HK40008973B - Cellular vamp cleavage assay - Google Patents

Description

Cellular VAMP cleavage assay

Invention Field

This invention relates to a cell-based assay for VAMP-mediated clostridium neurotoxin cleavage.

background

Bacteria in the genus Clostridium produce highly potent and specific protein toxins that can poison the neurons and other cells they are delivered to. Examples of such clostridial toxins include neurotoxins produced by Clostridium tetani (tetanus neurotoxin) and Clostridium botulinum (botulinum neurotoxin serotypes A to G), as well as neurotoxins produced by Clostridium baratii and Clostridium butyricum.

Clostridium neurotoxins act by inhibiting cholinergic transmission in the peripheral nervous system, particularly at the neuromuscular junction. In nature, clostridium neurotoxins are synthesized as single-chain polypeptides, which are post-translational modified through proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at specific cleavage sites, often referred to as activation sites, located between cysteine residues that provide the interchain disulfide bonds. It is this double-chain form that is the active form of the toxin. These two chains are called the heavy chain (H chain) (with a molecular weight of approximately 100 kDa) and the light chain (L chain) (with a molecular weight of approximately 50 kDa). The H chain contains an N-terminal translocation component (H _N domain) and a C-terminal targeting component (H _C domain). The cleavage site is located between the L chain and the H _N domain. After the H _C domain binds to its target neuron and internalizes the bound toxin into the cell via endosomes, the H _N domain transfers the L chain across the endosome membrane and into the cytosol, and the L chain provides protease function (also known as a non-cytotoxic protease).

Non-cytotoxic proteases function by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP25, VAMP, or syntaxin) – see Gerald K (2002), “Cell and Molecular Biology” (4th edition), John Wiley & Sons, Inc. The acronym SNARE comes from the term soluble NSF attachment receptor, where NSF stands for N-ethylmaleimide sensitive factor. The acronym SNAP25 comes from the term 25 kDalton synaptosome-associated protein. The acronym VAMP comes from the term vesicle-associated membrane protein. SNARE proteins are components of intracellular vesicle fusion, and thus components of molecules secreted from the cell via vesicle transport. The protease function is zinc-dependent endopeptidase activity, exhibiting high substrate specificity for SNARE proteins. Therefore, once delivered to the desired target cell, non-cytotoxic proteases are able to inhibit secretion from the target cell. Clostridium neurotoxin's L-chain protease is a non-cytotoxic protease that cleaves SNARE proteins. L-chain proteases of BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, and TeNT cleave VAMP (also known as synaptobrevins), L-chain proteases of BoNT/A and BoNT/E cleave SNAP25, and L-chain proteases of BoNT/C cleave SNAP25 and synaptic fusion proteins, leading to inhibition of neurotransmitter release and subsequent nerve paralysis (Rossetto, O. et al., “Botulinum neurotoxins: genetic, structural, and mechanistic insight”. Nature Reviews Microbiology 12.8 (2014): 535-549) (Zhang et al., “Identification and characterization of a novel botulinum neurotoxin”; Nature Communications, 2017, 8: 14130).

Clostridium neurotoxins target and enter neurons by binding to their specific receptors via their receptor-binding domains ( _HC ), which are well-defined in the literature (Schiavo, G., Matteoli, M. & Montecco, C. Neurotoxins affected neuroxocytosis, Physiol Rev., 2000, 80, 717-766). Receptor binding determines the efficacy and specificity of BoNTs in recognizing neurons. BoNT/B, DC, and G share two homologous synaptic vesicle proteins, synaptic binding proteins I and II (Syt I/II), as their receptors, while BoNT/A, E, D, F, and TeNT use another synaptic vesicle protein, SV2. In addition to protein receptors, all BoNTs require lipid co-receptors, gangliosides, which are abundant on the neuronal surface.

Clostridium neurotoxins are used as a therapy to treat motor and autonomic nervous system disorders. Several BoNT/A products (including [list of products]) and one BoNT/B product have been approved by regulatory agencies for human use.

Traditionally, the potency of BoNT drug products has been quantified in MLD50 (mice lethal dose 50), with one unit corresponding to the median lethal intraperitoneal dose in mice. However, the MLD50 unit used for botulinum toxin is not a standardized unit. In fact, different manufacturers of commercially available toxins use assays that differ significantly in their choice of dilution buffers (Straughan, DW, 2006, ATLA 34(3), 305-313; Hambleton and Pickett, Hambleton, P. and AMPickett., 1994, Journal of the Royal Society of Medicine 87.11:719). Furthermore, due to ethical concerns and recent regulations, animal-based potency assays are now preferred to be avoided. Cell-based potency assays avoid the requirements of animal testing and related ethical issues. After cell poisoning, the potency of clostridial neurotoxins can be measured by assessing the extent of SNARE cleavage within target cells, for example, by Western blotting. Alternatively, sandwich ELISA methods can be used to detect and quantify SNARE cleavage. These methods are applicable to SNAP25 and synaptic fusion protein cleavage (see, for example, Pellett, Sabine et al., "Comparison of the primary rat spinal cord cell (RSC) assay and the mouse bioassay for botulinum neurotoxin type: A potency determination." Journal of pharmacological and toxicological methods 61.3(2010):304-310; Fernández-Salas, Ester et al., "Botulinum neurotoxin serotype: A specific cell-based potency assay to replace the mouse bioassay." PLoS). One 7.11(2012):e49516; Kalandakanond S et al., “Cleavage of intracellular substrates of botulinum toxins A, C and D in mammalian target tissue” The Journal of Pharmacology and Experimental Therapeutics (2001):749-755; Peng L et al., “Cytotoxicity of botulinum neurotoxins reveals a direct role of syntaxin 1 and SNAP25 in neuron survival” Nature Communications (2013):4:1472). However, to date, VAMP cleavage products from cell lysates have been shown to be... It is extremely difficult to detect. In fact, although VAMP cleavage-specific antibodies that recognize cleaved VAMP are available and are suitable for detecting VAMP cleavage in extracellular or cellular fractionation assays (Hallis, Bassam, B.A. James, and Clifford C. Shone. "Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities." Journal of Clinical Microbiology 34.8 (1996): 1934-1938; Kegel, B. et al., "An in vitro assay for detection of tetanus neurotoxin activity: Using antibodies for recognizing The proteolytically generated cleavage product. Toxicology in Vitro 21.8 (2007): 1641-1649; Fujita-Yoshigaki, Junko et al., "Vesicle-associated Membrane Protein 2 Is Essential for cAMP-regulated Exocytosis in Rat Paraid Acinar Cells The Inhibition of cAMP-dependent Amylase Release by Botulinum Neurotoxin B." Journal of Biological Chemistry 271.22 (1996): 13130-13134). These antibodies did not detect cleaved VAMP in cell studies.

The prevailing consensus in the field to date is that cleaved VAMP products must degrade very rapidly in cells and therefore do not promote the duration of BoNT effects (Foran, Patrick G., et al., "Evaluation of the Therapeutic Usefulness of Botulinum Neurotoxin B, C1, E, and F Compared with the Long Lasting Type A Basis for Distinct Durations of Inhibition of Exocytosis in Central Neurons." Journal of Biological Chemistry 278.2(2003):1363-1371). However, Schiavo et al. have shown that two VAMPs are present in small synaptic vesicle fractions prepared from the rat cerebral cortex when treated with BoNT/B and TeNT using Coomassie blue staining. P-cleavage products (Schiavo G. et al. (1992), Tetanus and Botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359p832-835). This suggests that VAMP products from cell origin can exist, although synaptosomal formulations may not contain all the proteases present in total cell lysates. Dong et al. (2004) described that in PC12 cells expressing YFP-Syb(FL)-CFP, signals from both VAMP products were detectable after BoNT/B cleavage, and that the YFP-N-terminal cleaved VAMP product dispersed into the cytosol and redistributed itself to the nucleus, while the CFP-C-terminal product remained localized to vesicles (Dong M. et al. (2004), Using fluorescent sensors to detect bo Tulinum neurotoxin activity in vivo and in living cells. PNAS 101(41)p14701-14706). This evidence suggests the possible existence of two VAMP products, but unknown cellular processes hinder the recognition of antibodies against the N-terminal product. Therefore, standard practice is to measure VAMP cleavage solely by the disappearance of the full-length band (see, for example, Pellett, Sabine et al., "A neuronal cell-based botulinum neurotoxina assay for highly sensitive and specific detection of neutralizing serum antibodies." FEBS Letters 581.25(2007):4803-4808.; Whitemarsh, Regina CM, et al., "Novel application of human..." n neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection (Biological Sciences: Applied Biological Sciences). Toxicological Sciences, 2012, 126(2):426–435. However, signal loss-based assays convey the risk of error because total protein load can vary, leading to either an overestimation or underestimation of VAMP loss. Housekeeping proteins unchanged during BoNT treatment can be used to normalize the density of control proteins against VAMP loss. The drawback here is that the signals between antibodies need to be matched and on a linear scale to detect any differences for normalization purposes. While this may be a reasonable indication of BoNT activity qualitatively, it is not suitable for more detailed quantification, particularly for determining the potency of drug BoNT formulations.

Therefore, cell VAMP cutting assays based on signal reading gain are required.

Invention Overview

In a first aspect, the present invention provides an antigenic polypeptide comprising a VAMP epitope, wherein the antigenic polypeptide comprises 10 to 65 amino acid residues, wherein the VAMP epitope comprises an amino acid sequence having at least 90% identity with a VAMP sequence comprising at least 8 amino acid residues at the C-terminus of a clostridium neurotoxin cleavage site directly located in the VAMP.

On the other hand, the present invention relates to polypeptides comprising the antigenic polypeptide of the present invention, wherein the polypeptide does not contain a region having more than 17, preferably 16, more preferably 15 consecutive amino acids having 100% sequence identity with the naturally occurring VAMP amino acid sequence.

On the other hand, the present invention provides an antigen protein comprising a polypeptide according to the present invention covalently linked to a carrier.

On the other hand, the present invention provides the use of the antigenic peptide or protein according to the invention for generating antibodies against C-terminal VAMP cleavage products. In one embodiment, the epitope of the present invention is used to generate polyclonal antibodies against C-terminal VAMP cleavage products. In another embodiment, the epitope of the present invention is used to generate monoclonal antibodies against C-terminal VAMP cleavage products.

On the other hand, the present invention provides antibodies that bind to the antigenic peptides or proteins of the present invention.

On the other hand, the present invention provides the use of the antibody according to the invention in the assay of signal cells that enhance VAMP cleavage by VAMP cleavage of Clostridium neurotoxin.

On the other hand, the present invention provides a method for determining VAMP cleavage in cells by cleaving VAMP with a clostridium neurotoxin, comprising:

a) Contact the cells with clostridium neurotoxin under conditions suitable for the activity of clostridium neurotoxin;

b) After VAMP is cleaved by a clostridial neurotoxin that cleaves VAMP, the cytoplasmic contents of the cells are contacted with a first detection antibody against the C-terminal VAMP cleavage product under conditions suitable for the binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the invention; and

c) Detect the binding of the first detection antibody to the C-terminal VAMP cleavage product using a suitable method.

On the other hand, the present invention provides a method for determining immune tolerance to clostridium neurotoxins that cleave VAMP in subjects, comprising:

a) Adding a clostridium neurotoxin that cleaves VAMP to test samples obtained from subjects;

b) Under conditions suitable for Clostridium neurotoxin activity, bring the cells into contact with the test sample from step a);

c) After VAMP is cleaved by a clostridium neurotoxin that cleaves VAMP, the cytoplasmic contents of the cell are contacted with a first detection antibody against the C-terminal VAMP cleavage product under conditions suitable for the first detection antibody to bind to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the invention.

d) Detect the binding of the first detection antibody to the C-terminal VAMP cleavage product using a suitable method;

e) Quantify the amount of C-terminal VAMP cleavage product bound to the first detection antibody;

f) Repeat steps a) to e) using a negative control sample instead of the test sample; and

g) Compare the amount of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein the detection of a lower amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (e) relative to the amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (f) indicates the presence of a neutralizing antibody against the clostridium neurotoxin that cleaves VAMP.

On the other hand, the present invention provides a kit comprising cells susceptible to neurotoxin poisoning by cleaved VAMP; and a first detection antibody against cleaved VAMP, wherein the first detection antibody is an antibody according to the present invention.

Invention Details

This invention is based on the inventors’ discovery that antibodies suitable for cell VAMP cleavage assays can be generated based on the gain of signal readings.

In particular, the inventors have demonstrated that detecting epitopes located C-terminally at the BoNT cleavage site is crucial for in vitro detection of VAMP cleavage. Indeed, the inventors have demonstrated successful detection of neuronal VAMP2 cleavage products by Western blotting (WB) using antibodies binding to epitopes located C-terminally at BoNT/F and/or BoNT/D and/or BoNT/B cleavage sites adjacent to BoNT/D and/or BoNT/F and/or BoNT/B cleavage sites. Specifically, this antibody is capable of detecting both full-length VAMP and cleavage products in cell lysates. This tool enables the quantitative assessment of the potency of BoNT in signal gain cell assays by monitoring the appearance of the cleaved VAMP products.

In a first aspect, the present invention provides an antigenic polypeptide comprising a VAMP epitope, wherein the antigenic polypeptide comprises 10 to 65 amino acid residues, wherein the VAMP epitope comprises an amino acid sequence having at least 90% identity with a VAMP sequence comprising at least 8 amino acid residues located directly at the C-terminus of the clostridium neurotoxin cleavage site in the VAMP.

