Modified Clostridial neurotoxins and conjugate vaccine platforms as vaccines

Abstract

Provided herein are engineered non-catalytic, non-toxic tetanus toxin variants, methods of using the tetanus toxin variants at low doses, and protective vaccines that are non-toxic and more potent than their corresponding chemically inactivated toxoids. Additionally, provided herein are conjugate vaccine vehicles comprising engineered tetanus toxin variants, and methods of eliciting T cell-dependent immune memory responses with these conjugate vaccines that can target a broad spectrum of microbial pathogens as a single vaccine.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 599,444, filed on December 15, 2017, the entire contents of which are incorporated herein by reference.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Grant Nos. R01 AI030162 and AI118389 awarded by NIH-NIAID. The government has certain rights in this invention. Background Art

[0005] Botulinum neurotoxin (BoNT), the most toxic substance known to humans, is a protein toxin produced by Clostridium botulinum and selected strains of Clostridium butyricum and Clostridium baratii (Hill and Smith, 2013; Johnson and Montecucco, 2008). BoNT is synthesized as a 150 kDa two-chain protein consisting of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC) linked by disulfide bonds. The HC is further divided into an N-terminal domain (H N ) and the C-terminal domain (H C ), the N-terminal domain helps transport LC into the cytoplasm, and the C-terminal domain recognizes and binds to cell surface receptors on neuronal cells (Montal, 2010). Once inside the cell, LC specifically cleaves a portion of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), thereby inactivating neurotransmitter release (Montecucco and Schiavo, 1993, Trends in biochemical sciences 18, 324-327; Schiavo et al., 1995). Experimental vaccines were previously used to protect "at-risk" people against botulinum toxin, but due to their reduced performance, this chemically inactivated BoNT toxoid vaccine is no longer used. Moreover, conventional tetanus toxin fragment vaccines are not ideal due to their poor antigenicity and immunological efficacy. Therefore, there is still a need in the art for non-catalytic and non-toxic tetanus and botulinum toxin variants for use as adjuvants and conjugate vaccines. Summary of the invention

[0006] Provided herein is a recombinant non-catalytic non-toxic variant form of tetanus toxin, and the purposes of the variant toxin. The data described herein show significantly reduced toxicity compared to natural tetanus toxin and previously described tetanus variants. We envision several independent engineered mutations, which inactivate the inherent toxicity of tetanus toxin, and can be combined to produce safe and effective vaccines, including but not limited to eliminating catalytic activity by removing substrate affinity or reducing reaction rate, eliminating receptor binding, inhibiting shifting ability (potential), or interfering with toxin domain cutting or disulfide bond interruption, and other steps of other toxin poisoning, etc. Experiments are described herein, in which we engineered toxins with weakened host receptor binding and weakened catalysis. These data show the ability of the recombinant toxin comprising selected independent mutations, which makes it non-toxic and suitable for use as a vaccine or conjugate vaccine, without the need for chemical cross-linking to reduce toxicity.

[0007] In the first aspect, the present invention provides a modified tetanus toxin polypeptide comprising a sequence having at least 95% identity to SEQ ID NO: 1 and having mutations at each of positions R372 and Y375, and further comprising mutations at two or more positions selected from E234, K768, R1226 and W1289, wherein each position is numbered according to SEQ ID NO: 1, and the polypeptide has reduced catalytic activity, translocation and receptor binding compared to the toxicity and receptor binding of SEQ ID NO: 1. The amino acid R at position R372 can be replaced by amino acid A, and the Y at position Y375 can be replaced by amino acid F. Mutations may include R372A, Y375F, E234Q, R1226L, and W1289A. The modified polypeptide may also include a covalently linked carbohydrate, wherein the polypeptide is a polypeptide-sugar conjugate. The modified polypeptide may be encoded by SEQ ID NO: 2.

[0008] In some cases, the mutation may include R372A, Y375F, E234Q, K768A, R1226L, and W1289A. The modified polypeptide may be encoded by SEQ ID NO:5. In some cases, the modified polypeptide may also include mutations at one or both of positions L231 and Y26, wherein each position is numbered according to SEQ ID NO:1. Mutations at one or both of positions L231 and Y26 include L231K and Y26A. The modified polypeptide may be encoded by SEQ ID NO:6 or SEQ ID NO:7.

[0009] In another aspect, provided herein is a composition comprising a modified polypeptide described herein and a pharmaceutically acceptable carrier.

[0010] In one aspect, the present invention provides a method for reducing the risk of tetanus in a patient, comprising administering a therapeutically effective amount of a modified polypeptide as described herein to the patient to induce an immune response. In some cases, the modified polypeptide is used as an adjuvant. In some cases, the modified polypeptide is used as a vaccine.

[0011] The above and other aspects and advantages of the present invention will become apparent from the following description. In the following description, reference is made to the accompanying drawings which form a part of the specification, in which preferred embodiments of the present invention are shown in an illustrative manner. Such embodiments do not necessarily represent the full scope of the present invention, but are provided as references to illustrate the claims and to explain the scope of the present invention herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Recombinant proteins used to assess host immune response to vaccination are illustrated. (Top) Schematic representation of the BoNT derivatives used for this study is shown. When indicated, proteins were purified with two epitopes (His6 and Strep). 3XFLAG (3XF) and two consecutive hemagglutinin (2HA) epitopes were included for cellular studies. Domain connectivity was determined using the crystal structure of BoNT / A1 (PDB:3BTA). The single amino acid designation on each schematic represents the amino acid substitution introduced to reduce catalysis (LC) or receptor binding (HCC). Note that single-chain BoNT and LCHCN were used for immunization. (Bottom) 4 μg of the indicated proteins were subjected to SDS-PAGE and stained with Coomassie blue. Lanes: 1, M-BoNT / A1; 2, M-BoNT / A1 trypsin cleaved and reduced; 3, M-LCHCN / A1; 4. M-LCHCN / A1 trypsin cleaved and reduced; 5, LC / A1RY; 6, HCC / A1 W ; and 7, TeNTRY. Molecular weight marker proteins (kDa) migrate as shown in the left lane. Note that the cleaved HC in lane 2 runs at about 80 kDa, which is also shown in other experiments and is attributed to the band region of casein cleaved HC.

[0013] Figure 2 Is M-BoNT / A1 or M-BoNT / A1 W ELISA of sera from mice vaccinated and challenged with native BoNT / A2 (a heterologous subtype). W (Bottom) Mice were vaccinated and serum was collected before challenge with 106 LD50 natural BoNT / A2. Mice that survived (A) or did not survive (D) the challenge were shown. ELISA was performed to measure M-BoNT / A1; M-LCHCN / A1; LC / A1RY; HCC / A1 W; TeNTRY; and no protein control (Con) serum antibody titers (1:20,000 dilution). Bound mouse antibodies were detected using TMB reagent with goat α-mouse IgG-HRP (1:20,000 dilution). The reaction was stopped with dilute sulfuric acid and read at 450 nm. Data are presented as the mean of 5 (A) mice and 4 (D) mice after M-BoNT / A1 vaccination from two independent experiments performed in duplicate, as well as at M-BoNT / A1 W Mean values ​​of 3 (A) and 6 (D) mice after inoculation are shown with standard deviations. Statistical analysis was performed as described: P, .05 = *.

[0014] Figure 3 ELISA of serum from mice vaccinated with BoNT derivatives and challenged with native BoNT / A1. W (0.3μg),M-LCHCN / A1(0.2μg),M-LCHCN / A1(0.2μg)+HCC / A1 W (0.1μg), or HCC / A1 W (0.3 μg) was inoculated into mice. Serum was obtained before BoNT challenge. ELISA was used to measure M-BoNT / A1; M-LCHCN / A1; LC / A1RY; HCC / A1 W ; TeNTRY; and no protein control (Con) serum antibody titers (1:30,000 dilution). Bound mouse antibodies were detected with goat α-mouse IgG-HRP (1:20,000 dilution) using TMB reagent. The reaction was stopped with dilute sulfuric acid and read at 450 nm. Data are presented as the mean of 10 independent sera from mice surviving BoNT / A1 challenge from Experiment 3 of Table 1, analyzed in two independent experiments performed in duplicate, with standard deviations indicated; except for the use of HCC / A1 W Vaccinated mice, where data are from 7 mice that survived (A) or 3 mice that did not survive (D) the natural BoNT / A1 challenge. The variation in titer range is due to different antibody titers between individual mice, not to variation in ELISA replicates. Statistical analysis was performed as described in the Methods section: P < 0.05 = *, 0.01 = **, 0.001 = ***, and 0.0001 = ****.

[0015] Figure 4 ELISA of serum from individual mice vaccinated with M-BoNT derivatives and surviving challenge with native BoNT / A1. WAs antigen, BoNT / A1 was used in ELISA (1:30,000 dilution). W (#7 and #3), HCC / A1 W Serum obtained prior to BoNT challenge from individual mice vaccinated with (#78) and LCHCN / A1 (#21, #24, and #25) and surviving native BoNT / A1 challenge. Bound mouse antibodies were detected using TMB reagent with goat α-mouse IgG-HRP (1:20,000 dilution). The reaction was stopped with dilute sulfuric acid and read at 450 nm. Data are presented as the mean of two independent experiments performed in duplicate, with standard deviations indicated.

[0016] Figure 5 Serum neutralization of native BoNT / A1 cleavage of SNAP25 in human induced pluripotent stem cells (hiPSCs) was shown. W (#7 and #3), HCC / A1 W Neutralization of native BoNT / A1 by sera obtained from individual mice vaccinated with (#78) and M-LCHCN / A1 (#21, #24, and #25) and surviving BoNT / A1 challenge. Human induced pluripotent stem cell (hiPSC)-derived neurons were seeded at a density of 30,000-40,000 cells / well on plates coated with poly-L-ornithine and Matrigel, maintained in iCell Neuronal Medium for 7 days, and then subjected to neutralization assays. To detect neutralizing antibodies in mouse sera, 2 pM native BoNT / A1 was combined with serial dilutions of sterile-filtered serum and incubated in culture medium at 37°C for 1 hour. "No antibody" buffer was used as a control. 50 μl of each antibody-toxin mixture was added to each well of hiPSC-derived neurons in at least duplicate; cells were incubated at 37°C in 5% CO2 for 24 hours. Toxin / antibody was aspirated from cells and cell lysates were subjected to PAGE followed by Western blot analysis for SNAP-25 cleavage (Pellett et al., 2007; Pellett et al., 2010). Cleaved and uncleaved SNAP-25 were quantified by densitometry and % protection was determined by comparison to a "no antibody" control. IC50 values ​​were estimated using GraphPad Prism 6 software and nonlinear regression, variable slope, and four parameters.

[0017] Figure 6 It is M-BoNT / A1 W Representative ELISA of sera from mice vaccinated and surviving BoNT / A1 challenge. Serum from mice surviving BoNT / A1 challenge was analyzed by ELISA as described in the Methods section. WThe sera were inoculated with the indicated antigens. Values ​​are means of representative determinations performed in duplicate with standard deviations indicated.

[0018] Figure 7 M-BoNT / A1 is shown W Compared with M-LCHCN / A1 or LCHCN / A1+HCC / A1 W The immunogenicity of M-BoNT / A1 (0.3 μg) and M-LCHCN / A1 were analyzed by ELISA. W (0.2μg), or M-LCHCN / A1 (0.2μg)+HCC / A1 W (0.1 μg) and inoculated at 10 6 Serum of individual mice that survived the LD50 BoNT / A1 showed expression of M-BoNT / A1, M-LCHCN / A1, LC / A1RY, and HCC / A1 W , TeNTRY, or protein-free (Con) antibody. Data are presented as the mean of 10 independent sera from mice surviving BoNT / A1 challenge from Experiment 3 of Table 1, with duplicates performed in two independent experiments, with standard deviations indicated. Statistical analysis was performed as described in the Methods section: P<0.05=*, 0.01=**, 0.001=***, and 0.0001=****.

[0019] Figure 8 is the amino acid sequence encoding the wild-type Clostridium tetani toxin (SEQ ID NO: 1).

