845 to Neuro 2a cells without affecting HC967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both HC967-1296 and HN519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both HC967-1296 and HN519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K+) depletion buffer lowered the binding of both HC967-1296 and HN519-845 to the cells, but seemed to exert a more pronounced effect on the binding of HC967-1296 than on the binding of HN519-845. Results indicate that while both the HN and HC domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells.

The C-terminal heavy-chain domain of botulinum neurotoxin a is not the only site that binds neurons, as the N-terminal heavy-chain domain also plays a very active role in toxin-cell binding and interactions.

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin.

Recruitment of septin cytoskeletal proteins by botulinum toxin A protease determines its remarkable stability.

Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.

Identification of fibroblast growth factor receptor 3 (FGFR3) as a protein receptor for botulinum neurotoxin serotype A (BoNT/A).

Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

Stimulation of IgY responses in gene gun immunized laying hens by combined administration of vector DNA coding for the target antigen Botulinum toxin A1 and for avian cytokine adjuvants.

DNA immunization is a convenient and effective way of inducing a specific antibody response. In mammals, co-administration of vectors encoding immunostimulatory cytokines can enhance the humoral response resulting in elevated antibody titers. We therefore set out to investigate the effect using avian interleukin 1ß (IL-1ß) and avian interleukin 6 (IL-6) as genetic adjuvants when immunizing laying hens. A BoNT A1 holotoxoid DNA immunogen carrying two inactivating mutations was evaluated for its ability to induce a specific and sustained IgY antibody response. Both the holotoxoid and the cytokine sequences were codon-optimized. In vitro, the proteins were efficiently expressed in transfected HEK 293T cells and the cytokines were secreted into the culture supernatants. Whereas eggs from hens immunized via gene gun using a prime boost strategy showed no differences in their total IgY content, the specific αBoNT A1 response was slightly elevated up to 1.4× by the IL-1ß adjuvant vector and increased by 3.8× by the IL-6 vector. Finally, although hens receiving the IL-1ß adjuvant had laying capacities above the average, hens receiving the IL-6 adjuvant experienced laying problems.

Sublingual immunization with adenovirus F protein-based vaccines stimulates protective immunity against botulinum neurotoxin A intoxication.

Sublingual (s.l.) vaccination is an efficient way to induce elevated levels of systemic and mucosal immune responses. To mediate mucosal uptake, ovalbumin (OVA) was genetically fused to adenovirus 2 fiber protein (OVA-Ad2F) to assess whether s.l. immunization was as effective as an alternative route of vaccination. Ad2F-delivered vaccines were efficiently taken up by dendritic cells and migrated mostly to submaxillary gland lymph nodes, which could readily stimulate OVA-specific CD4(+) T cells. OVA-Ad2F + cholera toxin (CT)-immunized mice elicited significantly higher OVA-specific serum IgG, IgA and mucosal IgA antibodies among the tested immunization groups. These were supported by elevated OVA-specific IgG and IgA antibody-forming cells. A mixed T(h)-cell response was induced as evident by the enhanced IL-4, IL-10, IFN-γ and TNF-α-specific cytokine-forming cells. To assess whether this approach can stimulate neutralizing antibodies, immunizations were performed with the protein encumbering the ß-trefoil domain of C-terminus heavy chain (Hcßtre) from botulinum neurotoxin A (BoNT/A) as well as when fused to Ad2F. Hcßtre-Ad2F + CT-dosed mice showed the greatest serum IgG, IgA and mucosal IgA titers among the immunization groups. Hcßtre-Ad2F alone also induced elevated antibody production in contrast to Hcßtre alone. Plasma from Hcßtre + CT- and Hcßtre-Ad2F + CT-immunized groups neutralized BoNT/A and protected mice from BoNT/A intoxication. Most importantly, Hcßtre-Ad2F + CT-immunized mice were protected from BoNT/A intoxication relative to Hcßtre + CT-immunized mice, which only showed ∼60% protection. This study shows that s.l. immunization with Ad2F-based vaccines is effective in conferring protective immunity.

Immunization of mice with the non-toxic HC50 domain of botulinum neurotoxin presented by rabies virus particles induces a strong immune response affording protection against high-dose botulinum neurotoxin challenge.