As used herein, the term "clostridial neurotoxin" refers to any polypeptide that enters neurons and inhibits the release of neurotransmitters. This process involves the binding of the neurotoxin to low- or high-affinity receptors, the internalization of the neurotoxin, the translocation of the neurotoxin's endopeptidase portion into the cytoplasm, and the enzymatic modification of the neurotoxin substrate. More specifically, the term "clostridial neurotoxin" includes any polypeptide produced by Clostridium species that enters neurons and inhibits the release of neurotransmitters, as well as such polypeptides produced through recombinant or chemical techniques. It is a double-stranded form and is the active form of the neurotoxin. These two chains are called the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. Clostridial neurotoxins include botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT). BoNT serotypes A through G can be distinguished based on inactivation by specific neutralizing antisera; this serotype classification is related to percentage sequence identity at the amino acid level. Based on percentage sequence identity of amino acids, BoNT proteins of a given serotype are further subdivided into different subtypes.

An example of the BoNT/A neurotoxin amino acid sequence is provided as SEQ ID NO: 1 (UniProt accession number A5HZZ9). An example of the BoNT/B neurotoxin amino acid sequence is provided as SEQ ID NO: 2 (UniProt accession number B1INP5). An example of the BoNT/C neurotoxin amino acid sequence is provided as SEQ ID NO: 3 (UniProt accession number P18640). An example of the BoNT/D neurotoxin amino acid sequence is provided as SEQ ID NO: 4 (UniProt accession number P19321). An example of the BoNT/E neurotoxin amino acid sequence is provided as SEQ ID NO: 5 (NCBI RefSeq accession number WP_003372387). An example of the BoNT/F neurotoxin amino acid sequence is provided as SEQ ID NO: 6 (UniProt accession number Q57236). An example of the BoNT/G neurotoxin amino acid sequence is provided as SEQ ID NO: 7 (NCBI RefSeq accession number WP_039635782). An example of the BoNT/X neurotoxin amino acid sequence is provided as SEQ ID NO: 41 (Genbank accession number BAQ12790.1). An example of the TeNT amino acid sequence is provided as SEQ ID NO: 8 (UniProt accession number P04958).

The term "H_{_C}domain" as used in this article refers to the functionally distinct region of the neurotoxin heavy chain, with a molecular weight of approximately 50 kDa, which enables the neurotoxin to bind to receptors located on the surface of target cells. The H_{_C} domain consists of two structurally distinct subdomains: the "H _{_CN}subdomain" (the N-terminal portion of the H_{_C} domain) and the "H _{_CC}subdomain" (the C-terminal portion of the H_{_C} domain), each with a molecular weight of approximately 25 kDa.

The term “LH _N domain” as used in this article refers to neurotoxins that lack an H _C domain and consist of an endopeptidase domain (“L” or “light chain”) and a domain responsible for the translocation of endopeptidase into the cytoplasm (the H _N domain of the heavy chain).

Exemplary L, _HN , _HCN , and _HCC structural domains are shown in Table 1.

Table 1 - Exemplary L, _HN , _HCN , and _HCC structural domains

The reference sequences used for identification described above should be considered as guidelines, as slight variations may occur depending on the subserotype. For example, US 2007/0166332 (incorporated hereinth by reference in its entirety) cites a slightly different Clostridium sequence.

Vesicle-associated membrane proteins (VAMPs) belong to the SNARE protein family. They share similar structures and participate in vesicle fusion and exocytosis, particularly neurotransmitter release. VAMPs are members of the SNARE protein family, also known as the synaptic vesicle protein family, which includes members such as VAMP1, VAMP2 (also called synaptic vesicle protein), VAMP3 (also called cellubrevin), VAMP4, VAMP5, VAMP7 (also called SYBL1, or tetanus-insensitive VAMP), VAMP8 (also called endobrevin), YKT6, SEC22A, etc. VAMP1, VAMP2, and VAMP3 are cleaved by the light chains of BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, and TeNT. BoNT/X can also cleave VAMP4, VAMP5, and YKT6.

The term “VAMP epitope” as used in this article refers to a portion of the VAMP protein that the antibody binds to.

In a preferred embodiment, the antigenic polypeptide of the present invention comprises 10 to 65, 10 to 60, 10 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16 or 10 to 15 amino acid residues, preferably 10 to 15 amino acid residues. For example, the antigenic polypeptide of the present invention may be composed of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 amino acid residues.

In a preferred embodiment, the antigenic polypeptide of the present invention comprises or is composed of a VAMP epitope, the VAMP epitope comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a VAMP sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acid residues directly located at the C-terminus of the clostridial neurotoxin cleavage site in the VAMP.

The amino acid sequences of naturally occurring VAMPs, particularly rat and human VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and YKT6, and their corresponding clostridial neurotoxin VAMP cleavage sites are shown in Table 2 and Figure 1.

Table 2 - Clostridium neurotoxin VAMP cleavage sites

In one implementation, VAMP is selected from VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and/or YKT6.

In one implementation, VAMP is selected from VAMP1, VAMP2 and/or VAMP3.

In one implementation, VAMP is selected from VAMP4, VAMP5 and/or YKT6.

In a preferred embodiment, the VAMP is a human VAMP, more preferably human VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and/or YKT6.

In one implementation, VAMP is selected from human VAMP1, VAMP2 and/or VAMP3.

In one implementation, VAMP is selected from human VAMP4, VAMP5 and/or YKT6.

In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT/F cleaved VAMP epitope, wherein at least 8 amino acid residues are located directly at the C-terminus of the BoNT/F cleavage site in the VAMP.

BoNT/F VAMP epitopes, and more particularly instances of BoNT/F VAMP1, VAMP2, and/or VAMP3 epitopes, include:

·KLSELDDRADALQ(SEQ ID NO：15)

·QKLSELDDRADALQ(SEQ ID NO：16)

·KLSELDDRAD(SEQ ID NO：17)

·KLSELDDRADALQAGAS(SEQ ID NO：18)

·DQKLSELDDRADALQ (SEQ ID NO: 31).

In one embodiment, the BoNT/F VAMP epitope, particularly BoNT/F VAMP1, VAMP2, and/or VAMP3 epitopes, comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NO: 15 to SEQ ID NO: 18 and SEQ ID NO: 31. In a preferred embodiment, the BoNT/F VAMP epitope comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with KLSELDDRADALQ (SEQ ID NO: 15). In a more preferred embodiment, the BoNT/F VAMP epitope comprises or consists of KLSELDDRADALQ (SEQ ID NO: 15).

In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT/D VAMP epitope, wherein at least 8 amino acid residues are located directly at the C-terminus of the BoNT/D cleavage site in the VAMP.

BoNT/D VAMP epitopes, and more particularly instances of BoNT/D VAMP1, VAMP2, and/or VAMP3 epitopes, include:

·KLSELDDRADALQ(SEQ ID NO：15)

·LSELDDRADALQ(SEQ ID NO：19)

·LSELDDRADA(SEQ ID NO：20)

·LSELDDRADALQAGAS (SEQ ID NO: 21).

In one embodiment, the BoNT/D VAMP epitope, particularly BoNT/D VAMP1, VAMP2, and/or VAMP3 epitopes, comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with or consisting of a sequence selected from SEQ ID NO: 15 and SEQ ID NO: 19 to SEQ ID NO: 21. In a preferred embodiment, the BoNT/D VAMP epitope comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with or consisting of an amino acid sequence of KLSELDDRADALQ (SEQ ID NO: 15). In a more preferred embodiment, the BoNT/D VAMP epitope comprises or consists of KLSELDDRADALQ (SEQ ID NO: 15).

In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT/F5 or BoNT/FA cleaved VAMP epitope, wherein at least 8 amino acid residues are located directly at the C-terminus of the BoNT/F5 or BoNT/FA cleavage site in the VAMP.

BoNT/F5 or BoNT/FA VAMP epitopes, and more particularly instances of BoNT/F5 or BoNT/FA VAMP1, VAMP2 and/or VAMP3 epitopes, include:

·ERDQKLSELDDRA(SEQ ID NO：32)

·LERDQKLSELDDRA(SEQ ID NO：33)

·VLERDQKLSELDDRA (SEQ ID NO: 34).

In one embodiment, the BoNT/F5 or BoNT/FA VAMP epitope, particularly BoNT/F5 or BoNT/FAVAMP1, VAMP2 and/or VAMP3 epitopes, comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with or constitutes an amino acid sequence selected from SEQ ID NO: 32 to SEQ ID NO: 34. In a preferred embodiment, the BoNT/F5 or BoNT/FA VAMP epitope comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with or constitutes an amino acid sequence of ERDQKLSELDDRA (SEQ ID NO: 32). In a more preferred embodiment, the BoNT/F5 or BoNT/FAVAMP epitope comprises or consists of ERDQKLSELDDRA (SEQ ID NO: 32).

In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT/B or TeNT VAMP epitope, wherein at least 8 amino acid residues are located directly at the C-terminus of the BoNT/B or TeNT cleavage site in the VAMP.

BoNT/B or TeNT VAMP epitopes, and more particularly instances of BoNT/B or TeNT VAMP1, VAMP2 and/or VAMP3 epitopes, include:

·FETSAAKLKRKYW(SEQ ID NO：22)

·FESSAAKLKRKYW(SEQ ID NO：23)

·QFETSAAKLKRKYW(SEQ ID NO：24)

·FETSAAKLKR(SEQ ID NO：25)

·FETSAAKLKRKYWWKN(SEQ ID NO：26)

·ETSAAKLKRKYWWK(SEQ ID NO：48)

·FETSAAKLKRKYWWK(SEQ ID NO：49)

·QFESSAAKLKRKYW(SEQ ID NO：50)

·FESSAAKLKR(SEQ ID NO：51)

·FESSAAKLKRKYWWK (SEQ ID NO: 52).

In one embodiment, the BoNT/B or TeNT VAMP epitope, particularly BoNT/B or TeNT VAMP1, VAMP2 and/or VAMP3 epitopes, comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from SEQ ID NO: 22 to SEQ ID NO: 26 and SEQ ID NO: 48 to SEQ ID NO: 52. In a preferred embodiment, the BoNT/B or TeNT VAMP epitope comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with ...% or 100% identity with an amino acid sequence having at least 90%, 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 100% identity with an amino acid sequence having at least 90%, 98% or 100% identity with an amino acid sequence having at least 90%, 98% or 100% identity with an amino acid sequence having at least 90%, 98% or 99% or 100% identity In a more preferred embodiment, the BoNT/B or TeNT VAMP epitope comprises or consists of FETSAAKLKRKYW (SEQ ID NO: 22) or FETSAAKLKRKYWWK (SEQ ID NO: 49). Surprisingly, antibodies binding to the latter epitope allow for the detection of not only BoNT/B VAMP cleavage but also BoNT/F VAMP cleavage.

In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT/G VAMP epitope, wherein at least 8 amino acid residues are located directly at the C-terminus of the BoNT/G cleavage site in the VAMP.

BoNT/G VAMP epitopes, and more particularly instances of BoNT/G, VAMP1, VAMP2 and/or VAMP3 epitopes, include:

·AKLKRKYWWKN(SEQ ID NO：27)

·AAKLKRKYWWKN(SEQ ID NO：28)

·AKLKRKYWWKNCKM(SEQ ID NO：29)

·AKLKRKYWWKNLKM (SEQ ID NO: 30).

In one embodiment, the BoNT/G VAMP epitope, particularly BoNT/G VAMP1, VAMP2, and/or VAMP3 epitopes, comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NO: 27 to SEQ ID NO: 30. In a preferred embodiment, the BoNT/G VAMP epitope comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with AKLKRKYWWKN (SEQ ID NO: 27). In a more preferred embodiment, the BoNT/G VAMP epitope comprises or consists of AKLKRKYWWKN (SEQ ID NO: 27).

In one embodiment of the antigenic polypeptide of the present invention, the VAMP epitope is a BoNT/X VAMP epitope, wherein at least 8 amino acid residues are located directly at the C-terminus of the BoNT/X cleavage site in the VAMP.

BoNT/X VAMP epitopes, and more particularly instances of BoNT/X VAMP1, VAMP2, and/or VAMP3 epitopes, include:

·ADALQAGASQF(SEQ ID NO：53)

·ADALQAGASQ(SEQ ID NO：54)

·RADALQAGASQF(SEQ ID NO：55)

·ADALQAGASQFE(SEQ ID NO：56)

·ADALQAGASVF(SEQ ID NO：57)

·ADALQAGASV(SEQ ID NO：58)

·ADALQAGASVFE(SEQ ID NO：59)

·RADALQAGASVF(SEQ ID NO：60)

·RADALQAGAS (SEQ ID NO: 61).

In one embodiment, the BoNT/X VAMP epitope, particularly BoNT/X VAMP1, VAMP2, and/or VAMP3 epitopes, comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NO: 53 to SEQ ID NO: 61. In a preferred embodiment, the BoNT/X VAMP epitope comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ADALQAGASQF (SEQ ID NO: 53). In a more preferred embodiment, the BoNT/X VAMP epitope comprises or consists of ADALQAGASQF (SEQ ID NO: 53).

Other instances of the BoNT/X VAMP tabletop, and more particularly the BoNT/X VAMP4 tabletop, include:

·SESLSDNATAF(SEQ ID NO: 62)

·SESLSDNATA(SEQ ID NO：63)

·KSESLSDNATAF(SEQ ID NO：64)

·SESLSDNATAFS (SEQ ID NO: 65).