[0020] Fig. 9 is the amino acid sequence encoding 2M-TT (SEQ ID NO: 2).

[0021] Fig.10 is the amino acid sequence encoding 5M-TeNT (SEQ ID NO: 4).

[0022] Fig.11 is the amino acid sequence encoding 6M-TeNT (SEQ ID NO: 5).

[0023] Fig.12 is the amino acid sequence encoding 7M-TeNT (SEQ ID NO: 6).

[0024] Fig.13 is the amino acid sequence encoding 8M-TeNT (SEQ ID NO: 7).

[0025] Fig.14K768 mediates light chain translocation in tetanus toxin. (Left) TT767DKE (WT), TT767AAA, TT767RKK, or TT767DAE were incubated with neurons and translocation was measured using the reporter (β-lactamase) CCF2 cleavage. (Right) Alignment of TT and BT at 767DKE. Note the conserved lysine (K) between toxins.

[0026] Fig.15 It is the crystal structure of TeNTRY PDB: 5n0b. Four TT functions are inactivated: light chain E234Q, R372A, Y375F (Zn++ binding), L231K (VAMP-2 cleavage) and Y26A (VAMP-2 binding), K768A (LC transposition) and R1226L and W1289A (receptor binding). DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention has been described in terms of one or more preferred embodiments, but it should be understood that many equivalents, alternatives, variations and modifications besides those explicitly described are possible and within the scope of the present invention.

[0029] The methods and compositions described herein are based, at least in part, on the inventors' discovery of toxins that are both non-catalytic and incapable of binding to neuronal cells. As described in the following paragraphs and examples, tetanus toxin and botulinum toxin (BoNT) have engineered defects that impede catalysis and receptor binding, providing a platform for vaccine development for toxin-mediated diseases. For example, non-toxicity was detected with modified tetanus toxin, while BoNT has engineered mutations that render catalysis and receptor binding incapable. Additionally, such modified toxins are suitable as vaccines against botulinum neurotoxin challenge.

[0030] Composition

[0031] Preferably, the genetically modified toxins herein include genetic modifications at multiple target sites relative to the wild-type toxin, wherein such modifications eliminate residual toxicity and improve safety, as further described below. Although BoNTs and tetanus toxins act on different substrates and receptors, the inventors have determined that immune cells take up a non-catalytic, non-receptor binding BoNT form (referred to herein as "M-BoNT W ”) and produced a neutralizing immune response similar to that observed with non-catalytic BoNTs (“M-BoNTs”). Based on this observation, additional independent mutation sites were identified to engineer additional non-catalytic, non-receptor binding toxin variants. When modified as described herein, the resulting engineered non-catalytic, non-receptor binding toxins are useful as vaccines and conjugate vaccine platforms.

[0032] Without being limited to any particular theory of mechanism of action, mutations engineered at independent sites within tetanus toxin and botulinum toxin render the toxin proteins incapable of expressing toxicity by independent mechanisms, thereby providing fail-safe properties against accidental genetic reversion to toxicity. Such properties facilitate the use of variant toxins as vaccines against tetanus and botulism, and as combined vaccine platforms.

[0033] Therefore, the present invention provides a recombinant inactivated bacterial toxin, which is irreversibly non-toxic, more potent, and easier to produce and manipulate than the current chemically inactivated toxoids, thus providing improved vaccines and conjugate vaccine carriers. In the first aspect, the present invention provides a recombinant non-catalytic non-toxic modified form of a bacterial protein toxin (e.g., tetanus toxin and botulinum neurotoxin), wherein the modified toxin is a full-length toxin, comprising at least 4 amino acid substitutions, so that the protein cannot express toxicity by an independent mechanism. "Preparation" refers to a toxin polypeptide of any concentration that is enhanced or purified relative to its naturally occurring form. Preferably, the preparation is substantially pure, or is combined with other ingredients into a pharmaceutical preparation. In some cases, the preparation of the present invention may include one or more adjuvants or carriers, which can be coupled to the toxin polypeptide sequence to help stimulate the immune system. In other cases, the preparation itself has adjuvant activity, which can effectively enhance the immune response of the antigen bound or co-administered antigen.

[0034] As used herein, "toxin" refers to a harmful or toxic substance (e.g., a cytotoxin) that is formed or produced as an intrinsic part of a cell or tissue (e.g., an endotoxin), as an intracellular or extracellular product (exotoxin), or as a group thereof during the metabolism and growth of some microorganisms. As used herein, the term "modified toxin" refers to a non-catalytic, non-toxic variant form of a toxin, wherein the amino acid sequence of a modified polypeptide toxin is modified by genetic engineering (e.g., non-naturally occurring, artificial) to render the toxin non-catalytic and non-toxic. In an exemplary embodiment, the modified toxin is a genetically engineered variant produced by Clostridium bacteria (e.g., Clostridium difficile, Clostridium novyi, Clostridium sordellii, Clostridium perfringens, Clostridium tetani, and Clostridium botulinum). Toxins can be recombinant, synthetic, fusion protein portions (including, for example, antigens, or polypeptides (e.g., His6) that facilitate fusion protein purification), covalently coupled to antigens, and / or chemically cross-linked to antigens. In some cases, non-catalytic, non-toxic forms of toxins are referred to as toxoids. Toxoids lack toxicity but maintain their antigenicity and their immunogenicity.

[0035] As used herein, the term "reduced toxicity" means that relative to a first composition comprising a specific protein active component (e.g., wild-type TT), a second composition comprising a modified form of a specific protein active component can be administered to a mammal at the same or greater dose level than a dose that is lethal to the first composition without causing death to the mammal. Reduced toxicity includes partial or complete elimination of detectable toxicity using methods known to practitioners in the art. Additionally, reduced toxicity includes reduced systemic toxicity (i.e., after intravenous administration) or reduced toxicity after intramuscular administration.

[0036] In some embodiments, the preparation includes a modified tetanus toxin. Generally, the tetanus toxin modified as described herein exhibits one or more altered properties compared to the wild-type tetanus toxin polypeptide set forth in SEQ ID NO: 1, such as significantly reduced catalytic activity and receptor binding activity. In some embodiments, the modified tetanus toxin described herein is at least 1,000,000 times less toxic than wild-type tetanus toxin.

[0037] Table 1: Exemplary toxins M-BoNT / A1 as vaccines and conjugate vaccines W and 5M-TeNT have multiple functionally independent mutations

[0038] Mutated residues (inhibited function) <![CDATA[M-BoNT / A1 W ]]> 5M-TeNT E(Catalysis) E224Q E234Q R(Catalysis) R363A R372A Y(Catalysis) Y366F Y375F R (receptor binding) wild type R1226L W (receptor binding) W1266A W1289A

[0039] In some embodiments, the preparation comprises a modified tetanus toxin having mutations at amino acid residues 372 and 375, and also having mutations at one or more of residues 234, 1226, and 1289, wherein the residue positions are numbered relative to the full-length wild-type tetanus neurotoxin (Clostridium tetani CN3911; GenBank Accession No. X06214) set forth in SEQ ID NO: 1. In some embodiments, the amino acid mutations at residues 372 and 375 are R372A and Y375F, and the modified toxin further comprises at least one mutation selected from E234Q, R1226L, and W1289A. In some examples, the modified toxin comprises 5 mutations (R372A, Y375F, E234Q, R1226L, and W1289A), numbered according to SEQ ID NO: 1, and referred to herein as "5M-TeNT" or "5M-TT". See Table 1. In some cases, 5M-TeNT is encoded by the amino acid sequence shown in SEQ ID NO:4.

[0040] In some embodiments, the tetanus toxin has a modified translocation domain. For example, the lysine at position 768 is located in a loop connecting two long alpha helices. The single amino acid mutation to alanine (A) inactivates or prevents light chain translocation. In some cases, the K768A mutation is added to the 5M-TT modified toxin to produce 6M-TT, wherein the resulting modified toxin includes independent mutations at 6 positions (see Tables 2 and 3). In some examples, the modified toxin includes 6 mutations (R372A, Y375F, E234Q, K768A, R1226L, and W1289A), numbered according to SEQ ID NO: 1, and referred to herein as "6M-TeNT" or "6M-TT". In some cases, 6M-TeNT is encoded by the amino acid sequence shown in SEQ ID NO: 5. Without being limited by any particular mechanism or theory, the vaccine efficacy of 6M-TT is expected to be higher than that of 5M-TT, but the reversal rate should be lower than that of 5M-TT. Addition of mutations in one or more of D767, K768 or E769 of 5M-TT would render the engineered vaccine more completely inactivated, in addition to destroying the functionality of the catalytic and receptor binding domains, also inactivating the translocation domain function.

[0041] Table 2: Exemplary toxin 6M-TeNT containing multiple functionally independent mutations as vaccines and conjugate vaccines

[0042]

[0043] In some embodiments, the tetanus toxin is modified to inhibit VAMP-2 cleavage. For example, a mutation of the leucine residue at position 231 (e.g., a mutation of leucine to lysine (K)) inactivates the catalytic activity of the toxin for VAMP-2 cleavage. In some cases, the L231K mutation is added to the 6M-TT modified toxin to produce 7M-TT, wherein the resulting modified tetanus toxin includes independent mutations at 7 positions (Table 3). In some examples, the modified toxin includes 7 independent mutations (R372A, Y375F, E234Q, R1226L, W1289A, K768A, and L231K), numbered according to SEQ ID NO: 1, and referred to herein as "7M-TeNT" or "7M-TT". In some cases, 7M-TeNT is encoded by the amino acid sequence shown in SEQ ID NO: 6.

[0044] In some embodiments, the tetanus toxin is modified to inhibit VAMP-2 binding. For example, a mutation of the tyrosine (Y) residue at position 26 (e.g., a mutation of tyrosine to alanine (A)) inactivates the VAMP-2 binding ability of the toxin. In some cases, a Y26A mutation is added to a 7M-TT modified toxin to produce 8M-TT, wherein the resulting modified tetanus toxin includes independent mutations at 8 positions (Table 3). In some examples, the modified toxin includes 8 independent mutations (R372A, Y375F, E234Q, R1226L, W1289A, K768A, L231K, and Y26A), numbered according to SEQ ID NO: 1, and referred to herein as "8M-TeNT" or "8M-TT". In some cases, 8M-TeNT is encoded by the amino acid sequence shown in SEQ ID NO: 7.

[0045]

[0046] In some cases, the modified tetanus toxin includes additional amino acid substitutions at residue positions 372, 275, 234, 768, 1226, 1289, 231, and / or 26. For example, amino acids that may be substituted for the listed amino acids include substitutions that reverse the charge or hydrophobicity of the original residue, conservative amino acid substitutions, and substitutions that result in the deletion of the original residue.

[0047] As is well known to those skilled in the art, altering the primary structure of a polypeptide with conservative amino acid substitutions may not significantly alter the activity of the polypeptide because the side chains of the amino acids of the inserted sequence may form similar bonds and contacts as the substituted amino acid side chains, even when the substitution is in a region that is critical to determining the polypeptide configuration.

[0048] Conservative amino acid substitutions are art-recognized substitutions that substitute one amino acid for another amino acid with similar characteristics. Conservative amino acid substitutions can be achieved by modifying the nucleotide sequence to introduce nucleotide changes encoding conservative substitutions. For example, each amino acid can be described as having one or more of the following characteristics: positively charged, negatively charged, aliphatic, aromatic, polar, hydrophobic, and hydrophilic. Conservative substitutions include substitutions between amino acids in each group. Acidic amino acids include asparagine and glutamine. Basic amino acids include histidine, lysine, and arginine; aliphatic amino acids include isoleucine, leucine, and valine. Aromatic amino acids include phenylalanine, glycine, tyrosine, and tryptophan. Polar amino acids include asparagine, glutamine, histidine, lysine, arginine, serine, threonine, and tyrosine. Hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tyrosine. Amino acids may also be described in terms of relative size, with alanine, cysteine, asparagine, glycine, aspartic acid, proline, threonine, serine, and valine being considered small amino acids.