We previously showed that rabies virus (RABV) virions are excellent vehicles for antigen presentation. Here, a reverse genetic approach was applied to generate recombinant RABV that express a chimeric protein composed of the heavy chain carboxyterminal half (HC50) of botulinum neurotoxin type A (BoNT/A) and RABV glycoprotein (G). To promote surface expression and incorporation of HC50/A into RABV virions, the RABV glycoprotein (G) ER translocation sequence, various fragments of RABV ectodomain (ED) and cytoplasmic domain were fused to HC50/A. The HC50/A chimeric proteins were expressed on the surface of cells infected with all of the recombinant RABVs, however, the highest level of surface expression was detected by utilizing 30 amino acids of the RABV G ED (HV50/A-E30). Our results also indicated that this chimeric protein was effectively incorporated into RABV virions. Immunization of mice with inactivated RABV-HC50/A-E30 virions induced a robust anti-HC50/A IgG antibody response that efficiently neutralized circulating BoNT/A in vivo, and protected mice against 1000 fold the lethal dose of BoNT/A.

Basic tetrapeptides as potent intracellular inhibitors of type A botulinum neurotoxin protease activity.

Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to arginine-arginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose- and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200+ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development.

Strategy for treating motor neuron diseases using a fusion protein of botulinum toxin binding domain and streptavidin for viral vector access: work in progress.

Although advances in understanding of the pathogenesis of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) have suggested attractive treatment strategies, delivery of agents to motor neurons embedded within the spinal cord is problematic. We have designed a strategy based on the specificity of botulinum toxin, to direct entry of viral vectors carrying candidate therapeutic genes into motor neurons. We have engineered and expressed fusion proteins consisting of the binding domain of botulinum toxin type A fused to streptavidin (SAv). This fusion protein will direct biotinylated viral vectors carrying therapeutic genes into motor nerve terminals where they can enter the acidified endosomal compartments, be released and undergo retrograde transport, to deliver the genes to motor neurons. Both ends of the fusion proteins are shown to be functionally intact. The binding domain end binds to mammalian nerve terminals at neuromuscular junctions, ganglioside GT1b (a target of botulinum toxin), and a variety of neuronal cells including primary chick embryo motor neurons, N2A neuroblastoma cells, NG108-15 cells, but not to NG CR72 cells, which lack complex gangliosides. The streptavidin end binds to biotin, and to a biotinylated Alexa 488 fluorescent tag. Further studies are in progress to evaluate the delivery of genes to motor neurons in vivo, by the use of biotinylated viral vectors.

Identification and biochemical characterization of small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A.

An integrated strategy that combined in silico screening and tiered biochemical assays (enzymatic, in vitro, and ex vivo) was used to identify and characterize effective small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A (BoNT/A). Virtual screening was initially performed by computationally docking compounds of the National Cancer Institute (NCI) database into the active site of BoNT/A light chain (LC). A total of 100 high-scoring compounds were evaluated in a high-performance liquid chromatography (HPLC)-based protease assay using recombinant full-length BoNT/A LC. Seven compounds that significantly inhibited the BoNT/A protease activity were selected. Database search queries of the best candidate hit [7-((4-nitro-anilino)(phenyl)methyl)-8-quinolinol (NSC 1010)] were performed to mine its nontoxic analogs. Fifty-five analogs of NSC 1010 were synthesized and examined by the HPLC-based assay. Of these, five quinolinol derivatives that potently inhibited both full-length BoNT/A LC and truncated BoNT/A LC (residues 1 to 425) were selected for further inhibition studies in neuroblastoma (N2a) cell-based and tissue-based mouse phrenic nerve hemidiaphragm assays. Consistent with enzymatic assays, in vitro and ex vivo studies revealed that these five quinolinol-based analogs effectively neutralized BoNT/A toxicity, with CB 7969312 exhibiting ex vivo protection at 0.5 microM. To date, this is the most potent BoNT/A small-molecule inhibitor that showed activity in an ex vivo assay. The reduced toxicity and high potency demonstrated by these five compounds at the biochemical, cellular, and tissue levels are distinctive among the BoNT/A small-molecule inhibitors reported thus far. This study demonstrates the utility of a multidisciplinary approach (in silico screening coupled with biochemical testing) for identifying promising small-molecule BoNT/A inhibitors.

Bivalent recombinant vaccine for botulinum neurotoxin types A and B based on a polypeptide comprising their effector and translocation domains that is protective against the predominant A and B subtypes.