In one embodiment, the BoNT/X VAMP epitope, particularly the BoNT/X VAMP4 epitope, comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NO: 62 to SEQ ID NO: 65. In a preferred embodiment, the BoNT/X VAMP epitope comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SESLSDNATAF (SEQ ID NO: 62). In a more preferred embodiment, the BoNT/X VAMP epitope comprises or consists of SESLSDNATAF (SEQ ID NO: 62).

Other instances of the BoNT/X VAMP tabletop, and more particularly the BoNT/X VAMP5 tabletop, include:

·SDQLLDMSSTF(SEQ ID NO：66)

·SDQLLDMSST(SEQ ID NO: 67)

·RSDQLLDMSSTF(SEQ ID NO: 68)

·SDQLLDMSSTFN(SEQ ID NO: 69)

·SDQLLDMSSAF(SEQ ID NO：70)

·SDQLLDMSSA(SEQ ID NO：71)

·RSDQLLDMSSAF(SEQ ID NO：72)

·SDQLLDMSSAFS(SEQ ID NO：73)

·RSDQLLDMSS (SEQ ID NO: 74).

In one embodiment, the BoNT/X VAMP epitope, particularly the BoNT/X VAMP5 epitope, comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NO: 66 to SEQ ID NO: 74. In a preferred embodiment, the BoNT/X VAMP epitope comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SDQLLDMSSTF (SEQ ID NO: 66). In a more preferred embodiment, the BoNT/X VAMP epitope comprises or consists of SDQLLDMSSTF (SEQ ID NO: 66).

Other instances of the BoNT/X VAMP tabletop, and more particularly the BoNT/X YKT6 tabletop, include:

·SEVLGTQSKAF(SEQ ID NO：75)

·SEVLGTQSKA(SEQ ID NO：76)

·KSEVLGTQSKAF(SEQ ID NO：77)

·SEVLGTQSKAFY (SEQ ID NO: 78).

In one embodiment, the BoNT/X VAMP epitope, particularly the BoNT/X YKT6 epitope, comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence selected from SEQ ID NO: 75 to SEQ ID NO: 78. In a preferred embodiment, the BoNT/X VAMP epitope comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEVLGTQSKAF (SEQ ID NO: 75). In a more preferred embodiment, the BoNT/X VAMP epitope comprises or consists of SEVLGTQSKAF (SEQ ID NO: 75).

In this paper, the “percentage of sequence identity” between two or more nucleic acid or amino acid sequences is a function of the number of identical nucleotides or amino acids at the same positions shared by the aligned sequences. Therefore, % identity can be calculated as the number of identical nucleotides or amino acids at each position in the alignment divided by the total number of nucleotides or amino acids in the aligned sequence, multiplied by 100. The calculation of % sequence identity can also take into account the number of vacancies and the length of each vacancy that needs to be introduced to optimize the alignment of two or more sequences. Sequence comparisons and determination of percentage identity between two or more sequences can be performed using specific mathematical algorithms familiar to those skilled in the art, such as global alignment mathematical algorithms (e.g., described by Needleman and Wunsch, J. Mol. Biol. 48(3), 443-453, 1972).

On the other hand, the present invention relates to polypeptides comprising the antigenic polypeptide according to the invention, wherein the polypeptide does not contain more than 17, 16, 15, 14, 13, 12, 11, 10, preferably 16, more preferably 15 consecutive amino acids having 100% sequence identity with the naturally occurring VAMP amino acid sequence. It will be readily understood by those skilled in the art that such polypeptides are also antigenic.

In a preferred embodiment, the polypeptide comprises a covalent linker, preferably at its N-terminus and/or C-terminus. Examples of suitable covalent linkers according to the present invention are provided below.

On the other hand, the present invention provides an antigen protein comprising the polypeptide of the present invention covalently linked to a carrier.

Preferably, the carrier is a non-immunogenic or weakly immunogenic protein. Examples of suitable carriers include keyhole hemocyanin (KLH), ovalbumin (OVA), thyroglobulin (THY), bovine serum albumin (BSA), soybean trypsin inhibitor (STI), or multiple attachment peptide (MAP).

In one embodiment, the antigen protein comprises a covalent linker between the polypeptide of the present invention (which may already contain the linker as described above) and a carrier. The linker may be one or more natural or non-natural amino acids, as is well known in the art, which can form covalent bonds with other amino acids (of the polypeptide and/or carrier) due to the presence of active groups at their N-terminus, C-terminus, and/or side chains. Notably, amino acids having a primary amine group (-NH2) in their N-terminus and/or side chain (e.g., lysine) can react with amino acids having a carboxyl group (-COOH) in their C-terminus and/or side chain (e.g., aspartic acid or glutamic acid) to form covalent bonds; amino acids having a thiol group (-SH) in their side chain (e.g., cysteine or selenocysteine) can react with amino acids having a thiol group (-SH) in their side chain (e.g., cysteine or selenocysteine) to form covalent bonds. For example, the covalent linker may be a cysteine added to the C-terminus or N-terminus of the polypeptide of the present invention, which forms a disulfide bridge with another cysteine added to or present in the carrier. Alternatively, the covalent linker can be in the form of several amino acids forming the spacer; for example, the linker can be a peptide containing a non-charged amino acid (e.g., glycine, alanine, valine, leucine, or serine) with a small side-chain R group. Examples of suitable spacers of the present invention include G-spacers, such as GGG, GGGG, and GGGGS, or A-spacers, such as AAA, AAAA, and AAAAV. In one embodiment, the linker consists of about 1 to about 30 amino acid residues, preferably about 2 to about 25 amino acid residues, more preferably about 3 to about 20 amino acid residues, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues.

On the other hand, the present invention provides the use of the antigenic peptide or protein according to the invention for generating antibodies against C-terminal VAMP cleavage products. In one embodiment, the epitope of the present invention is used to generate polyclonal antibodies against C-terminal VAMP cleavage products. In another embodiment, the epitope of the present invention is used to generate monoclonal antibodies against C-terminal VAMP cleavage products.

Methods for generating antibodies are well known in the art; see, for example, Greenfield, Edward A., ed., Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, 2014; Leenars, Marlies, and Coenraad FM Hendriksen. "Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations." Ilar Journal 46.3 (2005): 269-279.

Polyclonal antibodies conjugated to VAMP epitopes as described herein can be produced by injecting animals, such as mammals like rabbits, goats, mice, hamsters, or monkeys, or eggs, such as chicken eggs, with the antigenic peptides or proteins of the present invention. Polyclonal antibodies against the VAMP epitopes disclosed herein can be isolated from animals (e.g., from blood) or eggs and further purified using well-known techniques, such as protein affinity chromatography to obtain IgG fractions, or by affinity purification against the VAMP epitopes used to generate the antibodies. Some contract research organizations offer customized antibody production services; for example, Eurogentec offers a “rapid 28-day program” in which immunization is performed on day 0, followed by three booster immunizations on days 7, 10, and 18, with blood collected in the middle of day 21 and finally on day 28. This is an example of a general technique for polyclonal antibody production well-known in the art.

Monoclonal antibodies binding to VAMP epitopes as described herein can be generated using hybridoma methods. See, for example, Chapter 7, Greenfield, Edward A., ed., Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, 2014. In short, a host animal, such as a mammal like a rabbit, goat, mouse, hamster, or monkey, is exposed to one or more injections of the antigenic peptides or proteins of the present invention to induce the production or production of lymphocytes that specifically bind to and cleave VAMPs. Antibody titers in the immunized animal can be monitored over time using standard techniques, such as ELISA (enzyme-linked immunosorbent assay). Alternatively, suitable cell culture lines can be used to immunize lymphocytes in vitro. At an appropriate time post-immunization, such as when antibody titers are at their highest, antibody-producing cells are isolated from the animal. Typically, peripheral blood lymphocytes are used if human cells are desired, or spleen cells or lymph node cells are used if non-human mammalian cells are desired. Hybridoma cells are formed by fusing isolated antibody-producing cells with immortalized cell lines using a suitable fusion agent (e.g., polyethylene glycol). Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human-derived myeloma cells. Typically, mouse myeloma cell lines are fused with spleen cells harvested from appropriately immunized mice to generate hybridomas. Preferred immortalized cell lines are mouse myeloma cell lines sensitive to a medium containing hypoxanthine, aminopterin, and thymidine (HAT). Any of many myeloma cell lines can be used as fusion partners according to standard techniques, such as P3-NS1/1-Ag4-1, P3-x63-Ag8.653, or Sp2/O-Ag14 myeloma lines. Hybridoma cells generated by the fusion are then selected using HAT medium, which kills unfused and non-productive fused myeloma cells (unfused spleen cells die after a few days of culture because they are not transformed). The presence of monoclonal antibodies binding to VAMP epitopes as described herein can then be determined in the medium in which the hybridoma cells are grown. For example, cleaved VAMP-positive medium can be used in immunoprecipitation assays, in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), or hybridoma supernatants can be screened in cell-based activity assays. The binding affinity of monoclonal antibodies can also be determined, for example, by Scatchard analysis. See, for example, Peter J. Munson and David Rodbard, Ligand: A Versatile Computerized Approach For Characterization of Ligand-Binding Systems, 107(1) Anal. Biochem. 220-239 (1980). After identifying the desired hybridoma cells, clones derived from single cells are isolated using a limiting dilution procedure until a clonal cell line expressing the desired monoclonal antibody is obtained. Alternatively, monoclonal antibodies binding to VAMP epitopes as described herein can be generated by screening recombinant combined immunoglobulin libraries, such as antibody phage display libraries, with the antigenic peptides, proteins, or peptides of the present invention. Kits for generating and screening phage display libraries are commercially available, such as recombinant phage antibody systems (Amersham GE Healthcare, Piscataway, NJ); and phage display kits (Stratagene, La JoIIa, CA). Additionally, examples of methods and reagents for generating and screening antibody display libraries can be found, for example, in U.S. Patents 5,223,409 to Ladner et al.; 5,712,089 to Borrebaeck et al.; 5,885,793 to Griffiths et al.; 5,962,255 to Griffiths et al.; 5,969,108 to McCafferty et al.; 6,010,884 to Griffiths et al.; 6,017,732 to Jespers et al.; 6,027,930 to Borrebaeck et al.; 6,140,471 to Johnson et al.; and 6,172,197 to McCafferty et al., each of which is incorporated herein by reference in its entirety.

On the other hand, the present invention provides antibodies that bind to the antigenic peptides or proteins of the present invention.

In one implementation, the antibody is a polyclonal antibody.

In one implementation, the antibody is a monoclonal antibody.

The binding affinity between an antibody and an antigenic peptide or protein can be assessed by measuring the equilibrium dissociation constant (K_{_d} ), which measures the rate at which a new antibody-antigen complex is formed, equal to the rate at which the antibody-antigen complex dissociates at equilibrium. The equilibrium dissociation constant is denoted by M and defined by the K_d/Ka ratio at equilibrium, where Ka is the antibody binding rate constant and K_d is the antibody dissociation rate constant. K _{_d} = [Ab] x [Ag] / [Ab + Ag], where [Ab] is the molar concentration of the antibody, [Ag] is the molar concentration of the antigen, and [Ab + Ag] is the molar concentration of the antibody-antigen complex, where all concentrations are those of the components at equilibrium. A smaller equilibrium dissociation constant indicates a stronger binding affinity between the antibody and its antigen, or a higher binding affinity between the antibody and antigen.

In one embodiment, the _KD between the antibody and the antigenic peptide or protein epitope of the present invention is less than ^10⁻⁶ M. In a preferred embodiment, the _KD between the antibody and the antigenic peptide or protein of the present invention is less than ^10⁻⁷ M. In a more preferred embodiment, the _KD between the antibody and the antigenic peptide or protein of the present invention is less than ^10⁻⁸ M. In a more preferred embodiment, the _KD between the antibody and the antigenic peptide or protein of the present invention is less than ^10⁻⁹ M. In a more preferred embodiment, the _KD between the antibody and the antigenic peptide or protein of the present invention is less than ^10⁻¹⁰ M. In a more preferred embodiment, the _KD between the antibody and the antigenic peptide or protein of the present invention is less than ^10⁻¹¹ M. In a more preferred embodiment, the _KD between the antibody and the antigenic peptide or protein of the present invention is less than ^10⁻¹² M.

On the other hand, the present invention provides the use of the antibody according to the invention in signal gain cell assays of VAMP cleavage via VAMP cleavage of Clostridium neurotoxin.

In one implementation, the use is in vitro or ex vivo.

On the other hand, the present invention provides a method for determining VAMP cleavage in cells by cleaving VAMP with a clostridium neurotoxin, comprising:

a) Under conditions suitable for the activity of Clostridium neurotoxin, the cells are brought into contact with Clostridium neurotoxin;

b) After VAMP is cleaved by a clostridial neurotoxin that cleaves VAMP, the cytoplasmic contents of the cells are contacted with a first detection antibody against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the invention; and

c) Detect the binding of the first detection antibody to the C-terminal VAMP cleavage product using a suitable method.

In one embodiment, the method according to the invention further includes d) quantifying the amount of C-terminal VAMP cleavage product bound to the first detection antibody by a suitable method.

In one embodiment of the method of the present invention, step b) includes contacting the cytoplasmic contents of the cell with the second detection antibody against the full-length VAMP under conditions suitable for the binding of the second detection antibody to the full-length VAMP; step c) includes detecting the binding of the second detection antibody to the full-length VAMP by a suitable method; and step d) includes quantifying the amount of full-length VAMP bound to the second detection antibody by a suitable method.