[0049] In some cases, non-conservative substitutions may be provided if such substitutions do not disrupt the tertiary structure of an epitope within a polypeptide, for example, do not disrupt the immunogenicity (eg, antigenicity) of the polypeptide and do not restore toxicity.

[0050] In some embodiments, the modified tetanus toxin comprises mutations at amino acid residues 372 and 375, and also comprises mutations at one or more of residues 234, 1226, and 1289, wherein the modified toxin is conjugated or coupled to another peptide, as described below, for a suitable therapeutic method. Advantageously, the modified tetanus toxin of the present disclosure does not require formalin detoxification when used as a vaccine or adjuvant. In some cases, a small amount of formalin (~0.04%) or another fixing or stabilizing agent (e.g., formalin, glutaraldehyde, β-propiolactone, etc.) is added to the modified tetanus toxin as a stabilizer, but the amount is smaller (e.g., one order of magnitude smaller) than that commonly used (~0.4%) to remove the toxicity of wild-type tetanus toxin (or tetanus toxin not modified as described herein) to form a "tetanus toxoid".

[0051] In some embodiments, the modified toxins described herein also include molecules such as carbohydrates, proteins or peptides (e.g., antigens) and chemical groups. In particular, recombinant non-catalytic non-toxic variant toxin forms (e.g., modified tetanus toxins and modified botulinum neurotoxins) are provided herein, which are further modified to include conjugated or chemically linked (e.g., cross-linked) carbohydrates. Thus, modified toxins conjugated to carbohydrates provide a platform for use as T cell-dependent immunogens. In some cases, other molecules or groups (e.g., antigens) can be further connected to the cross-linked molecules as "cargo".

[0052] In some instances, the modified carbohydrate-conjugated toxin comprises 5 mutations (R372A, Y375F, E234Q, R1226L, and W1289A), numbered according to SEQ ID NO: 1, and referred to herein as "TeNT (CB)". In some instances, the modified carbohydrate-conjugated BoNT toxin comprises 4 mutations (E224Q, R363A, Y366F, and W1266A), numbered according to UniProtKB / Swiss-Prot: P10845.4, and referred to herein as "BoNT (CB)". In some cases, the carbohydrate is conjugated to the modified toxin by chemical cross-linking. Common chemical reactions for covalently linking polysaccharides to polypeptides, such as toxins, include, but are not limited to, reductive amination, cyanide conjugation, and carbodiimide reactions. In other cases, carbohydrate-toxin conjugates are prepared using other synthetic methods, such as the protocol described in Chu et al., 1983. Infect and Immun. 40(1):245-256.

[0053] The term "sugar" as used herein refers to polysaccharides, oligosaccharides and other carbohydrate polymers, including monosaccharides. Typically polysaccharides have about 10 to 2,000 or more repeating units, preferably about 100-1900 repeating units. Oligosaccharides typically have about 2-10 repeating units, to about 15, 20, 25, 30, or 35 to about 40 or 45 repeating units. In some cases, sugars suitable for coupling with toxins provided herein include but are not limited to polysaccharides with carboxyl groups. In these cases, polysaccharides with carboxyl groups can be coupled to modified toxins through thiol derivatives of the carboxyl groups.

[0054] As used herein, the terms "polypeptide", "peptide" and "protein" refer to polymers comprising amino acid residues, primarily bound together by covalent amide bonds. The term "protein" is meant to include all of the above definitions. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which amino acids are linked by covalent peptide bonds. Proteins or peptides can be isolated from natural organisms, produced by recombinant technology, or produced by synthetic production techniques known to those skilled in the art.

[0055] In some cases, the spacer moiety can serve as a spacer arm bridge between the modified toxin and the attached molecule.The spacer moiety can have any of a variety of molecular structures, including but not limited to dextran, polyglutamic acid, and oligopeptides.

[0056] The sequence identity between amino acid sequences can be determined by comparing sequences. When the equivalent position in the compared sequence is occupied by the same amino acid, the molecule is the same at that position. The comparison is scored as a percentage of identity, which is a function of the number of identical amino acids in the common position of the compared sequences. When comparing sequences, the best comparison may require the introduction of gaps in one or more sequences to take into account possible insertions and deletions in the sequence. The sequence comparison method can use gap penalties, so that for the same number of identical molecules in the compared sequences, there are as few gaps as possible, showing that the sequences with higher correlation between the two compared sequences can achieve higher scores than sequences with many gaps. The calculation of the maximum percentage of identity involves producing the best comparison, and taking into account the gap penalty. As mentioned above, the percentage of sequence identity can be determined using the default parameter settings using the Needleman-Wunsch universal sequence alignment tool, which is provided to the public at blast.ncbi.nlm.nih.gov / Blast.cgi. The Needleman-Wunsch algorithm is published in J.Mol.Biol. (1970) Volume 48: 443-53.

[0057] The polypeptide and nucleic acid of the present invention can be prepared by conventional synthesizers. In addition, they can also be produced by recombinant DNA technology and incorporated into suitable expression vectors, which are then used to transform suitable host cells, such as prokaryotic cells such as Escherichia coli. The transformed host cells are cultured and the polypeptide is isolated therefrom.

[0058] In another embodiment, the present invention is a nucleic acid sequence encoding a modified toxin preparation and other nucleic acid sequences that hybridize with the nucleic acid molecule composed of the above nucleotide sequence under high stringency conditions. In a specific embodiment, the present invention provides a DNA sequence encoding a modified tetanus toxin, wherein the modified tetanus toxin has mutations at amino acid residues 372 and 375, and also has mutations at one or more of residues 234, 1226, and 1289, or a modified catalytic domain as described herein. In some cases, the amino acid sequence of the modified tetanus toxin is shown in SEQ ID NO:4.

[0059] The term "stringent conditions" used herein refers to parameters well known in the art. For example, nucleic acid hybridization parameters can be found in the literature compiling these methods, such as: "Molecular Cloning: A Laboratory Manual", J. Sambrook, et al., ed., 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or "Current Protocols in Molecular Biology", FM Ausubel et al., John Wiley & Sons, Inc., New York. More specifically, the high stringency conditions used herein refer to hybridization at 65°C in a hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 25mM NaH2PO4 (pH 7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride / 0.015M sodium citrate, pH 7; SDS is dodecyl sulfonate; EDTA is ethylenediaminetetraacetic acid. After hybridization, the membrane with the transferred DNA is washed in 2xSSC at room temperature and then washed in 0.1-0.5xSSC / 0.1xSDS at up to 68°C, for example, 55°C, 60°C, 65°C or 68°C. Alternatively, high stringency conditions can be obtained using commercially available hybridization buffers, such as ExpressHyb TM Buffer (Clontech) was used, and the hybridization and washing conditions provided by the manufacturer were used.

[0060] It should also be understood that the present invention utilizes sequences in expression vectors and transfected host cells and cell lines, whether these are prokaryotic (E. coli) or eukaryotic (e.g., dendritic cells, CHO cells, COS cells, yeast expression systems, recombinant baculovirus expression in insect cells). The expression vector requires the relevant sequences, i.e., the sequences described above, to be operably linked to a promoter.

[0061] In another aspect, provided herein are immunogenic compositions comprising the modified toxins described herein, which upon introduction into a host will render the host immune when the host is later challenged by the same microorganism that produced the protein (e.g., Clostridium tetani). In a preferred embodiment, the immunogenic composition is a vaccine comprising the modified toxins described herein, and further comprising an excipient and / or a diluent, suitable for the composition to be administered to an individual who needs to be immunized against a disease caused by Clostridium tetani or Clostridium botulinum or its purified toxins.

[0062] The term "vaccine" as used herein refers to a composition comprising an antigen. Vaccines may also include biological preparations that improve immunity to a particular disease. Vaccines may generally include a reagent, called an antigen, which represents a pathogenic microorganism, which may be prepared in a weakened or inactivated form of the microorganism, its toxin, or one of its surface proteins. Antigens may stimulate the body's immune system to recognize the reagent as a foreign object and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms encountered later. Similarly, modified toxin preparations, combined with vaccines for other pathogens, can act as vaccine adjuvants to "enhance" the immune response to the pathogen of interest. Adjuvants may be classified according to their physicochemical properties or mechanism of action. Two major categories of adjuvants include compounds that act directly on the immune system, such as bacterial toxins that stimulate immune responses, and molecules that can promote antigen presentation in a controlled form and serve as carriers.

[0063] The selection of suitable vaccine components is within the routine capabilities of those skilled in the art. For example, the vaccine composition of the present invention can be conventionally formulated with pharmaceutically acceptable excipients or diluents, such as aqueous solvents, non-aqueous solvents, non-toxic excipients such as salts, preservatives, buffers, etc. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate. Aqueous solvents include water, alcohol / aqueous solvents, saline solutions, intraperitoneal carriers such as sodium chloride, Ringer's dextrose, etc. Preservatives include antimicrobial agents, antioxidants, chelating agents, and inert gases. The pH and exact concentration of the various components of the vaccine composition can be adjusted by conventional techniques.

[0064] In some cases, the preparations described herein may include purified modified toxins, including partial toxin complexes. In some embodiments, the preparations may further include stabilizers known to stabilize BoNT and tetanus toxin proteins. Suitable stabilizers are known in the art and include, but are not limited to, for example, human or bovine serum albumin, gelatin, recombinant albumin, as described in U.S. Publication No. US2005 / 0238663 (the contents of which are incorporated herein by reference in their entirety), and the like.

[0065] The terms "subject" and "patient" are used interchangeably to refer to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein to refer to a human subject.

[0066] The preparation of the present invention can be administered in a therapeutically effective amount. The term "effective amount" or "therapeutically effective amount" refers to inducing an immune response in the subject receiving the antibody or vaccine, which is sufficient to prevent disease signs or symptoms caused by infection with pathogens (including viruses or bacteria), including adverse health effects or complications. Humoral immunity or cell-mediated immunity can be induced, or both can be induced simultaneously. The immunogenic response of animals to the vaccine can be evaluated, for example, directly by measuring antibody titers, lymphocyte proliferation tests, or directly by monitoring signs and symptoms after attack by wild-type strains. The protective immunity conferred by the vaccine can be evaluated by measuring, for example, clinical signs such as mortality, morbidity, temperature values, overall physiological conditions, and the overall health and performance of the object. The therapeutically effective vaccine amount can be changed according to the specific preparation used, or the condition of the object, and can also be determined by a physician.

[0067] The preparations of the present invention can be administered in a therapeutically effective amount, depending on the type of treatment required. Methods for determining the appropriate dose or dosage range for individual treatment are known to those skilled in the art. For the methods provided herein, the preparations of the present invention can be administered by achieving the intended purpose or any manner deemed suitable by those skilled in the art. In an exemplary embodiment, a modified toxin agent is administered in a single dose or in a continuous administration form when appropriate using, for example, a micropump system. In some cases, the modified toxin formulation is provided in a liquid dosage form or a lyophilized dosage form, which is, for example, reconstituted before administration.

[0068] As used herein, the term "protected" refers to immunizing a patient against a disease or condition. Immunity may be induced by administering a vaccine containing an antigen. In particular, in the present invention, the immunized patient is protected from tetanus or its symptoms.

[0069] In one embodiment, a suitable dose is about 1 μg to 20 μg.

[0070] Suitable routes of administration for the modified toxin formulations described herein include, but are not limited to, direct injection. In some embodiments, each dose is administered intramuscularly.

[0071] The dosage, toxicity and therapeutic efficacy of drugs provided by current technology can be determined by standard pharmaceutical methods in cell culture or experimental animals, such as determining LD 50 (lethal dose for 50% of the population) and ED 50 (The therapeutic dose that is effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD 50 / ED 50 .

[0072] As used herein, "therapeutically effective amount" refers to the amount of a compound that, when administered to a subject for treating a disease, is sufficient to achieve treatment of the disease. The "therapeutically effective amount" may vary depending on the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and mode of administration, the judgment of the attending physician or veterinary practitioner, and other factors. For purposes of the present invention, "treatment" or "treatment" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder. The term includes both prophylactic treatment or treatment (i.e., prevention) and palliative treatment or treatment.

[0073] The conjugate delivery platforms described herein can be tailored to target specific conditions or diseases by adjusting the conjugated modified Tet toxin platform administered.