The botulinum neurotoxins (BoNTs) are a large family of extremely potent, neuroparalytic, dichain proteins which act at the peripheral nervous system. The wide genetic diversity observed with this neurotoxin family poses a significant challenge for the development of an effective botulinum vaccine. The present study describes a vaccine development platform based on protein fragments representing the N-terminal two-thirds of each toxin molecule. These fragments, designated LH(N), comprise the light chain and translocation domains of each neurotoxin and are devoid of any neuron-binding activity. Using codon-optimized genes, LH(N) fragments derived from BoNT serotypes A and B were expressed in Escherichia coli in high yield with >1 g of purified, soluble fragment recoverable from 4.5 liter-scale fermentations. The protective efficacy of LH(N)/A was significantly enhanced by treatment with formaldehyde, which induced intramolecular cross-linking but virtually no aggregation of the fragment. A single immunization of the modified fragment protected mice from challenge with a 10(3) 50% lethal dose (LD(50)) of BoNT/A(1) with an 50% effective dose (ED(50)) of 50 ng of the vaccine. In similar experiments, the LH(N)/A vaccine was shown to protect mice against challenge with BoNT/A subtypes A(1), A(2), and A(3), which is the first demonstration of single-dose protection by a vaccine against the principal toxin subtypes of BoNT/A. The LH(N)/B vaccine was also highly efficacious, giving an ED(50) of approximately 140 ng to a challenge of 10(3) LD(50) of BoNT/B(1). In addition, LH(N)/B provided single-dose protection in mice against BoNT/B(4) (nonproteolytic toxin subtype).

The N-terminal half of the receptor domain of botulinum neurotoxin A binds to microdomains of the plasma membrane.

Botulinum neurotoxin type A (BoNT/A) is largely employed in human therapy because of its specific inhibition of peripheral cholinergic nerve terminals. BoNT/A binds to them rapidly and with high specificity via its receptor binding domain termed HC. Recent evidence indicate that BoNT/A interacts specifically with polysialogangliosides and with a luminal loop of the synaptic vesicle protein SV2 via the C-terminal half of HC. Here we show that the N-terminal half of HC binds to sphingomyelin-enriched membrane microdomains and that it has a defined interaction with phosphatidylinositol phosphates (PIP). We have identified a PIP binding site in this half of HC and we show how this interaction could predispose BoNT/A for membrane insertion, which is the step subsequent to binding, in the four-steps route leading BoNT/A inside nerve terminals.

Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins.

The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.

Trivalent vaccine against botulinum toxin serotypes A, B, and E that can be administered by the mucosal route.

Most reports dealing with vaccines against botulinum toxin have focused on the injection route of administration. This is unfortunate, because a mucosal vaccine is likely to be more efficacious for patients and pose fewer risks to health care workers and to the environment. Therefore, efforts were made to generate a mucosal vaccine that provides protection against the botulinum serotypes that typically cause human illness (serotypes A, B, and E). This work demonstrated that carboxy-terminal peptides derived from each of the three serotypes were able to bind to and penetrate human epithelial barriers in vitro, and there was no cross inhibition of membrane binding and transcytosis. The three polypeptides were then tested in vivo as a trivalent vaccine that could be administered to mice by the intranasal route. The results indicated that the mucosal vaccine evoked high secretory titers of immunoglobulin A (IgA), as well as high circulating titers of IgG and IgA, and it also evoked a high level of resistance to challenge with toxin. The immunoglobulin responses and the levels of resistance to challenge were increased by coadministration of adjuvants, such as chitosan and vitamin E. At least three mechanisms were identified to account for the antibody-induced resistance: (i) blockade of toxin absorption across epithelial cells, (ii) enhanced clearance of toxin from the circulation, and (iii) blockade of toxin action at the neuromuscular junction. These results are a compelling demonstration that a mucosal vaccine against multiple serotypes of botulinum toxin has been identified.

Mucosal vaccine targeting improves onset of mucosal and systemic immunity to botulinum neurotoxin A.

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 beta-trefoil (Hcbetatre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hcbetatre or Hcbetatre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hcbetatre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hcbetatre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hcbetatre-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 Hcbetatre-dosed mice. Mice immunized with adjuvanted Hcbetatre-Ad2F or Hcbetatre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hcbetatre-Ad2F or Hcbetatre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hcbetatre Ab titers even in the absence of adjuvant. This study shows that the Hcbetatre structure can confer protective immunity and that use of Hcbetatre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.