In one implementation, the method is an in vitro or ex vivo method.

Those skilled in the art will understand that an increase in the amount of C-terminal VAMP cleavage product bound to the first antibody and/or a decrease in the amount of full-length VAMP bound to the second detection antibody indicates an increase in VAMP cleavage caused by the clostridial neurotoxin that cleaves the VAMP.

In one implementation, the second detection antibody is the same as the first detection antibody and binds to the C-terminal VAMP cleavage product and the full-length VAMP.

In one alternative embodiment, the second detection antibody differs from the first detection antibody and binds to the full-length VAMP but not to the C-terminal VAMP cleavage product. Suitably, the second detection antibody binds to the VAMP epitope at the N-terminus of the clostridial neurotoxin cleavage site. Examples of suitable antibodies include commercially available antibodies such as ab3347 (Abcam) or ab181869 (Abcam).

In one specific implementation, a method for determining the VAMP cleavage of clostridium neurotoxins that cleave VAMP in cells includes:

a) Under conditions suitable for the activity of Clostridium neurotoxin, the cells are brought into contact with Clostridium neurotoxin;

b) To cause the cytoplasmic contents of the cells to undergo:

• After VAMP is cleaved by a clostridial neurotoxin that cleaves VAMP, the first detection antibody against the C-terminal VAMP cleavage product is contacted under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the invention that binds to both the C-terminal VAMP cleavage product and the full-length VAMP; and

• The second detection antibody is contacted, which binds to the full-length VAMP but not to the C-terminal VAMP cleavage product;

c) Detect using suitable methods

• The binding of the primary antibody to the C-terminal VAMP cleavage product and the full-length VAMP; and

• The second detection method involves the binding of the antibody to the full-length VAMP; and

d) Quantify in an appropriate way:

• The combined amount of the C-terminal VAMP cleavage product and the full-length VAMP bound to the first detection antibody; and

• The amount of full-length VAMP that binds to the second detection antibody.

Those skilled in the art will understand that the reduction in the amount of full-length VAMP bound to the second detection antibody, and the lack of change in the combined amount of full-length and C-terminal VAMP cleavage products bound to the first detection antibody, indicate VAMP cleavage by the clostridial neurotoxin that cleaves the VAMP.

In another specific implementation, the method for determining the VAMP cleavage of clostridium neurotoxin in cells by cleaving VAMP includes:

a) Contact the cells with clostridium neurotoxin under conditions suitable for the activity of clostridium neurotoxin;

b) After cleaving VAMP with a clostridium neurotoxin that cleaves VAMP, the cytoplasmic contents of the cells are contacted with a first detection antibody against the C-terminal VAMP cleavage product under conditions suitable for the first detection antibody to bind to the C-terminal VAMP, wherein the first detection antibody is an antibody according to the invention that binds the C-terminal VAMP cleavage product and the full-length VAMP.

c) Detect using suitable methods

• Binding of the primary antibody to the C-terminal VAMP cleavage product; and

• The first detection step is the binding of the antibody to the full-length VAMP;

The signal generated by the binding of the first detection antibody to the C-terminal VAMP cleavage product can be distinguished from the signal generated by the binding of the first detection antibody to the full-length VAMP; and

d) Quantify in an appropriate way:

• The amount of C-terminal VAMP cleavage product bound to the first detection antibody; and

• The amount of full-length VAMP that binds to the first detection antibody.

Those skilled in the art will understand that the increase in the amount of C-terminal VAMP cleavage product bound to the first antibody and the decrease in the amount of full-length VAMP bound to the first detection antibody indicate an increase in VAMP cleavage by clostridial neurotoxins that cleave the VAMP.

On the other hand, the present invention provides a method for determining immune tolerance to clostridium neurotoxin that cleaves VAMP in subjects, comprising:

a) Adding a clostridium neurotoxin that cleaves VAMP to test samples obtained from subjects;

b) Under conditions suitable for Clostridium neurotoxin activity, bring the cells into contact with the test sample from step a);

c) After VAMP is cleaved by a clostridium neurotoxin that cleaves VAMP, the cytoplasmic contents of the cell are contacted with a first detection antibody against the C-terminal VAMP cleavage product under conditions suitable for the first detection antibody to bind to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the present invention.

d) Detect the binding of the first detection antibody to the C-terminal VAMP cleavage product using a suitable method;

e) Quantify the amount of C-terminal VAMP cleavage product bound to the first detection antibody;

f) Repeat steps a) to e) using a negative control sample instead of the test sample; and

g) Compare the amount of C-terminal VAMP cleavage product bound to the first detection antibody in steps (e) and (f), wherein a lower amount of C-terminal VAMP cleavage product bound to the first detection antibody is detected in step (e) relative to the amount of C-terminal VAMP cleavage product bound to the first detection antibody in step (f), indicating the presence of a neutralizing antibody against the clostridium neurotoxin that cleaves VAMP.

In one implementation, step f) further includes repeating steps a) to e) with a positive control sample.

As used herein, the term "neutralizing antibody against clostridium neurotoxin cleaving VAMP" refers to any antibody that, under physiological conditions, binds to clostridium neurotoxin cleaving VAMP in a manner that reduces or prevents the therapeutic effect of clostridium neurotoxin cleaving VAMP in a subject.

In one implementation, the subject is a mammal. In a preferred implementation, the subject is a human.

In one implementation, the samples are selected from blood, plasma, serum, and lymph obtained from the subject.

Test samples can be obtained from subjects before exposure to clostridium neurotoxin cleaved by VAMP, after a single treatment with clostridium neurotoxin cleaved by VAMP, or after multiple treatments with clostridium neurotoxin cleaved by VAMP. In one specific embodiment, the test samples are obtained from subjects resistant to treatment with clostridium neurotoxin cleaved by VAMP.

As used herein, the term "control sample" means any sample in which the test sample is known to be present or absent, and includes both negative and positive control samples. Regarding neutralizing antibodies against clostridium neurotoxin cleaved by VAMP, negative control samples may be obtained from individuals who have never been exposed to clostridium neurotoxin cleaved by VAMP, and may include, but are not limited to, samples from the same individuals who provided the test sample but were obtained prior to treatment with clostridium neurotoxin cleaved by VAMP; samples from different individuals who have never been exposed to clostridium neurotoxin cleaved by VAMP; and pooled samples from multiple different individuals who have never been exposed to clostridium neurotoxin cleaved by VAMP.

Regarding neutralizing antibodies against clostridial neurotoxins that cleave VAMP, positive control samples can be obtained from individuals who exhibit immune resistance to clostridial neurotoxins that cleave VAMP, including but not limited to individuals who test positive in patient-based test assays; individuals who test positive in in vivo bioassays; and individuals who exhibit hyperimmunity, such as subjects vaccinated against clostridial neurotoxins that cleave VAMP.

In one implementation, the method is an in vitro or ex vivo method.

In one embodiment of the method for determining immune resistance, step c) includes contacting the cytoplasmic contents of the cell with the second detection antibody against the full-length VAMP under conditions suitable for the binding of the second detection antibody to the full-length VAMP; step d) includes detecting the binding of the second detection antibody to the full-length VAMP by a suitable method; and step e) includes quantifying the amount of full-length VAMP bound to the second detection antibody.

In one implementation, the second detection antibody is the same as the first detection antibody and binds to the C-terminal VAMP cleavage product and the full-length VAMP.

In one alternative embodiment, the second detection antibody differs from the first detection antibody and binds to the full-length VAMP but not to the C-terminal VAMP cleavage product. Suitably, the second detection antibody binds to the VAMP epitope at the N-terminus of the clostridial neurotoxin cleavage site. Examples of suitable antibodies include commercially available antibodies such as ab3347 (Abcam) or ab181869 (Abcam).

In one specific implementation, methods for determining immune tolerance to clostridial neurotoxins that cleave VAMP in subjects include:

a) Adding a clostridium neurotoxin that cleaves VAMP to test samples obtained from subjects;

b) Under conditions suitable for Clostridium neurotoxin activity, bring the cells into contact with the test sample from step a);

c) To cause the cytoplasmic contents of the cells to undergo...

• After VAMP cleavage by clostridial neurotoxin, the first detection antibody against the C-terminal VAMP cleavage product is contacted under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the invention that binds to both the C-terminal VAMP cleavage product and the full-length VAMP; and

• The second detection antibody is contacted, which binds to the full-length VAMP but not to the C-terminal VAMP cleavage product;

d) Detect using appropriate methods

• The binding of the primary antibody to the C-terminal VAMP cleavage product and the full-length VAMP; and

• The second detection method is to detect the binding of the antibody to the full-length VAMP;

e) Quantification

• The combined amount of the C-terminal VAMP cleavage product and the full-length VAMP bound to the first detection antibody; and

• The amount of full-length VAMP that binds to the second detection antibody;

f) Repeat steps a) to e) using a negative control sample instead of the test sample; and

g) Compare the amounts of C-terminal VAMP cleavage products bound to the first detection antibody in steps (e) and (f), wherein the lower amount of C-terminal VAMP cleavage products bound to the first detection antibody and full-length VAMP and/or the higher amount of full-length VAMP bound to the second detection antibody detected in step (e) relative to the corresponding amount in step (f) indicates the presence of a neutralizing antibody against the clostridial neurotoxin that cleaves the VAMP.

In another specific implementation, methods for determining immune tolerance to clostridium neurotoxins that cleave VAMP in subjects include:

a) Adding a clostridium neurotoxin that cleaves VAMP to test samples obtained from subjects;

b) Under conditions suitable for Clostridium neurotoxin activity, bring the cells into contact with the test sample from step a);

c) After VAMP cleavage by clostridium neurotoxin cleaving VAMP, the cytoplasmic contents of the cell are contacted with the first detection antibody against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is an antibody according to the invention that binds the C-terminal VAMP cleavage product and the full-length VAMP.

d) Detect using appropriate methods

• The binding of the primary antibody to the C-terminal VAMP cleavage product and the full-length VAMP; and

• The first detection step is the binding of the antibody to the full-length VAMP;

The signal generated by the binding of the first detection antibody to the C-terminal VAMP cleavage product can be distinguished from the signal generated by the binding of the first detection antibody to the full-length VAMP.

e) Quantification

• The amount of C-terminal VAMP cleavage product bound to the first detection antibody; and

• The amount of full-length VAMP that binds to the first detection antibody;

f) Repeat steps a) to e) using a negative control sample instead of the test sample;

g) Compare the amounts of C-terminal VAMP cleavage products bound to the first detection antibody in steps (e) and (f), wherein a lower amount of C-terminal VAMP cleavage products bound to the first detection antibody in step (e) and/or a higher amount of full-length VAMP bound to the first detection antibody, relative to the corresponding amount in step (f), indicates the presence of a neutralizing antibody against the clostridial neurotoxin that cleaves the VAMP.

In this article, "clostridial neurotoxin that cleaves VAMP" refers to clostridial neurotoxin that binds to receptors on target cells, transfers clostridial light chains (L) into the cytosol, and then proteases to cleave VAMP, thereby disrupting the secretion of the molecule via cellular vesicle transport.

Preferably, in the method or use of the present invention, the clostridial neurotoxin that cleaves VAMP comprises a BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, or TeNT light chain. Suitably, the BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, or TeNT light chain comprises a sequence selected from:

-Amino acid residues 1 to 441 of SEQ ID NO: 2, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it.

-Amino acid residues 1 to 442 of SEQ ID NO: 4, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it.

-Amino acid residues 1 to 439 of SEQ ID NO: 6, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it.

-Amino acid residues 1 to 446 of SEQ ID NO: 7, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it.

-Amino acid residues 1 to 439 of SEQ ID NO: 41, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it, and

-Amino acid residues 1 to 456 of SEQ ID NO: 8, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it.

It should be understood that the BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT light chains described herein have the ability to cleave VAMP.

In one embodiment of the method or use of the present invention, the clostridial neurotoxin that cleaves VAMP is selected from BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, and TeNT. Suitably, BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, or TeNT comprises a sequence selected from the following:

-SEQ ID NO: 2 or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it,

-SEQ ID NO: 4, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it,

-SEQ ID NO: 6, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it,

-SEQ ID NO: 7, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it,

-SEQ ID NO: 41, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it, and

-SEQ ID NO: 8, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with it.

It should be understood that the BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X or TeNT clostridium neurotoxins described herein have the ability to bind to receptors on target cells, transfer clostridium light chains to the cytoplasm, and cleave VAMP.

In one embodiment, the VAMP-cleaving clostridial neurotoxin is a mosaic neurotoxin. As used herein, the term "mosaic neurotoxin" refers to a naturally occurring clostridial neurotoxin that contains at least one functional domain from another clostridial neurotoxin (e.g., a different serotype of clostridial neurotoxin), which typically does not contain at least one functional domain. Examples of naturally occurring VAMP-cleaving mosaic neurotoxins are BoNT/DC and BoNT/FA. BoNT/DC contains the L chain and _HN domain of serotype D and the _HC domain of serotype C, Nakamura K et al., “Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan.” Vet. Microbiol. (2010):140:147–154. BoNT/FA, on the other hand, consists of the BoNT/F5 light chain, the _HN domain closely associated with subtype F1, and the BoNT/ _A1HC domain (Pellett, Sabine et al., “Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain.” mSphere 1.1 (2016):e00100-15).