[0074] method

[0075] In another aspect, provided herein is a method for engineering vaccines and conjugate vaccines with reduced toxicity and improved performance. The method includes specific amino acid residues in the domain of genetically modified multi-domain protein toxins, so that the toxin cannot express toxicity by catalytic activity or receptor binding activity on target cells (e.g., neurons), cannot translocate, and the ability to return to toxic form is weakened. By changing a variety of independent protein functions, the method provided herein advantageously provides a full-length toxin as an ideal candidate for a powerful vaccine and conjugate vaccine, which does not substantially have the ability to return protein to toxicity. The term "efficacy" used herein refers to a specific ability or ability of a vaccine that affects protective immunity as indicated by appropriate laboratory tests or by accurately controlled clinical data. In other words, efficacy is a measure of the strength of a vaccine.

[0076] In some cases, a method for obtaining an engineered bacterial protein toxin with improved efficacy as a vaccine comprises or consists essentially of the following steps: selecting one or more amino acid positions in each domain of an amino acid sequence encoding a multi-domain bacterial protein toxin, wherein each position is selected to inactivate a protein function associated with each domain, wherein the domains include two or more catalytic domains, a translocation domain, a receptor binding domain, and a substrate binding domain; replacing a natural amino acid residue at each selected position with a non-natural amino acid residue, such that the substitution inactivates one or more protein functions associated with the domain; and expressing a nucleic acid sequence encoding a full-length bacterial protein toxin comprising the substituted non-natural amino acid residues in a host cell, such that the expressed protein exhibits a partial or complete loss of catalytic activity, receptor binding activity, translocation activity or substrate binding activity relative to a full-length bacterial protein toxin comprising natural amino acids.

[0077] The amino acid residues selected for modification include analyzing the protein sequence (e.g., primary amino acid sequence) or structural information to identify the individual functional amino acid residues of each domain of the bacterial protein toxin. Preferably, the selected residues are one or more individual amino acid residues of each functional domain (e.g., catalytic domain, translocation domain, receptor binding domain, substrate binding domain), which can be modified without causing the full-length protein to lose stability or immunogenicity. Protein stability can be assessed by any suitable method. In some cases, by determining casein sensitivity 3 Testing the stability of the modified toxins and the immunogenicity of the modified toxins by measuring the immune response to vaccination with the modified toxins in a mouse model 4 .

[0078] Protein structure information can be obtained by any suitable method, such as x-ray crystallography, electron microscope, nuclear magnetic resonance mass spectrometry, computer protein structure modeling or its combination. In some cases, the crystal structure of the bacterial protein toxin of interest can be used to identify the interaction between the toxin functional domains and the functional conservative residues between the related protein toxins. For example, using diphtheria toxin as an example, the amino acid residues related to catalysis, substrate binding, translocation and receptor binding are identified from previous research documents, and the gene encoding diphtheria toxin is engineered to produce a mutated diphtheria toxin gene, which is encoded in multiple independent mutations in each of the four functional domains of the toxin, based on the amino acid sequence alignment in the diphtheria toxin crystal structure. The nucleic acid sequence encoding the mutated diphtheria toxin will be transformed into Escherichia coli, and the recombinant protein is produced and tested for its toxicity loss, the maintenance of stability, and the immunogenicity retained.

[0079] For example, the inter-domain and intra-domain molecular interactions can be determined by analyzing the crystal structure of wild-type bacterial protein toxins. Substitution mutations can be achieved using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis and total gene synthesis, and other methods known in the art. For example, an illustrative method of a mutagenesis scheme is shown in the following examples. In some cases, mutations at single amino acid residues are produced using site-directed mutagenesis. In some cases, oligonucleotide primers for site-specific mutagenesis can be designed using software programs such as PrimerX (available from bioinformatics.org / primerx / on the World Wide Web). Wild-type bacterial protein toxin genes can be cloned into expression vectors using any suitable method, such as polymerase chain reaction, as templates for mutagenesis. Mutagenesis can be confirmed using nucleic acid sequencing. In some cases, polynucleotides encoding modified protein toxins are located in expression vectors. In some cases, the vector is in a host cell (e.g., bacterial cell, yeast cell, eukaryotic cell). Many expression vectors and systems for prokaryotic and eukaryotic cells are known, and selecting a suitable system is just a matter of selection. Expression and purification of the modified protein product of the present invention can be easily achieved by those skilled in the art. See, e.g., Sambrook et al., "Molecular cloning—A Laboratory Manual, 2nd ed.."

[0080] The method can be used for almost any bacterial protein toxin, also known as exotoxin, which is a multi-domain protein secreted by bacteria, and in many cases, represents an enzyme with catalytic action and substrate specificity. Bacterial protein toxins include but are not limited to botulinum toxin, tetanus toxin, Shigella toxin, diphtheria toxin, Bordetella pertussis toxin, Escherichia coli heat-labile toxin LT, Bacillus anthracis toxin, Pseudomonas exotoxin A, anthrax toxin lethal factor (LF), cholera enterotoxin, and Staphylococcus aureus shedding protein B. Table 4 lists exemplary bacterial proteins, wherein crystal structure information can be used to design genetically engineered recombinant, non-toxic, high-performance toxoids, which are particularly advantageous for vaccines and conjugate vaccines.

[0081] Table 4: Bacterial protein toxins with multiple independent functional domains and resolved structures

[0082]

[0083] In some cases, independent inactivation modifications are selected to disrupt the catalytic domain, substrate binding domain, translocation domain, and receptor binding domain of one or more bacterial protein toxins. For example, diphtheria toxin is a bacterial protein toxin that is 535 amino acids long. Inactivation of amino acid residues involved in catalytic (E149S), substrate binding (H21A), translocation (E349K, E362K), and receptor binding (K516A, K526A, H391A) functions will result in a non-toxic but potent diphtheria vaccine.

[0084] However, it should be understood that the method can be applied to nearly any protein with a multifunctional domain (eg, an effector domain) for which amino acid sequence and / or crystal structure information is available and inactivating modifications can be identified based on known structure-function properties.

[0085] In some embodiments, at the heavy chain C-terminus (HC C ) introduces genetic modifications that hinder host receptor activity. In order to hinder catalytic activity, genetic modifications are introduced into the light chain via the SNARE complex at amino acid residues that are required or important for neurotransmitter release.

[0086] The techniques and methods described and referenced herein are generally well known and commonly used by those skilled in the art, such as the widely used molecular cloning methods described in Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Where appropriate, methods using commercially available kits and reagents are generally performed according to protocols and / or parameters determined by the manufacturer, unless otherwise stated.

[0087] Reagent test kit.

[0088] In another aspect, provided herein is a kit for administering a vaccine or adjuvant to a subject, comprising a modified toxin vaccine as described herein. In one embodiment, the kit comprises a certain form of a modified toxin (e.g., a modified tetanus toxin as described herein). The kit may also include instructions so that the user can vaccinate the subject against diseases caused by Clostridium tetani, particularly the occurrence of diseases caused by tetanus toxin. In one embodiment, the modified toxin of the present invention is formulated, delivered, and stored for use under physiological conditions. Suitable pharmaceutical carriers include, but are not limited to, for example, saline solutions (e.g., 0.9% sodium chloride), phosphate buffered saline, lactated Ringer's solution, and the like.

[0089] "Instructional material" means a publication, record, chart, or any other medium of expression that is used to convey the usefulness of the pharmaceutical composition of the present invention for one of the purposes described herein. For example, the instructional material of the kit can be attached to a container containing the present invention, or shipped with a container containing the present invention. In addition, the instructional material can also be shipped separately from the container, or provided in electronic contact form on a website, so that the instructional material and the biocompatible hydrogel can be used in conjunction with the recipient.

[0090] In this specification and claims, the terms "comprising" and "including" are open-ended terms and should be interpreted to mean "including, but not limited to." ... These terms encompass the more restrictive terms "consisting essentially of" and "consisting of.

[0091] Unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" as used herein and in the appended claims include plural references. Thus, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein. It should also be noted that the terms "including", "comprising", and "having" may be used interchangeably.

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. All publications and patents specifically mentioned herein are incorporated herein by reference in their entirety for all purposes, including describing and disclosing the chemical substances, equipment, statistical analysis and methods reported in the publications that can be used in conjunction with the present invention. All references cited in this specification should be regarded as an indication of the level of technology in the art. All contents herein should not be construed as admitting that the present invention is not a prior invention prior to these disclosures.

[0093] A more complete understanding of the present invention can be obtained upon consideration of the following non-limiting examples. Example

[0094] Example 1: M-BoNT / A1 and M-BoNT / A1 W Isolation and characterization of

[0095] This example describes a full-length BoNT engineered with defects in the catalytic and receptor structures that protect against 10 6 LD 50 These data suggest that genetically engineered toxins with multiple mutations that attenuate toxicity through independent mechanisms could serve as a platform strategy for developing vaccines against botulism and other toxin-mediated diseases.

[0096] Materials and methods:

[0097] Biosafety and Biosecurity: Experiments conducted at the University of Wisconsin-Madison were approved by the university's Biosafety Committee. In addition, experiments were conducted in a laboratory approved for this research under the federal Select Agent Program, and the experimental staff was assessed for suitability and followed university policies and practices. Animal experiments were approved and conducted according to the guidance of the University of Wisconsin-Madison Animal Care Committee. The Department of Health and Human Services determined that the BoNT / A gene and protein product encoding the three LC mutations ((E224Q / R363A / Y366F), referred to as M) does not meet the regulatory definition of a select agent, allowing the production of M-BoNT / A without select agent registration (§73.3 HHS Select Agents and Toxins 42 CFR 73.3(e)(1).

[0098] Botulinum neurotoxins were purified from Clostridium botulinum strains A-hyper, Kyoto-F, CDC A3 (provided by Susan Maslanka and Brian Raphael, Centers for Disease Control and Prevention), and A661222 by standard toxin purification methods (Jacobson et al., 2011; Lin et al., 2010; Malizio et al., 2000; Tepp et al., 2012). BoNT / A1, / A2, / A3, and / A5 were purified from CDC41370 B2tox - (Modified from strain CDC41370 to produce only BoNT / A6) Toxins BoNT / A6 was purified using previously described methods (Pellett et al., 2016). Toxin purity was determined by spectroscopic and SDS-PAGE analysis (Whitemarsh et al., 2013). Purified toxins were stored in phosphate-buffered saline containing 40% glycerol at -20°C until use. The activity of the five subtype preparations was determined using a standard intraperitoneal mouse bioassay (MBA) as previously described (Hatheway, 1988; Schantz, 1978). The median lethal dose of each toxin was defined as the LD50 unit (U) for 1 mouse. The specific activities of the BoNT / A subtypes were: 8 pg / U (A1), 7.9 pg / U (A2), 17 pg / U (A3), 7.3 pg / U (A5), and 5.9 pg / U (A6).

[0099] Recombinant BoNT derivatives: HC C / A1(W1266A)(HC C / A1 W ),LC / A1(R363A / Y366F)(LC / A1 RY ),LCHC N / A1(E224Q / R363A / Y366F)(M-LCHC N / A1),BoNT / A1(E224Q / R363A / Y366F)(M-BoNT / A1),BoNT / A1(E224Q / R363A / Y366F / W1266A)(M-BoNT / A1 W ) and non-catalytic tetanus toxin (R372A / Y375F) (TeNT RY ) was prepared as described previously (Przedpelski et al., 2013). Briefly, E. coli was grown overnight at 37°C on LB agar containing 50 μg / ml kanamycin. The culture was inoculated into LB medium (400 ml) containing kanamycin and shaken at 37°C for 3-6 hours to reach an OD600 of approximately 0.6, 1.0 mM IPTG was added, and then shaken at 16°C overnight. The cells were collected and the pellet was suspended in lysis buffer (20 mM Tris (pH 7.9), 500 mM NaCl, 5 mM imidazole, nuclease, DNase, and protease inhibitors) (Sigma). The cells were disrupted with a French press, clarified by centrifugation, and filtered through a 0.45 μm surfactant-free cellulose acetate membrane (Thermo Fischer). The lysate was further purified by tandem gravity flow chromatography using Ni 2 + -NTA resin (Qiagen), p-aminobenzylamidine agar (Sigma), and Strep-tactin superflow high-throughput resin (IBA-LifeSciences). The purified protein was dialyzed into 10 mM Tris (pH 7.9), 200 mM NaCl, and 40% glycerol and stored at -20°C. The recombinant proteins used in this study were Figure 1 Code M-BoNT / A1 W The nucleotide sequence is shown in SEQ ID NO: 4 (see Fig. 9 ).