Expression, purification and transduction of PEP-1-botulinum neurotoxin type A (PEP-1-BoNT/A) into skin.

Botulinum neurotoxin A (BoNT/A) has been used therapeutically to treat muscular hypercontractions and sudomotor hyperactivity and it has been reported that BoNT/A might have analgesic properties in headache. PEP-1 peptide is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In this study, a BoNT/A gene were fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-BoNT/A fusion protein. The expressed and purified PEP-1-BoNT/A fusion proteins were efficiently transduced into cells in a time- and dose-dependent manner when added exogenously in a culture medium. In addition, immunohistochemical analysis revealed that PEP-1-BoNT/A fusion protein efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin. These results suggest that PEP-1-BoNT/A fusion protein provide an efficient strategy for therapeutic delivery in various human diseases related to this protein.

Expression of an anti-botulinum toxin A neutralizing single-chain Fv recombinant antibody in transgenic tobacco.

Botulinum neurotoxins (BoNTs) are the most poisonous substances known and are thus classified as high-risk threats for use as bioterror agents. To examine the potential of transgenic plants as bioreactors for the production of BoNT antidotes, we transformed tobacco with an optimized, synthetic gene encoding a botulinum neurotoxin A (BoNT/A) neutralizing single-chain Fv (scFv) recombinant antibody fragment. In vitro mouse muscle twitch assays demonstrated the functional utility of this scFv extracted from tobacco for neutralizing the paralytic effects of BoNT/A at neuromuscular junctions. Based on the efficiency of the scFv capture process and the dose required to antidote a human being, 1-2 ha of this tobacco could yield up to 4 kg of scFv, which would be enough to contribute to the manufacture of 1,000,000 therapeutic doses of a monoclonal antibody (mAb) cocktail capable of neutralizing the effects of BoNT poisoning. Transgenic plants could provide an inexpensive production platform for expression of multiple mAbs toward the creation of polyclonal therapies (i.e. pooled mAbs) as the next improvement in recombinant antibody therapy.

Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications.

Clostridium botulinum neurotoxins are potently toxic proteins of 150 kDa with specific endopeptidase activity for SNARE proteins involved in vesicle docking and release. Following treatment with trypsin, a fragment of botulinum neurotoxin serotype A that lacks the C-terminal domain responsible for neuronal cell binding, but retains full catalytic activity, can be obtained. Known as the LH(N) fragment, we report the development of a recombinant expression and purification scheme for the isolation of comparable fragments of neurotoxin serotypes B and C. Expressed as maltose-binding protein fusions, both have specific proteolytic sites present between the fusion tag and the light chain to facilitate removal of the fusion, and between the light chain endopeptidase and the H(N) translocation domains to facilitate activation of the single polypeptide. We have also used this approach to prepare a new variant of LH(N)/A with a specific activation site that avoids the need to use trypsin. All three LH(N)s are enzymatically active and are of low toxicity. The production of specifically activatable LH(N)/A, LH(N)/B, and LH(N)/C extends the opportunities for exploitation of neurotoxin fragments. The potential utility of these fragments is discussed.

Modulation of botulinum neurotoxin A catalytic domain stability by tyrosine phosphorylation.

Botulinum neurotoxin A (BoNT A) is a substrate of the Src family of tyrosine kinases. Here, we report that the BoNT A light chain (LC) is phosphorylated in the tyrosine-71 located at N-terminus. Covalent modification of this residue notably increases the thermal stability of the endopeptidase activity, without affecting its catalytic efficacy. Similarly, mutation of this residue specifically affected the protein stability but not its endopeptidase function. Fusion of the Tat-translocating domain to the N-terminus of the enzyme produced a cell permeable, functional enzyme, as evidenced by immunocytochemistry and by the cleavage of cytosolic SNAP25 in intact PC12 cells. Noteworthy, truncation of cellular SNAP25 was reduced in cells when the Src kinase activity was inhibited with a specific antagonist, implying that tyrosine phosphorylation of BoNT A LC modulates the in vivo proteolytic activity of the neurotoxin. Taken together, these findings substantiate the tenet that tyrosine phosphorylation of BoNT A LC could be an important modulatory strategy of the neurotoxin stability and suggest that the phosphorylated neurotoxin may be a relevant molecule in vivo.