In one embodiment, the clostridial neurotoxin that cleaves the VAMP is a mosaic neurotoxin selected from BoNT/DC and BoNT/FA.

In one embodiment, the clostridial neurotoxin that cleaves VAMP is a chimeric neurotoxin. As used herein, the term "chimeric neurotoxin" refers to a neurotoxin comprising one or more domains derived from a first neurotoxin and one or more domains derived from a second neurotoxin. For example, a chimeric neurotoxin may comprise an LH_{_N} domain derived from a first neurotoxin and an H_{_C} domain derived from a second neurotoxin. Another example of a chimeric neurotoxin is a neurotoxin comprising an LH_{_N}H_{_CN} domain derived from a first neurotoxin and an H_{_CC} domain derived from a second neurotoxin. Examples of chimeric neurotoxins are provided in GB 1607901.4 (not yet disclosed), which is incorporated herein by reference.

In one embodiment, the clostridium neurotoxin that cleaves VAMP is a chimeric neurotoxin comprising:

- Light chains (L) from BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, or TeNT

- H _N structural domains from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, or TeNT.

- H _CN structural domains from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, or TeNT.

- _HCC structural domains from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, or TeNT.

At least two of the domains are derived from different Clostridium neurotoxins.

In one embodiment, the clostridium neurotoxin that cleaves VAMP is a chimeric neurotoxin comprising:

- The LH _N domain derived from the first clostridium neurotoxin selected from BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, or TeNT.

- The _{HCN- HCC} domain of a second clostridium neurotoxin selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, or TeNT.

The first and second Clostridium neurotoxins are different.

In one embodiment, the clostridium neurotoxin that cleaves VAMP is a chimeric neurotoxin comprising:

- The LH _N H _CN domain derived from the first clostridium neurotoxin selected from BoNT/B, BoNT/D, BoNT/F, BoNT/G, BoNT/X, or TeNT.

- The _HCC domain of a second clostridium neurotoxin selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, or TeNT.

The first and second Clostridium neurotoxins are different.

Clostridium neurotoxins that cleave VAMP can be modified neurotoxins or derivatives thereof, including but not limited to those described below. Modified neurotoxins or derivatives may contain one or more amino acids that have been modified compared to the natural (unmodified) form of the neurotoxin, or may contain one or more inserted amino acids that are not present in the natural (unmodified) form of the toxin. For example, a modified clostridium neurotoxin may have a modified amino acid sequence in one or more domains relative to the sequence of a natural (unmodified) clostridium neurotoxin. Such modifications can modify functional aspects of the neurotoxin, such as biological activity or persistence.

The modified clostridium neurotoxin that cleaves VAMP, as described herein, retains the ability to bind to receptors on target cells, transfer light chains into the cytoplasm, and cleave VAMP.

Modified VAMP-cleaving clostridium neurotoxins may have one or more modifications (e.g., modified H_{_C} domains) in the amino acid sequence of the heavy chain, wherein the modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) neurotoxin. Such modifications in the H_{_C} domain may include modifications to residues in the ganglioside-binding site or the protein receptor-binding site of the H _{_CC} domain, altering the binding to ganglioside receptors and/or protein receptors of target nerve cells. Examples of such modified neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are incorporated herein by reference in their entirety. For example, the H_{_CC} domain of the BoNT/B neurotoxin contains at least one substituted, added, or deleted amino acid residue, which has the effect of increasing the binding affinity of the BoNT/B H_CC domain to human Syt II compared to the native BoNT/B H _{_CC} sequence. Suitable amino acid residue substitutions, additions, or deletions in the BoNT/B _HCC subdomain have been disclosed in WO2013/180799 and the unpublished PCT/US2016/024211 (both incorporated herein by reference). Suitable amino acid residue substitutions, additions, or deletions in _{the HCC} subdomain include substitution mutations selected from the group consisting of: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinations thereof.

In one implementation, the clostridial neurotoxin that cleaves VAMP is a retargeted neurotoxin. As used herein, the term "retargeted neurotoxin" (also known as "targeted secretion inhibitor,""TSI,""TVEMP," or "TEM") refers to a clostridial neurotoxin containing a targeting moiety (TM) that binds to a non-clostridial receptor. The TM may replace part or all of the H_{_C} or H_{_CC} domains of the clostridial neurotoxin heavy chain. Examples of retargeted neurotoxins are disclosed in WO96/33273, WO98/07864, WO00/10598, WO01/21213, WO01/53336; WO02/07759, WO2005/023309, WO2006/026780, WO2006/099590, WO2006/056093, WO2006/059105, WO2006/059113, WO2007/138339, WO2007/106115, WO2007/106799, WO2009/150469, WO2009/150470, and WO2010/055358. , WO2010/020811, WO2010/138379, WO2010/138395, WO2010/138382, WO2011/020052, WO2011/020 056, WO2011/020114, WO2011/020117, WO2011/20119, WO2012/156743, WO2012/134900, WO2012/1 34897, WO2012/134904, WO2012/134902, WO2012/135343, WO2012/135448, WO2012/135304, WO2012/134902, WO2014/033441, WO2014/128497, WO2014/053651, WO2015/004464, all of these references are incorporated herein by reference.

Examples of cells suitable for the methods or uses of the present invention include prokaryotic cells, such as Escherichia coli cells, yeast cells, insect cells, animal cells, mammalian cells, human cells, mouse cells, primate cells, and/or neuronal cells. Preferably, the cells are neuronal cells, particularly cells highly sensitive to BoNT.

Cells highly sensitive to BoNT are cells susceptible to BoNT poisoning. In some embodiments, cells highly sensitive to BoNT are, for example, cells poisoned by, at approximately 500 pM or less, approximately 400 pM or less, approximately 300 pM or less, approximately 200 pM or less, approximately 100 pM or less, approximately 90 pM or less, approximately 80 pM or less, approximately 70 pM or less, approximately 60 pM or less, approximately 50 pM or less, approximately 40 pM or less, approximately 30 pM or less, approximately 20 pM or less, approximately 10 pM or less, approximately 9 pM or less, approximately 8 pM or less, approximately 7 pM or less, approximately 6 pM or less, approximately 5 pM or less, approximately 4 pM or less. Small, approximately 3 pM or less, approximately 2 pM or less, approximately 1 pM or less, approximately 0.9 pM or less, approximately 0.8 pM or less, approximately 0.7 pM or less, approximately 0.6 pM or less, approximately 0.5 pM or less, approximately 0.4 pM or less, approximately 0.3 pM or less, approximately 0.2 pM, approximately 0.1 pM or less, approximately 90 fM or less, approximately 80 fM or less, approximately 70 fM or less, approximately 60 fM or less, approximately 50 fM or less, approximately 40 fM or less, approximately 30 fM or less, approximately 20 fM or less, or approximately 10 fM or less of BoNT-sensitive cells.

Preferably, the cells are highly sensitive to BoNTs that cleave VAMP (as defined above).

In one implementation, the cell is a primary neuronal cell that is highly sensitive to BoNT, such as a cortical neuron, hippocampal neuron, and/or spinal neuron. For example, the cell is a rat cortical neuron.

In one implementation, the cells are derived from neuronal cell lines that are highly sensitive to BoNT, such as BE(2)-M17, Kelly, LA1-55n, N1E-115, N4TG3, N18, Neuro-2a, NG108-15, PC12, SH-SY5Y, SiMa and/or SK-N-BE(2)-C.

In one implementation, the cell is a neuronal cell derived from stem cells, particularly a neuronal cell derived from induced pluripotent stem cells (iPS cells), such as neurons, DopaNeurons iCell glutamatergic neurons, iCell MotoNeurons (Cell Dynamics) cortical neurons, neural stem cells (Axol Biosciences), Peri.4U neurons, CNS.4U neurons, Dopa.4UNeurons (Axiogenesis), MNP cells (Lonza), cortical neurons, motor neurons (iStem), and/or iPSC-derived neural cells (MTI-GlobalStem).

In one embodiment, cells can be modified using recombinant technology to express high levels of VAMP, such as VAMP1, VAMP2, VAMP3, VAMP4, VAMP5 and/or YKT6, more preferably VAMP1, VAMP2 and/or VAMP3.

In one embodiment where the neurotoxin that cleaves VAMP is BoNT/B, BoNT/DC, or BoNT/G, the cells express high levels of synaptokinetic protein I and/or synaptokinetic protein II (Syt I/Syt II). In another embodiment where the neurotoxin that cleaves VAMP is BoNT/B, BoNT/DC, or BoNT/G, the cells are modified using recombinant technology to express high levels of synaptokinetic protein I and/or synaptokinetic protein II (Syt I/Syt II).

In one embodiment where the neurotoxin that cleaves VAMP is BoNT/FA, BoNT/F, BoNT/D, or TeNT, the cells express high levels of synaptic vesicle protein (SV2). In another embodiment where the neurotoxin that cleaves VAMP is BoNT/FA, BoNT/F, BoNT/D, or TeNT, the cells are modified by recombinant technology to express high levels of synaptic vesicle protein (SV2).

As used in this article, "conditions suitable for clostridial neurotoxin activity" refers to the conditions (e.g., temperature, pH, cofactors, etc.) under which clostridial neurotoxins can bind to clostridial neurotoxin receptors present on the cell membrane, transfer the clostridial neurotoxin light chain to the cytoplasm, and cleave VAMP.

In one embodiment of the method of the present invention, conditions suitable for clostridial neurotoxin activity may include incubation at about 37°C for about 1 hour to about 48 hours. In one embodiment of the method of the present invention, conditions suitable for clostridial neurotoxin activity may include incubation at about 37°C for about 2 hours to about 36 hours. In one embodiment of the method of the present invention, conditions suitable for clostridial neurotoxin activity may include incubation at about 37°C for about 4 hours to about 24 hours.

For example, conditions suitable for clostridium neurotoxin activity may include incubation at 37°C for 24 hours.

As used herein, “conditions suitable for the binding of the first detection antibody to the cleaved VAMP” and “conditions suitable for the binding of the second detection antibody to the full-length VAMP” refer to the conditions (e.g., temperature, pH, cofactor, etc.) under which the first and/or second detection antibodies can bind to the cleaved VAMP and/or the full-length VAMP.

In one embodiment of the method of the present invention, conditions suitable for antibody binding may include incubation at about 4°C for about 8 hours to about 48 hours. In one embodiment of the method of the present invention, conditions suitable for antibody binding may include incubation at about 4°C for about 10 hours to about 24 hours. In one embodiment of the method of the present invention, conditions suitable for antibody binding may include incubation at about 4°C for about 12 hours to about 16 hours.

In one embodiment of the method of the present invention, suitable conditions for antibody binding may include incubation at about 25°C for about 30 minutes to about 8 hours. In one embodiment of the method of the present invention, suitable conditions for antibody binding may include incubation at about 25°C for about 1 hour to about 4 hours. In one embodiment of the method of the present invention, suitable conditions for antibody binding may include incubation at about 25°C for about 1.5 hours to about 3 hours.

Methods suitable for detecting and quantifying antibody binding to cleaved or full-length VAMP are well known in the art. For example, antibody binding to cleaved or full-length VAMP can be detected and quantified by Western blotting. When each protein is run by SDS-PAGE at a specific molecular weight, the cleaved VAMP will be detected at a lower molecular weight than the full-length VAMP. Analysis of the bands by densitography allows for the use of percentage cleavage readings of full-length and cleaved bands within the same lane on the gel. Alternatively, enzyme-linked immunosorbent assays (ELISA), such as sandwich ELISA, can be used to detect and quantify VAMP cleavage.

In one embodiment of the method of the present invention, the first detection antibody is a polyclonal antibody, and the binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in an enzyme-linked immunosorbent assay (ELISA).

In one embodiment of the method of the present invention, the first detection antibody is a polyclonal antibody, and the binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in a Western blot assay.

In one embodiment of the method of the present invention, the first detection antibody is a monoclonal antibody, and the binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in an enzyme-linked immunosorbent assay.

In one embodiment of the method of the present invention, the first detection antibody is a monoclonal antibody, and the binding of the first detection antibody to the C-terminal VAMP cleavage product is detected and quantified in a Western blot assay.

In one embodiment of the method of the present invention, cells are lysed before the cytoplasmic contents come into contact with the detection antibody.

In an alternative embodiment of the method of the present invention, the cells are permeabilized before their cytoplasmic contents come into contact with the detection antibody.

On the other hand, the present invention provides a kit comprising cells susceptible to neurotoxin poisoning by cleaved VAMP; and a first detection antibody against cleaved VAMP, wherein the first detection antibody is the antibody of the present invention.

In one embodiment, the kit further comprises a second detection antibody that binds to the full-length VAMP but not to the C-terminal VAMP cleavage product. Suitably, the second detection antibody binds to the VAMP epitope at the N-terminus of the clostridial neurotoxin cleavage site. Examples of suitable antibodies include commercially available antibodies such as ab3347 (Abcam) or ab181869 (Abcam).

This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used to practice or test embodiments of this disclosure. Numerical ranges include the numbers defining the range. Unless otherwise stated, any nucleic acid sequence is written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right, respectively, with the amino to carboxyl orientation.

In the case of a range of values provided, it should be understood that, unless the context explicitly specifies otherwise, each intermediate value between the upper and lower limits of the range, down to one-tenth of the lower limit unit, is also specifically disclosed. Each smaller range between any stated value or intermediate value within the range and any other stated value or intermediate value within the range is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range that includes any one, none, or both limits is also included in this disclosure, subject to any specifically excluded limits within the range. Where the range includes one or both limits, ranges that exclude one or both of those included limits are also included in this disclosure.