[0100] Vaccine challenge: with HC C / A1 W ,M-LCHC N / A1,M-BoNT / A1, or M-BoNT / A1 W Groups of female ICR mice (18-22 g) were immunized intraperitoneally with equal volumes of Alhydrogel as adjuvant at the indicated concentrations. WAs vaccines. Vaccines were administered on days 1 and 14, blood was collected by maxillary bleeding on day 21, and mice were challenged on day 26 with BoNT / A1, BoNT / A2, or BoNT- / A2, / A3, / A5,A6 mixtures as indicated. At least eight mice per group were used in each experiment as indicated. The statistical relevance of the results was assessed using a two-tailed paired Student's t-test, p=0.05.

[0101] ELISA: BoNT derivatives or TeNT RY (250 ng / well) was added to a high protein binding 96-well plate (Corning) in 0.1 ml coating buffer, 50 mM Na2CO3 (pH 9.6) and incubated overnight at 4°C. The plate was then washed three times with 0.3 ml phosphate buffered saline (PBS) containing 0.05% Tween 20 and blocked for 30 minutes at room temperature (RT) with 0.2 ml PBS containing 1% (wt / vol) bovine serum albumin (BSA). The plate was incubated for 1 hour at RT, and the indicated dilutions of serum from each vaccinated mouse (1:20,000 or 1:30,000) were added, dissolved in PBS containing 1% (wt / vol) BSA (0.1 ml). After washing three times with 0.3 ml PBS containing 0.05% Tween 20, the plates were incubated with goat α-mouse IgG-horseradish peroxidase (IgG-HRP dilution 1:20,000; Thermo) in PBS containing 1% (wt / vol) BSA for 1 hour at RT. The plates were washed three times with 0.3 ml PBS containing 0.05% Tween 20 and then incubated with 0.1 ml / well tetramethylbenzidine (TMB; Thermo Ultra TMB) as substrate. After 10 minutes, the reaction was stopped with 0.1 ml 0.1 M H2SO4 and the absorbance was read at 450 nm. Control ELISAs using α-HA and αFLAG antibodies to determine bound antigens showed the presence of the correct epitope within each antigen to within 15% (data not shown). For ELISA, statistical analysis was performed using a two-tailed unpaired Student's t-test based on immunization and / or challenge conditions for each serum group analyzed (N=10), P<0.05=*, 0.01=**, 0.001=***, and 0.0001=**** (GraphPad Prism 7). Each serum was analyzed by at least two independent ELISAs with duplicates. Mouse sera were analyzed at dilutions of 1:20,000-1:30,000 based on evaluation of several individual sera. Serum dilutions were established from a dose-dependent ELISA of mice vaccinated with M-BoNT / A1 that survived a BoNT / A1 challenge. Representative ELISAs are shown in Figure 2. Figure 6 shown.

[0102] Cell-based assay for detecting neutralizing antibodies: Cell-based neutralization assays were performed as previously described (Whitemarsh et al., 2012). Briefly, human induced pluripotent stem cell (hiPSC)-derived neurons (Cellular Dynamics International, Wisconsin) were seeded onto cells coated with poly-L-ornithine and Matrigel according to the manufacturer's instructions. TM Cells were plated on 96-well TPP plates (Midwest Scientific, MO) at a density of approximately 35,000-40,000 cells / well, maintained in iCel neuronal medium (Cellular Dynamics International, WI) and used for neutralization assays 7 days later. To detect neutralizing antibodies in mouse serum, 2 pM BoNT / A1 was combined with serially diluted sterile-filtered serum and incubated in culture medium at 37°C for 1 hour. BoNT / A1 without serum was used as a "no antibody" reference, and serum from immune-deficient mice was used as a control. Serum without toxin was used as a negative control. 50 μl of each antibody-toxin mixture was added to each well of hiPSC-derived neurons, at least in duplicate; cells were incubated at 37°C in 5% CO2 for 24 hours. Toxin / antibody was aspirated from the cells, and cell lysates were prepared in 50 μl lithium dodecyl sulfate (LDS) sample buffer (Life Technologies). Cell lysates were analyzed for SNAP-25 cleavage by Western blot as previously described (Pellett et al., 2007; Pellett et al., 2010). Images were acquired using the PhosphaGlo reagent (KPL, Gaithersburg, MD) and the Fotodyne / FOTO / Analyst FX imaging system (Harland, WI). Cleaved (24 kDa) versus uncleaved (25 kDa) SNAP-25 signal was analyzed by densitometry using TotalLab Quant software (Fotodyne, Harland, WI). Percent protection was determined by comparison to a “no antibody” control, and IC was estimated using GraphPad Prism 6 software and nonlinear regression with variable slope and four parameters. 50 .

[0103] result

[0104] M-BoNT / A1 was not toxic to outbred mice or cells in culture.

[0105] Intraperitoneal injection of 10 μg of trypsinized or untrypsinized M-BoNT / A1 / mouse (ICR) resulted in no observable signs of botulism, indicating that M-BoNT / A1 is at least one million times less toxic than native BoNT / A1. In addition, incubation of human iPSC-derived neurons with 80 nM M-BoNT / A1 resulted in no detectable cleavage of SNAP-25, whereas incubation with 50 fM native BoNT / A1 cleaved SNAP-25, indicating at least one million times less toxicity based on cell-based assays (data not shown).

[0106] M-BoNT / A1 and M-BoNT / A1 W is better than HC C / A1 W It is a more protective vaccine.

[0107] Outbred ICR mice (n=8-10) were vaccinated to reflect the natural immune differences within the host (Rai et al., 2009), using a primary immunization followed by a booster immunization. C / A1(W1266A)(HC C / A1 W ) in a mouse model of botulism and HC C / A1 has similar vaccine efficacy (Przedpelski et al., 2013), M-BoNT / A1 W Engineering was also performed. 0.3 μg / mouse single-chain M-BoNT / A1 W or 0.2 μg / mouse M-LCHC N / A1-vaccinated mice were protected against 10 6 LD 50 Natural BoNT / A1 or 10 5 LD 50 BoNT / A subtype mixture (2.5x 10 4 LD 50 Each A2, A3, A5, A6) attack, or for 10 6 LD 50 Challenge with native BoNT / A2 (a heterologous subtype) provided partial protection (Table 4). C / A1 W The mice vaccinated were 10 3 LD 50 These data suggest that at equimolar doses, M-BoNT / A1 W and M-LCHC N / A1 vaccine compared to HC C / A1W The vaccine is 1000 times more protective against toxins. When the vaccine is used in equal doses, 0.3 μg / mouse HC C / A1 W The mice vaccinated were 10 5 LD 50 Natural BoNT / A1 or 10 5 LD 50 The natural BoNT / A subtype mixture had a partial protective effect. This indicates that even at equal concentrations (HC C 3-fold molar excess), M-BoNT / A1 W and M-LCHC N / A1 has better protection against homologous and heterologous BoNT / A challenges. M-BoNT / A1 W The M-BoNT / A1 vaccine showed no difference in protection, indicating that the additional "receptor binding" mutation did not affect the vaccine efficacy. W and M-LCHC N / A1 vaccine vs HC C / A1 W The duration of effect on cultured neurons was further investigated for human iPSC-derived neurons by exposing neurons to serial dilutions of BoNT / A1 or BoNT / A6 for 72 h, followed by complete removal of the extracellular toxin and further incubation in culture medium. Cells from each dilution series were harvested at days 3, 39, and 70 in triplicate, and the EC of SNAP-25 cleavage was determined at each time point. 50 For BoNT / A6, at days 3, 39, and 70, the EC 50 The values ​​were approximately 0.04, 0.7 and 1 U / 50 μl / well (32, 560 and 800 fM) ( Figure 2 ). For BoNT / A1, on days 3, 39, and 70, EC 50 The values ​​were approximately 0.7, 6.3, and 28 U / 50 μl / well (313, 2940, and 12,880 fM, respectively) ( Figure 2 ). From the time-varying EC 50 The half-lives of BoNT / A1 and / A6 in these hiPSC-derived neurons determined by the RT-PCR assay were similar for BoNT / A1 and / A6, approximately 12 and 14 days, respectively ( Figure 2 ). Taken together, these results suggest that BoNT / A6 has a long duration of action, similar to other BoNT / A subtypes.

[0108] Antibody responses to BoNT vaccines in outbred mice vary both qualitatively and quantitatively.

[0109] M-BoNT / A1 W and M-BoNT / A1 immunization provided protection against 10 5 LD 50 Complete protection of BoNT / A2 and 10 6 LD 50 Partial protection against native BoNT / A2 (a heterologous subtype) (Table 5). ELISA analysis of the antibody response of vaccinated mice showed that mice that survived or did not survive the native BoNT / A2 challenge had resistance to BoNT and LCHC. N Similar dominant antibody titers, not statistically significantly different ( Figure 2 ). Therefore, partial protection against native BoNT / A2 challenge may be due to specific differences in the composition of neutralizing epitopes between BoNT / A subtypes rather than to differences in the ability of vaccinated mice to generate immune effectors in response to the delivered vaccine.

[0110]

[0111]

[0112] Serum ELISA was used to analyze the antibody response of vaccinated mice using LCA1 RY ,HC C / A1 W ,M-LCHC N / A1, or M-BoNT / A1 holotoxin as binding substrate. The antibody responses in each group of mice vaccinated varied in both quantity and quality. W of mice ( Figure 3 Bottom left) shows the titer of BoNT (mean titer 2.2 (range 1.3-2.6)) and LCHC N The significant antibody titer of HC was 1.7 (range 0.8-2.4). C The titers to LC did not exceed those to controls, indicating that the majority of the antibody response was directed to HC. The variance in titer range was due to varying antibody titers between individual mice and not to variation in ELISA replicates. Similar immune responses were observed to M-BoNT / A1 immunizations (data not shown). N / A1-inoculated mice ( Figure 3 Upper left) also for BoNT and LCHC N There was a significant antibody titer, but the average titer was lower than that of M-BoNT / A1 W Mice immunized with M-LCHCN / A1+HC C / A1 W Mice vaccinated with M-BoNT / A1 W Mice immunized with M-LCHC had qualitatively similar antibody titer profiles and quantitatively similar N / A1 alone immunized mice were equivalent to ( Figure 7 ). Use HC C / A1 W Vaccinated mice have resistance to HC C of antibody titers, which correlated with survival against BoNT / A1 challenge ( Figure 3 , lower right). With M-BoNT / A1 W Vaccinated mice have resistance to TeNT RY Limited antibody titer ( Figure 3 ), indicating that the observed antibody response was BoNT-specific.

[0113] Serum properties of individual vaccinated mice that survived BoNT challenge were similar to those of mice vaccinated with M-BoNT / A1 W ,M-LCHC N / A1, or HC C / A1 W Analysis of individual sera from mice that were vaccinated and survived challenge with native BoNT / A1 revealed several representative immune responses to vaccination ( Figure 4 Since our previous study (Przedpelski et al., 2013 Infect Immun. 81(7):2638-44) did not investigate M-LCHC N Characterization of antibody responses to / A1 immunization, analysis of N / A1 inoculated mice. ELISA results showed that M-BoNT / A1 W Vaccinated mice have resistance to BoNT and LCHC N (#7) or for BoNT, LCHC N , and HC C (Mouse #3) with significant antibody titers. N / A1-vaccinated mice showed resistance to BoNT and LCHC N (mice #21, #24, and #25) showed significant antibody responses, while HC C / A1 W Mice immunized and surviving BoNT / A1 challenge had resistance to HC C (mouse #78). Overall, the RY The antibody response was low, indicating that the immunoreactivity detected in the ELISA was specific for BoNT.

