Epitope characterization of sero-specific monoclonal antibody to Clostridium botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/A (H(C)50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant H(C)50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the N-terminal subdomain of H(C)50/A (H(CN)25/A) comprising amino acid residues (985)WTLQDTQEIKQRVVF(999), an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis

Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

Background: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components o

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

Evidence That Botulinum Toxin Receptors on Epithelial Cells and Neuronal Cells Are Not Identical: Implications for Development of a Non-Neurotropic Vaccine

Botulinum toxin typically interacts with two types of cells to cause the disease botulism. The toxin initially interacts with epithelial cells in the gut or airway to undergo binding, transcytosis, and delivery to the general circulation. The toxin then interacts with peripheral cholinergic nerve endings to undergo binding, endocytosis, and delivery to the cytosol. The receptors for botulinum toxin on nerve cells have been identified, but receptors on epithelial cells remain unknown. The initial toxin binding site on nerve cells is a polysialoganglioside, so experiments were performed to determine whether polysialogangliosides are also receptors on epithelial cells. A series of single mutant and dimutant forms of the botulinum toxin type A binding domain (HC50) were cloned and expressed. One of these (dimutant HC50 AW1266L,Y1267S) was shown to have lost its ability to bind nerve cells (phrenic nerve-hemidiaphragm preparation), yet it retained its ability to bind and cross human epithelial monolayers (T-84 cells). In addition, the wild-type HC50 and the dimutant HC50 displayed the same ability to undergo binding and transcytosis (absorption) in a mouse model. The fact that the dimutant retained the ability to cross epithelial barriers but did not possess the ability to bind to nerve cells was exploited to create a mucosal vaccine that was non-neurotropic. The wild-type HC50 and non-neurotropic HC50 proved to be comparable in their abilities to: 1) evoke a circulating IgA and IgG response and 2) evoke protection against a substantial challenge dose of botulinum toxin

BoNT neutralization activity of the 4LCA human monoclonal antibody in the mouse protection assay.

*<p>mice were healthy at 30 days.</p

Kinetic exclusion analysis (KinExA) of the solution binding properties of the 4LCA antibody and BoNT/A LC.

<p>(A) The equilibrium binding affinity was determined by titrating recombinant BoNT/A LC into solutions of fixed 4LCA concentration. At equilibrium, free 4LCA was captured by passage through a bead matrix conjugated to LC and measured by binding of a fluorescent secondary antibody. The data were fit using the manufacturer's software to a bimolecular binding model, giving an optimized KD value of 31±5×10⁻¹² M. (B) The association rate constant (k_on) was measured by mixing 4LCA (200 pM) with LC (10 pM) and measuring the concentration of free antibody concentration over time. The average k_on value for 4LCA was estimated to be 2.3±0.1×10⁶ M⁻¹ s⁻¹, which gives a calculated k_off value of 7.1×10⁻⁵ s⁻¹.</p

4LCA variable domain usage, CDR3 sequences, and mutation status.

<p>CDR3, complementary determining region 3. Muts, mutations in the entire IgG HC and LC variable domains.</p

Inhibition of the catalytic activity of the BoNT/A metalloprotease by the 4LCA antibody <i>in vitro</i>.

<p>In this fluorescence resonance energy transfer assay (FRET), cleavage of the SNAPtide by BoNT/A LC un-quenches FITC fluorescence, which is quantified as arbitrary fluorescence units. In triplicate samples, the SNAPtide was incubated with BoNT/A alone or with the 6A or 4LCA antibodies and fluorescence was measured. Standard deviation (s.d.) is indicated by error bars.</p

The effect of BoNT/A-specific antibodies on endocytosis of BoNT/A by Neuro-2a cells.

<p>Neuro-2a cells were cultured with fluorescently-labeled BoNT/A (red), with or without human antibody (green), and visualized by confocal microscopy. Panel (A) No antibody. (B) 6A antibody. (C) 4LCA antibody. In each panel, 4 images of each field are shown: BoNT/A (BT), Antibody (AB), merged (M), and phase contrast (P).</p