It is important to note that, unless the context clearly indicates otherwise, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, reference to “a clostridium neurotoxin” includes a plurality of such candidates, and reference to “the clostridium neurotoxin” includes reference to one or more clostridium neurotoxins and their equivalents known to those skilled in the art, etc.

The invention will now be described by way of example only with reference to the following figures and embodiments.

Attached Figure

Figure 1 - VAMP sequences with clostridial neurotoxin cleavage sites. (A) Human and rat VAMP1, VAMP2, and VAMP3 sequences with BoNT/F5 and BoNT/FA, BoNT/F, BoNT/D and BoNT/DC, BoNT/B, BoNT/G, TeNT, and BoNT/X cleavage sites. (B) Human and rat VAMP4, VAMP5, and YKT6 sequences with BoNT/X cleavage sites.

Figure 2 – VAMP sequences with Ab epitopes (i.e., immunogenic epitope regions) and BoNT/F, BoNT/D, and BoN/B cleavage sites. Human and rat VAMP1, VAMP2, and VAMP3 sequences are shown for comparison. The rat and human VAMP2 sequences are identical in the selected epitope regions. Cleavage sites are indicated by arrows: the VAMP2 cleavage sites for BoNT/F and BoNT/D are located at adjacent amino acids Q58-K59 and K59-L60, respectively, while the cleavage site for BoNT/B is located at amino acids Q76-F77 (based on the amino acid positions in the human VAMP2 sequence).

Figure 3 - Cell-free cleavage of recombinant VAMP2-GFP using MBP-LF and _LHND . Recombinant VAMP2-GFP was incubated with 0.01 μg/μl _LHND or MBP-LF at 37°C for 1 hour. An equal volume of sample buffer was added, and 0.5 μg (Coomassie) and 0.3 μg (Western blot) of protein were run by SDS-PAGE, stained with Coomassie or blotted with various anti-VAMP2 antibodies. A sketch shows the location of the antibody epitopes. The representation of the recombinant protein and the line lengths of the epitopes are not to scale. 1-BSA, 2-VAMP2-GFP, 3-Cleavage of VAMP2-GFP (aa59/60-terminus), 4-Cleavage of VAMP2-GFP (aa1-58/59).

Figure 4 – In vitro VAMP cleavage after BoNT/F and BoNT/D treatment. Rat cortical neurons grown in 96-well plates up to DIV18-21 were treated with BoNT/F (A) or BoNT/D (B) for 24 hours. Lysates were electrophoresed by SDS-PAGE and blotted with custom anti-VAMP2 antibodies: anti-Pep1, anti-Pep2, or anti-Pep3, or with commercial antibody ab1818691 – full-length VAMP2, 2-cleaved VAMP2. Anti-pep2 data showed the dose-dependent disappearance of full-length VAMP2 and the appearance of low molecular weight cleavage fragments. Two bands were used to quantify the dose-dependent percentage of VAMP2 cleavage by BoNT/F (C) and BoNT/D (D).

Figure 5-(A) Rat cortical neurons treated with native BoNT/F1(), native BoNT/A1(●), or recombinant BoNT/FA(Δ) for 24 hours. Cells were lysed, electrophoresed on SDS-PAGE, and blotted for VAMP-2 or SNAP-25 cleavage. The percentage of SNARE cleavage was determined by densitometry from the ratio of full-length to cleaved protein. Data were fitted using a four-parameter logistic equation and the BoNT concentration required for 50% maximum SNARE cleavage (pEC50) was determined (B). Data are mean ± s.e.m. (n = 3 (BoNT/F1 and BoNT/A1) or 4 (BoNT/FA) independent experiments, in triplicate).

Figure 6 - In vitro VAMP cleavage after BoNT/B and BoNT/F treatment. Rat cortical neurons grown to DIV18-21 were treated with BoNT/F or BoNT/B for 24 hours. Lysates were electrophoresed by SDS-PAGE and blotted with a newly customized anti-VAMP2 antibody (anti-Pep4), BoNT/B cleavage-specific anti-VAMP2 antibody or anti-Pep1, anti-Pep2 or anti-Pep3 antibody.

Example

Example 1: Detection of VAMP protein hydrolysis cleavage by BoNT/D and BoNT/F

A-method

1. Antibody production

Eurogentec used their Speedy 28-Day Program (https://secure.eurogentec.com/product/research-anti-protein-28-day-speedy-polyclonal-packages.html?country=gbr) to generate antibodies. The following peptides were used to immunize two rabbits per peptide:

-VAMP PEP1: H2N-SNR RLQ QTQ AQV DEC-CONH2 (SEQ ID NO: 39);

-VAMP PEP2: AcNH-KLS ELD DRA DAL Q-CONH2 (SEQ ID NO: 15); or

-VAMP PEP3: H2N-CLQ AGA SQ-CONH2 (SEQ ID NO: 40).

Animals receive their first immunization and three subsequent booster immunizations. Blood is collected before, during, and at the end of each immunization.

2. Recombinant protein cleavage

As previously described, an active construct containing a light chain and translocation domain of BoNT/D or fused with maltose-binding protein (MPB) and an equivalent BoNT/F domain was generated (Masuyer et al., “Structure and activity of a functional derivative of Clostridium botulinum neurotoxin BJ Struct Biol”, 174, p52-57, 2011; Sutton et al., “Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications. Protein Expression and Purification 40:31–41, 2005). In short, the fusion protein LH _N D (SEQ ID NO: 35) or MBP-LF (SEQ ID NO: 36) (the latter being a fusion of MBP with the BoNT/F1 light chain and C-terminal 6-histidine motif, and MPB and the 6-histidine motif are commonly known affinity tags) was assayed in a 50 mM buffer. Dilute to 0.01 μg/μl in HEPES pH 7.2, 200 μM ZnCl2, 1 μg/μl BSA, 10 mM DTT. Dilute VAMP2-GFP (SEQ ID NO: 37) (a fusion protein of amino acids 2-94 of human VAMP2 and detectable labeled green fluorescent protein (GFP)) to 8 μM in assay buffer (50 mM HEPES pH 7.2, 200 μM ZnCl2, 1 μg/μl BSA, 10 mM DTT). Combine equal volumes of _LHND or MBP-LF and VAMP2-GFP (SEQ ID NO: 37) (8 μM) and incubate at 37 °C for 1 hour. Terminate the reaction by adding 2x reduction sample buffer (NuPage LDS sample buffer, 100 mM DTT).

3. Rat cortical neuron cell culture

Rat cortical neurons were prepared from E17-E18CD rat embryos. Dissected cortical tissue was collected in ice-cold Hank's balanced salt solution (HBSS) with no Ca2+ or Mg2+, and then dissociated in papain solution at 37°C for 40 min according to the manufacturer's instructions (Worthington Biochemical, NJ, US). Cortical cells were seeded at a density of 20,000 cells/well in poly-L-ornithine (PLO)-coated 96-well plates in 125 μl of Neurobasal medium containing 2% B27 supplement, 0.5 mM GlutaMAX, 1% fetal bovine serum (FBS), and 100 U/ml penicillin/streptomycin. Cells were maintained at 37°C in a humid atmosphere with 5% CO2. At DIV (day 4 in vitro), an additional 125 μl of Neurobasal medium containing 2% B27 and 0.5 mM GlutaMAX was added. Cells were maintained by changing the half-medium twice weekly. At DIV11, 1.5 μM cytosine β-D-arabinofuranol (AraC) was added to the culture medium to prevent the proliferation of non-neuronal cells.

4. BoNT treatment

Rat cortical neurons of DIV18-21 were treated with natural BoNT/F1 (Metabiologics, US) (1 nM–0.1 pM) or BoNT/D (Metabiologics, US) (10 nM–1 pM) in triplicate wells at 37°C for 24 hours. The culture medium was removed, and the cells were washed once with PBS. The cells were then lysed in 40 μl of LRS sample buffer (NuPage LDS buffer, 1 mM DTT, 1:500 Benzosinase) at room temperature for 10 minutes.

5. SDS-PAGE and Western blotting

Neuronal lysates were boiled at 90°C for 5 minutes. 15 μl of lysate was loaded into each lane of a 12% Bis-Tris gel and run at 200 μV MES buffer for 50 minutes. Proteins were transferred to nitrocellulose membranes via Transblot Turbo (Biorad) using a low MW program. The membranes were blocked at room temperature with 5% low-fat emulsion/PBS-Tween for 1 hour, then incubated overnight at 4°C with a custom-designed anti-Pep1, anti-Pep2, or anti-Pep3 anti-VAMP2 primary antibody or with commercial anti-VAMP2 antibodies (Abcam ab3347 and ab181869). The membranes were washed three times in PBS-Tween and incubated at room temperature for 1 hour with an anti-rabbit-HRP secondary antibody. The membranes were washed 3 times in PBS-Tween for 5 minutes, then developed with SuperSignal West Femto chemiluminescent substrate and visualized using the Syngene PXi system.

B-Results

Evaluation of recombinant protein detection

Figure 2 shows the regions of three selected peptide epitopes from VAMP2 relative to the BoNT cleavage sites. Sequences of human and rat VAMP1, VAMP2, and VAMP3 are shown for comparison. The rat and human VAMP2 sequences are identical in the selected epitope regions. The BoNT/B and BoNT/D cleavage sites are located on adjacent amino acids.

Initially, the antibody was tested in a cell-free assay using recombinant VAMP2-GFP. BoNT/F and BoNT/D substitutes (MBP-LF and _LHND ) containing the toxin's enzymatic light chain domain were used to cleave the VAMP protein (Figure 3). Additionally, two other commercially available VAMP2 antibodies, ab3347 (epitopes aa1-18) and ab181869 (epitopes within aa1-100), were used for comparison. Figure 3 shows that the anti-Pep1 antibody detected the full-length VAMP2 and the N-terminal cleavage fragment (aa1-58/59), with a significantly reduced signal. As expected, this antibody did not detect the C-terminal cleavage product because its epitope is not located on that fragment. The anti-Pep2 and anti-Pep3 antibodies detected both the full-length VAMP2-GFP protein and the C-terminal cleavage product. Ab3347 detected only the full-length VAMP2 and not the N-terminal cleavage fragment, while ab181869 detected both.

These initial results indicate that the antibody is able to detect both the full-length recombinant VAMP2 and the expected cleavage product. An exception is ab3347, which detected only the full-length VAMP2 and not the N-terminal cleavage fragment.

Evaluation of endogenous protein detection

The next question was whether these antibodies could detect any cleavage products in a neuronal assay, in which endogenous proteases would be present. Rat primary cortical neurons were treated with BoNT/F or BoNT/D and lysed for Western blotting analysis (Figure 4). The anti-Pep1 antibody recognized only the full-length protein and no detectable cleavage products were found. The anti-Pep2 antibody detected both the full-length and C-terminal cleavage products. The anti-Pep3 antibody showed a weak signal in the cell lysate with very poor affinity for the monomeric VAMP and detected the most likely higher molecular weight species of dimers and other proteins (data not shown). The full-length monomeric signal was very low, but bands were present for the C-terminal products of BoNT/F and BoNT/D cleavage. In other words, anti-Pep3 did not detect the full-length VAMP but weakly detected the C-terminal fragments cleaved by BoNT/F and BoNT/D. This contrasts with earlier cell-free results, which showed strong signals from both the full-length and cleaved recombinant VAMP. The commercial antibody Ab3347 was not tested in vitro because cleavage protein detection was not possible in the cell-free assay. Although positive binding to the N-terminal cleaved recombinant fragment was observed in cell-free assays, the commercial antibody ab181869 detected full-length VAMP2 in the cortical lysate but not the cleaved fragment. Pep 2 data were used to quantify the dose-dependent cleavage of VAMP2 by BoNT/F (Fig. 4C) and BoNT/D (Fig. 4D).

The inventors initially demonstrated that two recombinant VAMP cleavage products could be detected in a cell-free system. However, upon transfer to cell lysates, the inventors also demonstrated that the N-terminal product was undetectable, possibly due to other unknown mechanisms besides degradation. Conversely, the inventors have shown that the C-terminal VAMP fragment, still bound to the vesicle membrane, is not degraded or altered in a manner that prevents antibody binding and Western blot detection. The Pep2 epitope is adjacent to the BoNT/D and BoNT/F cleavage sites, and antibodies generated against this peptide detect both the full-length VAMP and the cleavage products. Conversely, antibodies against the shorter epitope, which is further away from the BoNT F/D cleavage site, also detected the cleavage products, albeit weakly.

Example 2: Detection of VAMP protein hydrolysis cleavage of BoNT/FA and BoNT/F1 in rat cortical neurons

A-method

1. Rat cortical neuron cell culture

Rat cortical neurons were prepared as detailed in Example 1.

2. BoNT processing

In triplicate, rat cortical neurons of DIV18-21 were treated for 24 hours at 37°C with recombinant BoNT/FA (SEQ ID NO: 38) at a concentration range of 1 pM–1 fM, or with natural BoNT/F1 (Metabiologics, US) at a concentration range of 1 nM–1 pM, or with natural BoNT/A1 (List Biological Laboratories Inc., US) at a concentration range of 1 nM–1 fM. The culture medium was removed, and the cells were washed once with PBS. The cells were lysed in 40 μl of LRS sample buffer (NuPage LDS buffer, 1 mM DTT, 1:500 Benzosonease) at room temperature for 10 minutes.