[0114] BoNT / A and HC C Vaccine triggers LCHC N Stronger neutralizing antibody response from the vaccine.

[0115] Neutralizing antibodies were determined in vaccinated mice using a cell-based assay using hiPSC-derived neurons. Ten sera were pooled from each immunization group of equimolar immune challenge and tested for their ability to neutralize BoNT / A1-induced cleavage of SNAP25 in a cell-based assay. W The pooled pool was the most potent in neutralization, with an IC of 0.004. 50 value, which is higher than HC C / A1 W Inoculation of pooled libraries and M-LCHC N / A1+HC C / A1 W The pooled pool of immune cells was approximately 2-fold lower than that of M-LCHC N The pooled pool inoculated with HC / A1 was approximately 5-fold lower (data not shown). C The similarity in neutralizing antibody titers in mice vaccinated with HC and M-BoNT / A1 was surprising, since mice protected by M-BoNT / A1 vaccine were more protected against virulent challenge than mice vaccinated with HC C To further investigate this, the ability of sera from six representative individuals to neutralize BoNT / A1 cleavage of SNAP25 in a cell-based assay was also determined ( Figure 5 ). Overall, there was approximately a 10-fold difference in serum titers for each of the six sera neutralizing BoNT / A1. C / A1 W Immunization (mouse #78) and M-BoNT / A1 W The serum from immune (mouse #3) (containing HC C Significant antibody response ( Figure 4 ) is the strongest inhibitor of SNAP-25 cleavage by BoNT / A1. Lack of detectable HC C The sera with antibody responses (mouse #7, #21, #24, and #25) were poorly effective in cleaving known SNAP-25. C Epitope vaccines are better than LCHC N The vaccine elicited a higher "neutralizing / blocking antibody" response. Overall, these data suggest that the HC of BoNT / A1 C The domain elicited stronger neutralizing / blocking antibody responses than the HC or LC domains, but the HC N and (probably) the LC domain plays a major role in in vivo protection.

[0116] discuss

[0117] In an outbred mouse model of botulism, M-BoNT / A1, M-BoNT / A1 W and M-LCHC N / A1 vaccine vs HC C / A1 W Evaluation of sera from vaccinated mice that survived the BoNT / A1 challenge showed that the N The presence of an internal neutralizing epitope is consistent with LCHC N Common reactions. M-BoNT / A1 W The ability to elicit a protective immune response similar to that of M-BoNT / A1 suggests that reduced host cell binding does not adversely affect vaccine efficacy. Therefore, engineered full-length BoNTs defective in both the catalytic and receptor binding domains represent a new platform strategy for developing vaccines against botulism and other toxin-mediated diseases. Collier et al. (Killeen et al., 1992) showed that second-site mutations could be generated that partially reversed genetically inactivated diphtheria toxin for use in vaccine development testing. Additionally, recent work by Smith et al. revealed the need for more attenuation than simply reducing the catalytic capacity of several BoNT-based vaccine serotypes (Webb et al., 2017).

[0118] In previous studies, LCHC N Considered a candidate for BoNT vaccine (Shone et al., 2009). LCHC is produced in large quantities by fermentation in E. coli N For low-dose BoNT challenge (10 3 LD 50 BoNT) is an effective single-dose vaccine. In this report, we observed that LCHC N It was a strong vaccine directly compared to other BoNT vaccine candidates after primary immunization and one booster, and confirmed the LCHC N The presence of neutralizing epitopes within LCs (Shone et al., 2009). Dolly and colleagues identified LC-specific monoclonal antibodies (Mab) that prevent BoNT / A action (Cenci Di Bello et al., 1994), whereas Marks and colleagues identified BoNT / A neutralizing mAbs that possess LC function, inhibit SNARE cleavage (Cheng et al., 2009), and target HCs. N and neutralizing mAbs against several BoNT serotypes (Garcia-Rodriguez et al., 2011). Overall, these studies suggest that BoNT immunization elicits neutralization of LCHC NAntibody generation against epitopes within the domain. W M-LCHC N / A1 elicits a higher antibody response ( Figure 7 ), and determined HC C Producing antibodies with higher neutralizing / blocking titers in cultured cells, expected to include HC C Vaccines such as M-BoNT / A1 W More effective than LCHC in "high-dose" exposure scenarios N or HC C Vaccine derivatives are more protective.

[0119] HC C It is a common domain for the development of vaccines against botulism, such as DNA and viral vectors and protein-based vaccines constructed in previous studies, showing that HC C Neutralization titer (Clayton et al., 1995) and ease of production (Baldwin et al., 2008). Smith and colleagues expressed HC in Pichia pastoris C , and reported protective immunity induced by HC(c) (Byrne and Smith, 2000), and subsequently reported the protective immunity induced by recombinant HC C / A and HC C A bivalent vaccine consisting of rBVA / B (rBVA / B) is currently in clinical trials (Webb and Smith, 2013). Escherichia coli (E. coli) can also be used as a heterologous host for BoNT vaccine development, including the production of several serotypes (AG) HC against BoNT challenge. C - vaccine (Baldwin et al., 2008). In order to enhance the efficacy of the vaccine, C Mutations were introduced to block host receptor binding, where HC C W Preserve vaccine efficacy (Przedpelski et al., 2013). Although the convenience of production makes HC C has become an attractive vaccine platform, but current studies have shown that M-BoNT / A1 W is better than HC C / A1 W A stronger vaccine. This is supported by the findings of Atassi et al., who tested LC and HC using sera from patients with cervical dystonia resistant to BoNT treatment. N The immune epitopes in the LCHC of BoNT N The immunogenicity was consistent.

[0120] A recent study by Smith and colleagues (Webb et al., 2017) reported that catalytically inactivated BoNTs were more potent than corresponding HCs after a single immunization. C Display for 1000LD 50 The challenge experiment described by Smith and colleagues measured the threshold of toxin challenge, which is different from the current study which measured protection at the endpoint of toxin challenge. The data show that in both cases (for threshold or endpoint determination of protection to toxin challenge), the full-length BoNT vaccine was superior to its HC counterpart. C Subunits are strong. Catalysis and host receptor binding defective M-BoNT / A1 W Effective in endpoint toxin challenge of subunits. By inactivating multiple functional sites to reduce potential toxicity due to cell binding or import, M-BoNT / A1 W As a vaccine candidate, it overcomes concerns that genetic inactivation of catalytic function alone may not provide adequate safety for full-length BoNT vaccine development (Webb et al., 2017).

[0121] Although using HC C As a vaccine candidate against botulism, BoNT has been established (Baldwin et al., 2008; Henderson, 2006). The current study shows that multi-domain derivatives of BoNT are more effective than HC. C Stronger. M-BoNT / A1 W Triggering LCHC N The common significant antibody response of HC in outbred mice C The ability to simultaneously reduce catalysis and receptor binding supports the use of M-BoNT / A1 W As a vaccine platform against botulism. Protection against a mixture of BoNT / A subtypes confirms the broad-spectrum neutralizing capacity of the vaccine. W It was not processed into an activated two-chain form and no toxicity was detected in mice or cells, suggesting that single-chain M-BoNT / A1 W It is a safe and effective vaccine.

[0122] Example 2 - Characterization of disrupted light chain translocation in the lysine 768 TeNT variant

[0123] This section describes the first identification and characterization of a single amino acid point mutation in the TT translocation domain that blocks light chain (LC) translocation. The identification of the role of K768 in LC translocation provided the first opportunity to inactivate independent activities of TT and, by analogy with BT (see Table 6), catalysis, translocation and receptor binding, for recombinant vaccine development.

[0124] We recently identified the rate-limiting step in translocation of the light chain (LC) encoded in the translocation domain using tetanus toxin. Lysine (K) 768 is located in a loop connecting two long α-helices (helices 12-13 and helices 16-17). Site-directed mutagenesis identified a point mutation, K768A, located within the loop connecting the two long α-helices of the translocation domain, which inhibits translocation ( Fig.12 ). Cell studies showed that M-TT(K768A) did not bind neuronal membranes, supporting a role for the loop in membrane permeabilization. Comparative experiments showed that the K768A mutation did not inhibit TT binding, entry, trafficking, or pore formation in host cells, and did not inhibit cleavage of the light chain (LC) and heavy chain (HC) by trypsin, indicating that the mutation did not perturb the overall M-TT structure and suggesting a direct role for the loop in light chain translocation. Additional experiments showed that K768A is not a component of the pH trigger. This is the first single amino acid required for light chain translocation within the translocation domain, suggesting a role for two long α-helices (helices 12-13 and helices 16-17) in the toxin-membrane interaction. Additional experiments demonstrated that the D767 / E769A mutation also results in a translocation defect in tetanus toxin, allowing D767 / E769 to compensate for K768 and / or additionally inhibit LC translocation in tetanus toxin.

[0125] Engineering independent mutations in botulinum toxin (BT) and TT vaccines (see Table 6) will inactivate each of the three functions of toxin action: catalysis, translocation and receptor binding, thereby enhancing vaccine safety. Multiple independent mutations index to reduce toxin potency, enhance vaccine safety without interfering with protein structure and possible immunogenicity, and reduce the possibility of reversal in large-scale production.

[0126]

[0127] Example 3 - Engineered M-Tetanus Toxin (M-TT) as a Low-Dose Protective Vaccine

[0128] Similar to BT structure, tetanus toxin (TT) is an AB toxin that contains an N-terminal domain (catalytic light chain, LC) and a C-terminal domain (translocation and receptor binding heavy chain, HC). ++ Two amino acid mutations (R372A, Y375F) within the binding pocket resulted in 2M-TT, which inhibits Zn ++ The amino acid sequence of 2M-TT is listed in SEQ ID NO:2 and the nucleotide sequence is listed in SEQ ID NO:3. In the initial experiments to further reduce the toxicity, the addition of H233 based on E234 stabilized the Zn ++ An additional mutation (E234Q) was combined, which directly coordinates Zn ++Next, we engineered two independent mutations (R1226L, W1289A) to inhibit neuronal binding to dual ganglioside receptors, generating 5M-TT (see Table 7). As proof of principle, we engineered 6M-TT with an additional mutation to inhibit LC translocation (K768A) ( Fig.14 ). 6M-TT contains mutations in each of the following tetanus toxin functions: catalysis, translocation, and receptor binding. 6M-TT was purified from E. coli at approximately 6 mg / liter culture batches. Each of four mice injected with 20 μg of single-chain or double-chain 6M-TT did not show any tetanus symptoms. Therefore, 6M-TT is a soluble, well-expressed, non-toxic protein.

[0129]

[0130] 6M-TT will in turn be further engineered to inhibit VAMP-2 binding and cleavage by introducing mutations at positions Y26 and L231 to generate 7M-TT and 8M-TT. L231 was chosen based on previous studies showing that the L231 mutation reduces kcat without affecting VAMP-2 affinity, and that the L231K mutation does not affect the overall structure of the LC. 9 Y26 was selected based on the site within the S7 pocket of HCR / T, demonstrating that the Y26A mutation reduces the affinity of LC / T for VAMP-2 9 (See Table 7.) 8M-TT and intermediates (6M-TT and 7M-TT) were subjected to circular dichroism to determine secondary structure, trypsin sensitivity to determine overall relative stability, and mass spectrometry to determine protein composition. 42 .

[0131] Since we now know that 6M-TT is produced as a soluble protein, is highly expressed, and is nontoxic to mice when injected with 20 μg, the toxicity of 6M-TT, 7M-TT, and 8M-TT was first evaluated in human neuronal cell experiments, analyzing VAMP-2 cleavage after cell entry and analyzing VAMP-2 cleavage in cell lysates. 64 If no cleavage or cytotoxicity was detected, the absence of in vivo toxicity was determined in the mouse model using female ICR mice (18-22 g, 5 mice / group) (Table 8).