3. SDS-PAGE and Western blots of rat cortical neurons

Rat cortical neurons were lysed for 10 min at room temperature in 40 μl of lysis buffer (NuPage LDS sample buffer, 1 mM DTT, and 1:500 Benzonase). The samples were boiled at 90 °C for 5 min, and 15 μl of lysate was added to each lane of a 12% Bis-Tris gel. The gel was then run at 200 V for 80 min in MOPS buffer (SNAP-25) or at 200 V for 50 min in MES buffer (VAMP2). Proteins were transferred to nitrocellulose membranes via TransblotTurbo (Biorad) using either a mixed MW (SNAP25) or low MW (VAMP2) program. The membrane was blocked with 5% low-fat emulsion/PBS-Tween for 1 hour at room temperature, and then incubated with either anti-SNAP25 antibody (Sigma S9684 1:4000) or anti-Pep2 (1:500), a custom antibody as described in Example 1—VAMP2 (Eurogentec) antibody; each primary antibody was incubated overnight at 4°C. The membrane was washed three times in PBS-Tween and incubated with anti-rabbit-HRP secondary antibody for 1 hour at room temperature. The membrane was washed 3 times in PBS-Tween for 5 min, and then developed with SuperSignal West Dura or West Femto chemiluminescent substrates using a Syngene Pxi system. Band densitometric analysis was performed using Genetools software, and the percentage protein cleavage was determined using the ratio of full-length SNAP-25 and VAMP2 protein to cleavage products.

B-Results

After treatment with BoNT/F1, BoNT/A1, or BoNT/FA for 24 hours, rat cortical neurons were lysed, electrophoresed on SDS-PAGE, and Western blots were performed on VAMP-2 (BoNT/F1 and BoNT/FA) or SNAP-25 (BoNT/A1). The percentage of SNARE cleavage was determined by densitometry analysis of the ratio of full-length to cleaved protein.

The results are shown in Figure 5. The recombinant BoNT/FA-cut VAMP-2 had a potency of pEC50 = 12.75 ± 0.14, n = 4. The natural BoNT/F1-cut VAMP-2 had a potency of pEC50 = 10.77 ± 0.12, n = 3. The natural BoNT/A1-cut SNAP-25 had a potency of pEC50 = 12.38 ± 0.14, n = 3.

Example 3: Detection of VAMP protein hydrolysis of BoNT/B in rat cortical neurons

A-method

1. Antibody production

Abcam used rabbits immunized with peptide Pep4:FETSAAKLKRKYWWK (SEQ ID NO: 49) to produce monoclonal antibodies.

BoNT/B cleavage-specific anti-VAMP2 antibodies (Kegel et al., Toxicology in Vitro; 2007, 21: p1641-1649) were used for comparative studies.

2. Rat cortical neuron cell culture

Rat cortical neurons were prepared from E17-E18CD rat embryos. Dissected cortical tissue was collected in ice-cold Hank's balanced salt solution (HBSS) with no Ca2+ or Mg2+, and then dissociated in papain solution at 37°C for 40 min according to the manufacturer's instructions (Worthington Biochemical, NJ, US). Cortical cells were seeded at a density of 20,000 cells/well in poly-L-ornithine (PLO)-coated 96-well plates in 125 μl of Neurobasal medium containing 2% B27 supplement, 0.5 mM GlutaMAX, 1% fetal bovine serum (FBS), and 100 U/ml penicillin/streptomycin. Cells were maintained at 37°C in a humid atmosphere with 5% CO2. At DIV (day 4 in vitro), an additional 125 μl of Neurobasal medium containing 2% B27 and 0.5 mM GlutaMAX was added. Cells were maintained by changing the half-medium twice weekly. At DIV11, 1.5 μM cytosine β-D-arabinofuranol (AraC) was added to the culture medium to prevent the proliferation of non-neuronal cells.

3. BoNT treatment

Rat cortical neurons were cultured in T25 flasks and treated with 1 nM and 10 pM BoNT/B (provided by List Biological Laboratories, Inc.) (SEQ ID NO: 2) at 37°C for 24 hours. The culture medium was removed, and the cells were washed once with PBS. The cells were lysed in 1.5 ml of NuPage sample buffer (NuPage LDS buffer, 1 mM DTT, 1:500 Benzosinase) at room temperature for 10 minutes.

4. SDS-PAGE and Western blot

Neuronal lysates were boiled at 90°C for 5 minutes. 15 μl of lysate was added to each lane of a 12% Bis-Tris gel and run at 200 μV in MES buffer for 50 minutes. Proteins were transferred to nitrocellulose membranes via Transblot Turbo (Biorad) using a low MW program. The membranes were blocked at room temperature with 5% low-fat emulsion/PBS-Tween for 1 hour, then incubated overnight at 4°C with a custom anti-Pep1, anti-Pep2, anti-Pep3, or anti-Pep4 antibody or with a BoNT-B cleavage-specific antibody. The membranes were washed three times in PBS-Tween and incubated at room temperature for 1 hour with an anti-rabbit-HRP secondary antibody. The membranes were washed 3 times in PBS-Tween for 5 minutes, then developed with SuperSignal West Femto chemiluminescent substrate and visualized using the Syngene PXi system.

B-Results

Based on the results obtained in Examples 1 and 2 above, it is suggested that the location of the epitope is key to the in vitro detection of cleaved VAMP, generating new monoclonal antibodies against epitopes located near the BoNT/B cleavage site on the C-terminal side.

Following BoNT/B and BoNT/F treatments, the antibody was tested in the same rat cortical assay and compared with anti-Pep1, anti-Pep2, anti-Pep3, and BoNT/B cleavage-specific antibodies (Figure 6). The epitope regions of all compared antibodies were located N-terminally to the BoNT/B cleavage site. Figure 6 shows the location of the novel antibody epitope against Pep4, enabling the detection of full-length VAMP2, as well as the cleavage products from BoNT/B and BoNT/F treatments. Conversely, the anti-Pep2 and anti-Pep3 antibodies detected only BoNT/F cleavage products, but not BoNT/B cleavage products. The anti-Pep1 antibody did not detect any expected cleavage products. The BoNT/B cleavage-specific antibody also did not detect any BoNT/B cleavage products in these cell lysates.

In summary, this data demonstrates that the location of the antibody epitope is a crucial consideration in the detection of cleaved VAMP. Only antibodies targeting epitopes on membrane-bound VAMP fragments after cleavage can detect these fragments. By localizing the monoclonal antibody epitope to the C-terminus of the VAMP, it was assumed that this region should be present in VAMP fragments generated from cleaved neurotoxin serotypes B, D, and F. This proved to be the case, enabling the production of a single antibody (anti-Pep4Mab) that provided positive results for neurons treated with BoNT/B and BoNT/F. Furthermore, since TeNT shares the same cleavage site as BoNT/B, and the BoNT/D cleavage site is closely adjacent to the BoNT/F cleavage site, this antibody was expected to be applicable to TeNT and BoNT/D cleavage as well.

Another advantage of the Pep4 epitope region is that antibodies targeting this region can detect both full-length and cleaved VAMPs with similar sensitivity. The ability to simultaneously detect two protein forms in the same sample provides a powerful tool for normalization without the need for additional housekeeping protein blotting. This provides a very useful and direct signal-gain blot analysis for quantifying BoNT potency in cell models.

This data also shows a difference in VAMP detection between cell-free recombinant protein assays and whole-cell models. It is this inability to detect cell-cut VAMP that forms the basis of the hypothesis that VAMP degradation occurs very rapidly in cells (Foran et al., “Evaluation of the therapeutic usefulness of botulinum neurotoxin B, C1, E and F compared with the long-lasting type A”. J. Biol Chem 278(2) pp1363–1371 2003). In contrast to the antibodies of this invention, most commercially available VAMP antibodies target epitopes in the N-terminal region of proteins, thus N-terminal VAMP fragments were the focus of those earlier studies. Although this paper shows that smaller C-terminal VAMP fragments do not degrade in cells, larger N-terminal fragments were also not detected. Interestingly, however, cell-free results show that not all commercial antibodies were able to detect the expected N-terminal fragment, even when present in cell-free systems lacking any proteases. Based on the current data, we can conclude that the VAMP degradation hypothesis is most certainly related only to the N-terminal fragment, and that the C-terminal VAMP fragment does not degrade and remains bound to the vesicle membrane.

Sequence information

• SEQ ID NO: 1-BoNT/A1-UniProtKB Registry Number P10845 (Botulinum toxin)

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQ FECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLS EDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTN IEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLY KDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLED FDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNS GWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYM YLKGPRGSVMTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

• SEQ ID NO: 2-BoNT/B1-UniProtKB Registry Number P10844 (Botulinum toxin)

MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIF GPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNK FKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIEN DFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIA GASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLL NYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGN RIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEY YMFNAGNKNSYIKLKKDSPVGEILTRSKYNQ NSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

• SEQ ID NO: 3-BoNT/C1-UniProtKB Registry Number P18640 (Botulinum toxin)

MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGP RENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYK QKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLRKDINE ETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGN FTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFD RNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGWS IGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNMKIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQ IVFNTRRNNNDFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYAIGLREQTKDINDNIIFQIQPMNNTYYYASQ IFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFVPVSE

• SEQ ID NO: 4-BoNT/D-UniProtKB Registry Number P19321 (Botulinum toxin)

MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIF GPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNI DKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIIT DETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSA LRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVID ELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTIYTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIE QNSGWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYINGELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQILRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESN VLVLVQYPDRSKLYTGNPITIKSVSDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNYGIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLSTSSFWKFISRDPGWVE

• SEQ ID NO:5-BoNT/E – Registry Number WP_003372387 (Botulinum toxin)

MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPD LFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDA SGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLD QVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDALE LLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKLINEVKINKL REYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTI INCMRDNNSGWKVSLNHNEIIWTLQDNAGINQKLAFNYGNANGISDYINKWIFVTITNDDRLGDSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYLLNVLKPN NFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTMNFKNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

• SEQ ID NO: 6-BoNT/F-UniProtKB Registry Number YP_001390123 (Botulinum toxin)

MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAG PDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDY FQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNL NNNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFFYLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIRDFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKEN FKEAFELLGAGILLEFVPELLIPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRKEQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERFITESSIFYLMKLINEAKV SKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNNSIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIYSTRNNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTII DCIRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGNSRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETLYSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQ NSNFLNINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKLIRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQEN

• SEQ ID NO: 7-BoNT/G-UniProtKB Registry Number WP_039635782 (Botulinum toxin)

MPVNIKNFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFNASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLGNASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFG PGPVLSDNFTDSMIMNGHSPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLYGIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIANRLNIVSSAQGSGIDISLYKQIYK NKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNLAGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAYEEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYNTQNNTI ENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGDSLFEYLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWVKGVIDDFTSESTQKSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAF EIGGAAILMEFIPELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTIKERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFINQCSISYLMNRMIPLAVKKLKDFDDDNLKRD LLEYIDTNELYLLDEVNILKSKVNRHLKDSIPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSSGYGATMNVGSDVIFNDIGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTIISCIKNDSGWKVS IKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLGNANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIIFGRELNATEVSSLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTN FNNAAINYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQLFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYDNYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE

• SEQ ID NO: 8-TeNT-UniProtKB Registry Number P04958 (Clostridium tetani)

MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGP VLNKNEVRGIVLRVDNKNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDIDSYKQIYQQK YQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLTFIAEKNSFSEEPFQDEI VSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVISKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYE GNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKLVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKNKLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDT QSKNILMQYIKANSKFIGITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYL TNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNN LDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

• SEQ ID NO: 9-VAMP1_Rat (Q63666)

MSAPAQPPAEGTEGAAPGGGPPGPPPNTTSNRRLQQTQAQVEEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASVFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYIFT

•SEQ ID NO:10-VAMP1_人(P23763)

MSAPAQPPAEGTEGTAPGGGPPGPPPNMTSNRRLQQTQAQVEEVVDIIRVNVDKVLERDQKLSELDDRADALQAGASQFESSAAKLKRKYWWKNCKMMIMLGAICAIIVVVIVIYFFT

• SEQ ID NO: 11-VAMP2_rat (P63045)

MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST

•SEQ ID NO:12-VAMP2_人(P63027)

MSATAATAAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST

• SEQ ID NO: 13-VAMP3_rat (P63025)

MSTGVPSGSSAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGISVLVIIVIIIIVWCVS

•SEQ ID NO:14-VAMP3_人(Q15836)

MSTGPTAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMWAIGITVLVIFIIIIIVWVVSS

• SEQ ID NO:15 – VAMP epitope

KLSELDDRADALQ

• SEQ ID NO:16 – VAMP epitope

QKLSELDDRADALQ

• SEQ ID NO:17 – VAMP epitope

KLSELDDRAD

• SEQ ID NO:18 – VAMP epitope

KLSELDDRADALQAGAS

• SEQ ID NO:19 – VAMP epitope

LSELDDRADALQ

• SEQ ID NO:20 – VAMP epitope

LSELDDRADA

• SEQ ID NO:21 – VAMP epitope

LSELDDRADALQAGAS

• SEQ ID NO:22 – VAMP epitope

FETSAAKLKRKYW

• SEQ ID NO:23 – VAMP epitope

FESSAAKLKRKYW

• SEQ ID NO:24 – VAMP epitope

QFETSAAKLKRKYW

• SEQ ID NO:25 – VAMP epitope

FETSAAKLKR

• SEQ ID NO:26 – VAMP epitope FETSAAKLKRKYWWKN

• SEQ ID NO:27 – VAMP epitope

AKLKRKYWWKN

• SEQ ID NO:28 – VAMP epitope

AAKLKRKYWWKN

• SEQ ID NO:29 – VAMP epitope

AKLKRKYWWKNCKM

• SEQ ID NO:30 – VAMP epitope

AKLKRKYWWKNLKM

• SEQ ID NO:31 – VAMP epitope

DQKLSELDDRADALQ

• SEQ ID NO:32 – VAMP epitope

ERDQKLSELDDRA

• SEQ ID NO:33 – VAMP epitope

LERDQKLSELDDRA

• SEQ ID NO:34 – VAMP epitope

VLERDQKLSELDDRA

·SEQ ID NO:35-LH _N D

MGSMTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLIN YLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIF CMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKY NFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCVDKS EEKLYDDDDKDRWGSSLQCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNS YYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVA FLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSL DVKISEAMNNIKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNLEAHHHHHHHHHH

·SEQ ID NO:36-MBP-LF

MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADT DYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELA KDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISEFGSMPVAINSFNYNDPVNDDTILYMQIP YEEKSKKYYKAFEIMRNVWIIPERNTIGTNPSDFDPPASLKNGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGKVLLQEISYAKPYLGNDHTPIDEFSPVTRTTSVNIKLSTNVESSMLLNLLVLGAGPDIFESCCYPVRKLIDPDVVYDPSNYGFGSINIVTFSPEYEYTFNDISGGHNSSTESFIADPAISLAHELIHALH GLYGARGVTYEETIEVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSEVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNEN KFNEIYKKLYSFTESDLANKFKVKCRNTYFIKYEFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQSIKLNPKIIDSIPDKGLVEKIVKFAVDKLAAALEHHHHHH

• SEQ ID NO:37 (Recombinant VAMP2-GFP)

GPLGSSATAATAAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKLENVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGV QCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

• SEQ ID NO:38 - Recombinant BoNT/FA

MPVVINSFNYDDPVNDNTIIYIRPPYYETSNTYFKAFQIMDNVWIIPERYRLGIDPSLFNPPVSLKAGSDGYFDPNYLSTNTEKNKYLQIMIKLFKRINSKPAGQILLEEIKNAIPYLGNSYTQEEQFTTNNRTVSFNVKLANGNIVQQMANLIIWGPGPDLT TNKTGGIIYSPYQSMEATPYKDGFGSIMTVEFSPEYATAFNDISIASHSPSLFIKDPALILMHELIHVLHGLYGTYITEYKITPNVVQSYMKVTKPITSAEFLTFGGRDRNIVPQSIQSQLYNKVLSDYKRIASRLNKVNTATALINIDEFKNLYEWKYQFAK DSNGVYSVDLNKFEQLYKKIYSFTEFNLAYEFKIKTRLGYLAENFGPFYLPNLLDDSIYTEVDGFNIGALSINYQGQNIGSDINSIKKLQGQGVVSRVVRLCKSVIPRKGTKAPPRLCITVNNRDLFFIASQESYGENTINTYKEIDDTTTLDPSFEDILDKV ILNFNEQVIPQMPNRNVSTDIQKDNYIPKYDYNRTDIIDSYEVGRNYNTFFYLNAQKFSPNESNITLTSSFDTGLLEGSKVYTFFSSDFINNINKPVQALLFIEWVKQVIRDFTTEATKTSTVDKLKDISLVVPYIGLALNIGDEIYKQHFAEAVELVGAGLL LEFSPEFLIPTLLIFTIKGYLTGSIRDKDKIIKTLDNALNVRDQKWKELYRWVVSKWLTTINTQFNKRKEQMYKALKNQATAIKKIIENKYNNYTTDEKSKIDSSYNINEIERTLNEKINLAMKNIEQFITESSIAYLINIINNETIQKLKSYDDLVRRYLLG YIRNHSSILGNSVEELNSKVNNHLDNGIPFELSSYTNDSLLIRYFNKNYGELKYNCILNIKYEMDRDKLVDSSGYRSRINIGTGVKFSEIDKNQVQLSNLESSKIEVILNNGVIYNSMYENFSTSFWIRIPKYFRNINNEYKIISCMQNNSGWEVSLNFSNMN SKIIWTLQDTEGIKKTVVFQYTQNINISDYINRWIFVTITNNRLSNSKIYINGRLINEESISDLGNIHASNNIMFKLDGCRDPHRYIWIKYFNLFDKELNKKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYLDVNNVGIRGYMYLKGPRGRI VTTNIYLNSTLYMGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSAVEIPDVGNLSQVVVMKSENDQGIRNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIGKASRTFGCSWEFIPVDDGWGESSLHHHHHHHHHH

·SEQ ID NO:39–Pep1

SNRRLQQTQAQVDEC

·SEQ ID NO:40–Pep3

CLQAGASQ

• SEQ ID NO:41–BoNT/X Genbank accession number BAQ12790 (Botulinum toxin)

MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIPLPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIA NNATYGLYSTPISNGEGTLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPVQGRLG NFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISNKDSLNDINL SEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPY IGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWWGTIHLQ INTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYL TKEMIPKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSISFWIKHPKPTNLLNNGI EYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED

• SEQ ID NO:42-VAMP4_Rat (D4A560)

MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALAAAILLLMIITQIILHLKK

·SEQ ID NO:43-VAMP4_人(O75379)

MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGPRFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKSESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALVAAILLLVIIILIVMKYRT

•SEQ ID NO:44-VAMP5_Rat(Q9Z2J5)

MAGKELERCQRQADQVTEIMLNNFDKVLERDGKLSELQQRSDQLLDMSSAFSKTTKTLAQQKRWENIRCRVYLGLAVAGGLLLILVVLLVIFLPSGEDSSKP

·SEQ ID NO:45-VAMP5_人(O95183)

MAGIELERCQQQANEVTEIMRNNFGKVLERGVKLAELQQRSDQLLDMSSTFNKTTQNLAQKKCWENIRYRICVGLVVVGVLLIILIVLLVVFLPQSSDSSSAPRTQDAGIASGPGN

•SEQ ID NO:46-YKT6_Rat(Q5EGY4)

MKLYSLSVFYKGEPKAVLLKAAYDVSFSFFQRSSVQEFMTFTSQLIVERSAKGSRASVKEQEYLCHVYVRSDSLAGVVIADSEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYTALDGHLSRYQNPREADPMSKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQNSCCAIM

·SEQ ID NO:47-YKT6_人(O15498)

MKLYSLSVLYKGEAKVVLLKAAYDVSSFSFFQRSSVQEFMTFTSQLIVERSSKGTRASVKEQDYLCHVYVRNDSLAGVVIADNEYPSRVAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYPALDGHLSRYQNPREADPMTKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEVLGTQSKAFYKTARKQNSCCAIM

• SEQ ID NO:48-VAMP epitope

ETSAAKLKRKYWWK

• SEQ ID NO:49-VAMP epitope

FETSAAKLKRKYWWK

• SEQ ID NO:50-VAMP epitope

QFESSAAKLKRKYW

• SEQ ID NO:51-VAMP epitope

FESSAAKLKR

• SEQ ID NO:52-VAMP epitope

FESSAAKLKRKYWWK

• SEQ ID NO:53-VAMP epitope

ADALQAGASQF

• SEQ ID NO:54-VAMP epitope

ADALQAGASQ

• SEQ ID NO:55-VAMP epitope

RADALQAGASQF

• SEQ ID NO:56-VAMP epitope

ADALQAGASQFE

• SEQ ID NO:57-VAMP epitope

ADALQAGASVF

• SEQ ID NO:58-VAMP epitope

ADALQAGASV

• SEQ ID NO:59-VAMP epitope

ADALQAGASVFE

• SEQ ID NO: 60-VAMP epitope

RADALQAGASVF

• SEQ ID NO: 61-VAMP epitope

RADALQAGAS

• SEQ ID NO: 62-VAMP epitope

SESLSDNATAF

• SEQ ID NO: 63-VAMP epitope

SESLSDNATA

• SEQ ID NO: 64-VAMP epitope

KSESLSDNATAF

• SEQ ID NO: 65-VAMP epitope

SESLSDNATAFS

• SEQ ID NO: 66-VAMP epitope

SDQLLDMSSTF

• SEQ ID NO: 67-VAMP epitope

SDQLLDMSST

• SEQ ID NO: 68-VAMP epitope

RSDQLLDMSSTF

• SEQ ID NO: 69-VAMP epitope

SDQLLDMSSTFN

• SEQ ID NO:70-VAMP epitope

SDQLLDMSSAF

• SEQ ID NO:71-VAMP epitope

SDQLLDMSSA

• SEQ ID NO:72-VAMP epitope

RSDQLLDMSSAF

• SEQ ID NO:73-VAMP epitope

SDQLLDMSSAFS

• SEQ ID NO:74-VAMP epitope

RSDQLLDMSS

• SEQ ID NO:75-VAMP epitope

SEVLGTQSKAF

• SEQ ID NO:76-VAMP epitope

SEVLGTQSKA

• SEQ ID NO:77-VAMP epitope

KSEVLGTQSKAF

• SEQ ID NO:78-VAMP epitope

SEVLGTQSKAFY

Claims (13)

1. An antigenic polypeptide having an amino acid sequence as shown in AcNH-KLSELDDRADALQ-CONH2, SEQ ID NO:15, or SEQ ID NO:49. 2. A method for generating a polyclonal antibody against a C-terminal VAMP cleavage product, wherein the C-terminal VAMP cleavage product is generated by cleaving VAMP with botulinum neurotoxin F (BoNT/F), BoNT/D, or BoNT/DC. The method includes isolating and purifying polyclonal antibodies from: (a) Blood obtained from an animal that has been injected with an antigenic peptide, wherein the amino acid sequence of the antigenic peptide is as shown in AcNH-KLSELDDRADALQ-CONH2 or SEQ ID NO:15. (b) Eggs obtained from an animal, wherein the eggs have been injected with an antigenic polypeptide, wherein the amino acid sequence of the antigenic polypeptide is such as AcNH-KLSELDDRADALQ-CONH2 or SEQ ID NO: As shown in Figure 15. 3. A method for generating a polyclonal antibody against a C-terminal VAMP cleavage product, wherein the C-terminal VAMP cleavage product is generated by cleavage of VAMP with BoNT/B, BoNT/F, BoNT/D, BoNT/DC, or tetanus neurotoxin (TeNT). The method includes isolating and purifying polyclonal antibodies from: (a) Blood obtained from an animal that has been injected with an antigenic polypeptide, wherein the amino acid sequence of the antigenic polypeptide is shown in SEQ ID NO:49. (b) Eggs obtained from an animal, wherein the eggs have been injected with an antigenic polypeptide, wherein the amino acid sequence of the antigenic polypeptide is shown in SEQ ID NO:49. 4. An antibody obtainable by the method of claim 2, wherein the antibody is a polyclonal antibody. 5. An antibody obtainable by the method of claim 3, wherein the antibody is a polyclonal antibody. 6. The antibody according to claim 4 or 5, wherein the _KD between the antibody and its epitope is less than ^10⁻⁷ M. 7. Use of the antibody of claim 4 in a signal gain cell assay, wherein the cell assay is used for VAMP cleavage via BoNT/F, BoNT/D, or BoNT/DC. 8. Use of the antibody of claim 5 in a signal gain cell assay, wherein the cell assay is used for VAMP cleavage by BoNT/B, BoNT/F, BoNT/D, BoNT/DC, or TeNT. 9. A method for determining the cleavage of VAMP by BoNT/F, BoNT/D, or BoNT/DC in cells, comprising: (a) Contact cells with BoNT/F, BoNT/D, or BoNT/DC under conditions suitable for clostridium neurotoxin activity; (b) After VAMP is cleaved by BoNT/F, BoNT/D, or BoNT/DC, the cytoplasmic contents of the cells are contacted with a first detection antibody against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is the antibody according to claim 4; and (c) Detect the binding of the first detection antibody to the C-terminal VAMP cleavage product using appropriate means. 10. A method for determining the cleavage of VAMP by BoNT/B, BoNT/F, BoNT/D, BoNT/DC, or TeNT in cells, comprising: (a) Contact cells with BoNT/B, BoNT/F, BoNT/D, BoNT/DC, or TeNT under conditions suitable for clostridium neurotoxin activity; (b) After VAMP is cleaved by BoNT/B, BoNT/F, BoNT/D, BoNT/DC, or TeNT, the cytoplasmic contents of the cells are contacted with a first detection antibody against the C-terminal VAMP cleavage product under conditions suitable for binding of the first detection antibody to the C-terminal VAMP cleavage product, wherein the first detection antibody is the antibody according to claim 5; and (c) Detect the binding of the first detection antibody to the C-terminal VAMP cleavage product using appropriate means. 11. The method of claim 9 or 10, further comprising (d) quantifying the amount of C-terminal VAMP cleavage product bound to the first detection antibody by suitable means. 12. A kit comprising cells susceptible to BoNT/F, BoNT/D, or BoNT/DC poisoning; and a first detection antibody against cleaved VAMP, wherein the first detection antibody is the antibody of claim 4. 13. A kit comprising cells susceptible to BoNT/B, BoNT/F, BoNT/D, BoNT/DC, or TeNT poisoning; and a first detection antibody against cleaved VAMP, wherein the first detection antibody is the antibody of claim 5.