[0132]

[0133] Initial experiments will be performed by intraperitoneal injection of 20, 50, 250, or 1000 μg 8M / TT / mouse (1000 μg weight is equivalent to 4×10 7 LD 50 Wild-type tetanus toxin) 65Injected mice will be scored for 3-day survival in the Mouse Bioassay and observed for up to 14 days for any signs of TT pathology, including lack of weight gain, signs of stress, organ damage, and tetanus symptoms. Male mice will be tested to investigate sex differences.

[0134] Outbred female ICR mice (8 per group) were immunized with 0.01-0.1 μg optimized M-TT or an equivalent amount of chemically inactivated tetanus toxoid and boosted on day 14 (Table 9). On day 26, mice were bled and on day 30, 10 3 -10 6 To investigate long-term protection, mice were challenged with tetanus toxin. To investigate long-term protection, vaccinated mice were maintained for 180 days and challenged with TT to test the persistence of the immune response. Mice surviving 3 days were scored as protected. 6 Sera obtained before challenge were tested by ELISA for anti-TT and neutralization capacity against tetanus toxin intoxication of cultured neurons as an inhibitor of VAMP-2 cleavage 71-72 Male mice were tested to ensure no gender differences.

[0135]

[0136] We expect that each introduced independent LC point mutation will cause a fold decrease in catalytic activity and that the decrease will also be reflected in M-TT toxicity, as the LC mutations inhibit independent steps in catalysis. We do not expect these LC or HC mutations to affect protein stability or immunogenicity, as their effects have been previously studied as individual mutations in LC-TT or HC-TT. Injection of 1000 μg of 8M-TT was not toxic in our mouse model, and 8M-TT may have a stronger neutralizing immune response than chemically inactivated TT, thereby enabling low-dose immunization with 8M-TT.

[0137] Recent reviews estimate that current combination vaccines achieve only a fraction of their potential immune efficacy 4 Research on microbial pathogens continues to identify other immunogens that need to be combined with protein toxoids to generate effective T cell-dependent immune responses. These include, but are not limited to, the capsule of meningococci, 15 Fungi, 16 , and pneumococcus 17-18 In addition, synthetic glycans offer a promising future alternative to natural polysaccharide vaccines, which vary considerably in purity and content. 20-22 Tetanus toxoid is an immunogenic carrier protein of polysaccharide 23Based on our understanding of the structural-functional properties of TT and the basic information about tetanus toxin as a chemically inactivated toxoid, and the ongoing worldwide need for tetanus vaccine, tetanus toxin is one of the best candidates for the development of these next generation recombinant conjugate vaccines. The generation of a safe, easily manufactured and protective recombinant TT vaccine has enabled the first analysis of recombinant full-length avirulent TT as a conjugate vaccine vector. There is currently a lack of understanding of the protective properties of conjugate vaccine vectors, including tetanus toxoid. 73 Recombinant non-toxic M-TT can be used as a carrier for several commonly used antigens to ensure an enhanced immune response to the antigen when combined with M-TT relative to chemically inactivated TT.

[0138] The protocol for conjugation of oligosaccharides to M-TT followed the published protocol of Lees. 74 Briefly, polysaccharide (PS) was reduced to a molecular weight of 100-300 kDa using an LV-1 microfluidizer. PS was prepared at 5 mg / ml in water and activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP, 0.5 mg / mg). 74 . An equal weight of protein (5 mg / ml) was added and the solution was maintained at pH 9. The reaction was monitored by size exclusion chromatography (SEC) HPLC and quenched with excess glycine. The conjugates were purified by SEC and the molecular weights were determined by SEC-Multi-Angle light scattering. The identified polysaccharides and peptides were cross-linked with 8M-TT: (i) Group B Streptococcus (GBS) polysaccharides serotypes Ia, Ib, II, III, IV, and IV; (2) poly-β-(1-6)-N-acetyl-glucosamine (PNAG), which is a candidate broad-spectrum vaccine against Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomonas maltophilia, and Burkholderia cepacia complex (BCC) mediated biofilm formation 45 ; (3) Peptides are currently being tested in influenza vaccines.

[0139] Outbred female ICR mice (8 per group) were immunized with 0.01-0.1 μg of conjugated optimized M-TT or an equivalent amount of chemically inactivated tetanus toxoid and boosted on day 14 (Table 10). On day 26, mice were bled and on day 30, 10 3 -10 6 To investigate long-term protection, the vaccinated mice were maintained for 180 days and challenged with TT to test the persistence of the immune response. Mice surviving 3 days were scored as protected. Serum obtained before challenge was tested for anti-conjugate and anti-TT by ELISA as described above. 6 74. Male mice were tested for sex differences.

[0140]

[0141] PNAG oligosaccharides, GBS polysaccharides and influenza peptides will be conjugated to 8M-TT vaccine separately. Subsequent experiments will combine GBS and PNAG to produce multiple polysaccharide conjugates-8M-TT vaccine. Next, influenza peptides and PNAG will be combined to produce multiple peptide-polysaccharide conjugates-8M-TT vaccine. These experiments will test the efficacy of 8M-TT as a vaccine carrier.

[0142] It is expected that each of the conjugate vaccines, PNAG-8M-TT, GBS-8M-TT, and peptide-8M-TT, will elicit an immune response to the conjugate similar to that of the conjugate-chemically inactivated TT vaccine and a stronger immune response to TT. We also expect that the immune response to the polysaccharide and peptide within the 8M-TT vaccine will elicit an immune response similar to that when each antigen is conjugated to 8M-TT. We expect that the immune response to 8M-TT will be associated with protection against native TT challenge.

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[0218] It should be understood that the above description, drawings and their description parts are for illustration rather than for limiting the present invention. Many themes and variations of the present invention are enlightening to those skilled in the art according to the present disclosure. All these themes and variations are within the consideration of this article. For example, when the present invention is described in conjunction with various exemplary embodiments described above, various alternative forms, modifications, variants, improvements and / or basic equivalents, whether known or rare, or currently unforeseen, are clear to at least ordinary capable personnel in the art. Various changes can be made without being limited to the spirit and scope of the present invention. Therefore, the present invention is intended to include all known and later developed alternative forms, modifications, changes, improvements and / or embodiments that are substantially equivalent to the exemplary embodiments. Sequence Listing <110> THE MEDICAL COLLEGE OF WISCONSIN, INC Barbieri, Joseph T <120> Modified Clostridial neurotoxins and conjugate vaccine platforms as vaccines <130> 650053.00580 <150> US 62 / 599,444 <151> 2017-12-15 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 1315 <212> PRT <213> Clostridium tetani <400> 1 Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn 1 5 10 15 Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr 65 70 75 80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95 Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110 Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125 Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe 145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190 Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220 Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His 225 230 235 240 Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp 305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350 Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365 Ile Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys 370 375 380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe 385 390 395 400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435 440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly 450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 465 470 475 480 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485 490 495 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525 Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 545 550 555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile 565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 595 600 605 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser 625 630 635 640 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650 655 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680 685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile 690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 705 710 715 720 Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys 755 760 765 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775 780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile 820 825 830 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845 Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 865 870 875 880 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890 895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 945 950 955 960 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975 Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe 1010 1015 1020 Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn 1025 1030 1035 Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met 1040 1045 1050 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn 1055 1060 1065 Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr 1070 1075 1080 Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro 1085 1090 1095 Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 1100 1105 1110 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr 1115 1120 1125 Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys 1130 1135 1140 Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr 1145 1150 1155 Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu 1160 1165 1170 Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser 1175 1180 1185 Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn 1190 1195 1200 Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe 1205 1210 1215 Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly 1220 1225 1230 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala 1250 1255 1260 Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp 1265 1270 1275 Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His 1280 1285 1290 Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr 1295 1300 1305 Asp Glu Gly Trp Thr Asn Asp 1310 1315 <210> 2 <211> 1315 <212> PRT <213> Clostridium tetani <400> 2 Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn 1 5 10 15 Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr 65 70 75 80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95 Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110 Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125 Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe 145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190 Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220 Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His 225 230 235 240 Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp 305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350 Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365 Ile Lys Thr Ala Leu Ser Phe Phe Ser Met Asn His Asp Pro Val Lys 370 375 380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe 385 390 395 400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435 440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly 450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 465 470 475 480 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485 490 495 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525 Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 545 550 555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile 565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 595 600 605 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser 625 630 635 640 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650 655 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680 685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile 690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 705 710 715 720 Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys 755 760 765 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775 780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile 820 825 830 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845 Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 865 870 875 880 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890 895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 945 950 955 960 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975 Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe 1010 1015 1020 Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn 1025 1030 1035 Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met 1040 1045 1050 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn 1055 1060 1065 Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr 1070 1075 1080 Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro 1085 1090 1095 Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 1100 1105 1110 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr 1115 1120 1125 Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys 1130 1135 1140 Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr 1145 1150 1155 Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu 1160 1165 1170 Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser 1175 1180 1185 Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn 1190 1195 1200 Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe 1205 1210 1215 Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly 1220 1225 1230 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala 1250 1255 1260 Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp 1265 1270 1275 Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His 1280 1285 1290 Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr 1295 1300 1305 Asp Glu Gly Trp Thr Asn Asp 1310 1315 <210> 3 <211> 3945 <212> DNA <213> Clostridium tetani <400> 3 atgccgatta ccattaacaa ctttcgttat agcgatccgg tgaacaacga taccattatt 60 atgatggaac cgccgtattg caaaggcctg gatatttatt ataaagcgtt taaagttacc 120 gatcgtattt ggattgtgcc ggaacgttat gaatttggca ccaaaccgga agatttcaac 180 ccgccgagca gcctgattga aggcgcgagc gaatattatg atccgaacta tctgcgtacc 240 gatagcgata aagatcgttt cctgcagacc atggtgaaac tgtttaaccg tattaagaac 300 aacgtggcgg gcgaagcgct gctggataaa attattaacg cgattccgta tctgggcaac 360 agctatagcc tgctggataa atttgatacc aacagcaaca gcgtgagctt taacctgctg 420 gaacaagatc cgagcggcgc gaccaccaaa agcgcgatgc tgaccaacct gattattttc 480 ggcccgggcc cggtgctgaa caaaaacgaa gtgcgtggca ttgtgctgcg tgtggataac 540 aagaactatt tcccgtgccg tgatggcttt ggcagcatta tgcagatggc gttttgcccg 600 gaatatgtgc cgacctttga taacgtgatt gaaaacatta ccagcctgac cattggcaaa 660 agcaaatatt tccaagatcc ggcgctgctg ctgatgcatg aactgattca tgtgctgcat 720 ggcctgtatg gcatgcaggt gagcagccat gaaattattc cgagcaaaca ggaaatttat 780 atgcagcata cctatccgat tagcgcggaa gaactgttta cctttggcgg ccaggatgcg 840 aacctgatta gcattgatat taagaacgat ctgtatgaaa agaccctgaa cgattataaa 900 gcgattgcga acaaactgag ccaggtgacc agctgcaacg atccgaacat tgatattgat 960 agctataaac agatttatca gcagaaatat cagtttgata aagatagcaa cggccagtat 1020 attgtgaacg aagataaatt tcagattctg tataacagca ttatgtatgg ctttaccgaa 1080 attgaactgg gcaagaaatt taacattaaa accgcgctga gcttttttag catgaaccat 1140 gatccggtga aaattccgaa cctgctggat gataccattt ataacgatac cgaaggcttt 1200 aacattgaaa gcaaagacct gaaaagcgaa tataaaggcc agaacatgcg tgtgaacacc 1260 aacgcgtttc gtaacgtgga tggatccggc ctggtgagca aactgattgg cctgtgcaag 1320 aagattattc cgccgaccaa cattcgtgag aacctgtata accgtaccgc gagcctgacc gatctgggcg gcgaactgtg cattaagatt aagaacgaag atctgacctt tattgcggag aagaacagct ttagcgaga accgtttcag gatgaaattg tgagctaa caccaagaac aaaccgctga actttaacta tagcctggat aaaattattg tggattataa cctgcagagc aagattaccc tgccgaacga tcgtaccacc ccggtgacca aaggcattcc gtatgcgccg 1620 gcaacgcggc gagcaccatt gaattcata acattgatga taacaccatt tatcagtatc tgtatgcgca gaagagcccg accaccctgc agcgtattac catgaccaac agcgtggatg atgcgctgat taacagcacc aaaatttata gctattttcc gagcgtgatt agcaaagtga accagggcgc gcagggcatt ctgtttctgc agtgggtgcg tgatattatt gatgatttta ccaacgaaag cagccagaa accaccattg ataaaattag cgatgtgagc accattgtgc cgtatattgg cccggcgctg aacattgtga aacagggcta tgaaggcaac tttattggcg cgctggaaac caccggcgtg gtgctgctgc tggaatatat tccggaaatt accctgccgg tgattgcggc gctgagcatt gcggaaagca gcacccagaa agagaagatt 2100 attaaaacca ttgataactt tctggagaaa cgttatgaga aatggattga agtgtataaa 2160 ctggtgaaag cgaaatggct gggcaccgtg aacacccagt ttcagaaacg tagctatcag 2220 atgtatcgta gcctggaata tcaggtggat gcgattaaga aaattattga ttatgaatat 2280 aagatttata gcggcccgga taaagaacag attgcggatg aaattaacaa cctgaaaaac 2340 aaactggaag agaaagcgaa caaagcgatg attaacatta acatctttat gcgtgaaagc 2400 agccgtagct ttctggtgaa ccagatgatt aacgaagcga agaaacagct gctgggaattt 2460 gatacccaga gcaagaacat tctgatgcag tatattaaag cgaacagcaa atttattggc 2520 attaccgaac tgaagaaact ggaaagcaaa attaacaaag tgtttagcac cccgattccg 2580 tttagctata gcaagaacct ggattgctgg gtggataacg aagaagatat tgatgtgatt 2640 ctgaagaaga gcaccattct gaacctggat attaacaacg atattattag cgatattagc 2700 ggcttcaaca gcagcgtgat tacctatccg gatgcgcagc tggtaccggg cattaacggc 2760 aaagcgattc atctggtgaa caacgaaagc agcgaagtga ttgtgcataa agcgatggat 2820 attgaatata acgatatgtt caacaacttt accgtgagct tttggctgcg tgtgccgaaa 2880 gtgagcgcga gccatctgga acagtatggc accaacgaat atagcattat tagcagcatg 2940 aagaaacata gcctgagcat tggcagcggc tggagcgtga gcctgaaagg caacaacctg 3000 atttggaccc tgaaagatag cgcgggcgaa gtgcgtcaga ttaccttcg tgatctgccg 3060 gataagttta acgcgtatct ggcgaacaaa tgggtgttta ttaccattac caacgatcgt 3120 ctgagcagcg cgaacctgta tattaacggc gtgctgatgg gcagcgcgga aattaccggc 3180 ctgggcgcga ttcgtgaaga taacaacatt accctgaaac tggatcgttg caacaataac 3240 aaccagtatg tgagcattga taaatttcgt atttttgca aagcgctgaa cccgaaagaa 3300 attgaaaaac tgtataccag ctatctgagc attacctttc tgcgtgattt ttggggcaac 3360 ccgctgcgtt atgataccga atattatctg attccggtgg cgagcagtag caaagatgtg 3420 cagctgaaga acattaccga ttatatgtat ctgaccaacg cgccgagcta taccaacggc 3480 aaactgaaca tttactatcg tcgtctgtat aacggcctga aattcattat taaacgttat 3540 accccgaata acgaaattga tagctttgtg aaaagcggcg attttattaa actgtatgtg 3600 agctataaca ataacgaaca tattgtgggc tatccgaaag atggcaacgc gtttaataac 3660 ctggatcgta ttctgcgtgt gggctataac gcgccgggca ttccgctgta taagaagatg 3720 gaagcggtga aactgcgtga tctgaaaacc tatagcgtgc agctgaaact gtatgatgat 3780 aagaacgcga gcctgggcct ggttggaacc cataacggtc agattggcaa cgatccaaac 3840 cgtgatattc tgattgcgag caactggtat tttaaccatc tgaaagacaa gatcctgggc 3900 tgtgattggt acttcgttcc gacagatgaa ggctggacca acgat 3945 <210> 4 <211> 1315 <212> PRT <213> Clostridium tetani <400> 4 Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn 1 5 10 15 Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr 65 70 75 80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95 Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110 Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125 Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe 145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190 Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220 Gln Asp Pro Ala Leu Leu Leu Met His Gln Leu Ile His Val Leu His 225 230 235 240 Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp 305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350 Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365 Ile Lys Thr Ala Leu Ser Phe Phe Ser Met Asn His Asp Pro Val Lys 370 375 380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe 385 390 395 400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435 440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly 450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 465 470 475 480 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485 490 495 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525 Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 545 550 555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile 565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 595 600 605 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser 625 630 635 640 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650 655 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680 685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile 690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 705 710 715 720 Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys 755 760 765 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775 780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile 820 825 830 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845 Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 865 870 875 880 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890 895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 945 950 955 960 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975 Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe 1010 1015 1020 Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn 1025 1030 1035 Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met 1040 1045 1050 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn 1055 1060 1065 Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr 1070 1075 1080 Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro 1085 1090 1095 Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 1100 1105 1110 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr 1115 1120 1125 Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys 1130 1135 1140 Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr 1145 1150 1155 Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu 1160 1165 1170 Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser 1175 1180 1185 Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn 1190 1195 1200 Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe 1205 1210 1215 Asn Asn Leu Asp Arg Ile Leu Leu Val Gly Tyr Asn Ala Pro Gly 1220 1225 1230 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala 1250 1255 1260 Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp 1265 1270 1275 Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Ala Tyr Phe Asn His 1280 1285 1290 Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr 1295 1300 1305 Asp Glu Gly Trp Thr Asn Asp 1310 1315 <210> 5 <211> 1315 <212> PRT <213> Clostridium tetani <400> 5 Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn 1 5 10 15 Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr 65 70 75 80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95 Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110 Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125 Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe 145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190 Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220 Gln Asp Pro Ala Leu Leu Leu Met His Gln Leu Ile His Val Leu His 225 230 235 240 Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp 305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350 Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365 Ile Lys Thr Ala Leu Ser Phe Phe Ser Met Asn His Asp Pro Val Lys 370 375 380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe 385 390 395 400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435 440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly 450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 465 470 475 480 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485 490 495 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525 Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 545 550 555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile 565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 595 600 605 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser 625 630 635 640 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650 655 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680 685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile 690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 705 710 715 720 Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Ala 755 760 765 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775 780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile 820 825 830 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845 Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 865 870 875 880 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890 895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 945 950 955 960 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975 Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe 1010 1015 1020 Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn 1025 1030 1035 Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met 1040 1045 1050 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn 1055 1060 1065 Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr 1070 1075 1080 Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro 1085 1090 1095 Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 1100 1105 1110 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr 1115 1120 1125 Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys 1130 1135 1140 Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr 1145 1150 1155 Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu 1160 1165 1170 Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser 1175 1180 1185 Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn 1190 1195 1200 Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe 1205 1210 1215 Asn Asn Leu Asp Arg Ile Leu Leu Val Gly Tyr Asn Ala Pro Gly 1220 1225 1230 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala 1250 1255 1260 Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp 1265 1270 1275 Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Ala Tyr Phe Asn His 1280 1285 1290 Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr 1295 1300 1305 Asp Glu Gly Trp Thr Asn Asp 1310 1315 <210> 6 <211> 1315 <212> PRT <213> Clostridium tetani <400> 6 Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn 1 5 10 15 Asp Thr Ile Ile Met Met Glu Pro Pro Ala Cys Lys Gly Leu Asp Ile 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr 65 70 75 80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95 Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110 Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125 Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe 145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190 Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220 Gln Asp Pro Ala Leu Leu Leu Met His Gln Leu Ile His Val Leu His 225 230 235 240 Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp 305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350 Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365 Ile Lys Thr Ala Leu Ser Phe Phe Ser Met Asn His Asp Pro Val Lys 370 375 380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe 385 390 395 400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435 440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly 450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 465 470 475 480 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485 490 495 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525 Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 545 550 555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile 565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 595 600 605 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser 625 630 635 640 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650 655 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680 685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile 690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 705 710 715 720 Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Ala 755 760 765 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775 780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile 820 825 830 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845 Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 865 870 875 880 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890 895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 945 950 955 960 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975 Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe 1010 1015 1020 Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn 1025 1030 1035 Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met 1040 1045 1050 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn 1055 1060 1065 Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr 1070 1075 1080 Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro 1085 1090 1095 Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 1100 1105 1110 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr 1115 1120 1125 Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys 1130 1135 1140 Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr 1145 1150 1155 Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu 1160 1165 1170 Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser 1175 1180 1185 Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn 1190 1195 1200 Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe 1205 1210 1215 Asn Asn Leu Asp Arg Ile Leu Leu Val Gly Tyr Asn Ala Pro Gly 1220 1225 1230 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala 1250 1255 1260 Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp 1265 1270 1275 Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Ala Tyr Phe Asn His 1280 1285 1290 Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr 1295 1300 1305 Asp Glu Gly Trp Thr Asn Asp 1310 1315 <210> 7 <211> 1315 <212> PRT <213> Clostridium tetani <400> 7 Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn 1 5 10 15 Asp Thr Ile Ile Met Met Glu Pro Pro Ala Cys Lys Gly Leu Asp Ile 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr 65 70 75 80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95 Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110 Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125 Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe 145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190 Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220 Gln Asp Pro Ala Leu Leu Lys Met His Gln Leu Ile His Val Leu His 225 230 235 240 Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp 305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350 Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365 Ile Lys Thr Ala Leu Ser Phe Phe Ser Met Asn His Asp Pro Val Lys 370 375 380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe 385 390 395 400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435 440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly 450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 465 470 475 480 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485 490 495 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525 Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 545 550 555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile 565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 595 600 605 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser 625 630 635 640 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650 655 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680 685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile 690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 705 710 715 720 Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Ala 755 760 765 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775 780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile 820 825 830 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845 Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 865 870 875 880 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890 895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys 945 950 955 960 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975 Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe 1010 1015 1020 Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn 1025 1030 1035 Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met 1040 1045 1050 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn 1055 1060 1065 Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr 1070 1075 1080 Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro 1085 1090 1095 Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 1100 1105 1110 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr 1115 1120 1125 Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys 1130 1135 1140 Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr 1145 1150 1155 Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu 1160 1165 1170 Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser 1175 1180 1185 Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn 1190 1195 1200 Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe 1205 1210 1215 Asn Asn Leu Asp Arg Ile Leu Leu Val Gly Tyr Asn Ala Pro Gly 1220 1225 1230 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala 1250 1255 1260 Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp 1265 1270 1275 Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Ala Tyr Phe Asn His 1280 1285 1290 Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr 1295 1300 1305 Asp Glu Gly Trp Thr Asn Asp 1310 1315

Claims

1. A modified tetanus polypeptide, which is obtained by performing the following mutations on the polypeptide shown in SEQ ID NO:1: R372A, Y375F, E234Q, R1226L, and W1289A, and also includes at least one mutation selected from D767A, K768A and E769A, and the polypeptide has reduced toxicity and receptor binding compared with the toxicity and receptor binding of SEQ ID NO:

1.

2. The modified polypeptide of claim 1, wherein the mutations are R372A, Y375F, E234Q, R1226L, K768A, and W1289A.

3. The modified polypeptide of claim 1, wherein the mutation is R372A, Y375F, E234Q, R1226L, W1289A, K768A, and D767A or E769A.

4. The modified polypeptide of claim 1, wherein the mutations are R372A, Y375F, E234Q, D767A, E769A, K768A, R1226L, and W1289A.

5. The modified polypeptide as claimed in claim 1, wherein the modified polypeptide is as shown in SEQ ID NO:

5.

6. The modified polypeptide of claim 1, further comprising a mutation at Y26A, wherein the position is numbered according to SEQ ID NO:

1.

7. The modified polypeptide as claimed in claim 6, wherein the modified polypeptide is as shown in SEQ ID NO:

6.

8. The modified polypeptide according to any one of claims 1 to 7, wherein the modified polypeptide is covalently linked to a carbohydrate or a peptide.

9. A composition comprising the modified polypeptide according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. Use of the modified polypeptide according to any one of claims 1 to 8 for the preparation of a medicament for reducing the risk of tetanus in a subject.

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