Modified Clostridial Neurotoxins as Vaccines and Conjugate Vaccine Platforms

Abstract

To provide means for inactivating intrinsic toxicity of tetanus toxin and producing safe and effective vaccines.SOLUTION: Provided herein are engineered non-catalytic, non-toxic tetanus toxin variants and methods of using such engineered tetanus toxin variants as low dose, protective vaccines that are non-toxic and more potent than their respective chemically inactivated toxoids. In addition, provided herein are conjugate-vaccine carriers comprising engineered tetanus toxin variants and methods of using such conjugate-vaccines to elicit T-cell dependent immune memory responses which can target a broad spectrum of microbial pathogens as a single vaccine.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 599,444, filed December 15, 2017, the entire contents of which are incorporated herein by reference as if fully set forth herein.

[0002] Statement of Federally Funded Research This invention was made with government support under grant numbers R01 AI030162 and AI118389 awarded by the NIH-NIAID. The government has certain rights in this invention. [Background technology]

[0003] Botulinum neurotoxin (BoNT), the most toxic substance to humans, is a protein toxin produced by selected strains of Clostridium botulinum, 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 a disulfide bond. The HC further contains an N-terminal domain (H) that aids in the translocation of the LC into the cytosol. N ) and a C-terminal domain (H) that recognizes and binds to cell surface receptors on neurons. C) (Montal, 2010). Once inside the cell, LC specifically cleaves portions of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby inactivating neurotransmitter release (Montecucco and Schiavo, 1993, Trends in biochemical sciences 18, 324-327; Schiavo et al., 1995). Experimental vaccines have previously been used to protect "at-risk" populations from botulism, but the use of chemically inactivated BoNT toxoid vaccines has been discontinued due to declining efficacy. Furthermore, conventional tetanus toxin fragment vaccines are not ideal due to issues associated with low antigenicity and immunopotency. Thus, there remains a need in the art for non-catalytic, non-toxic variants of tetanus and botulinum toxins for use as adjuvants and conjugate vaccines. Summary of the Invention

[0004] Disclosure Overview Provided herein are recombinant, non-catalytic, non-toxic mutant forms of tetanus toxin and uses of such mutant toxins. The data described herein demonstrate a significant reduction in toxicity compared to native tetanus toxin and compared to previously described tetanus mutants. The inventors envision several independent, engineered mutations, including, but not limited to, eliminating catalytic activity by eliminating substrate affinity or reducing reaction rate, eliminating receptor binding, inhibiting translocation, or interfering with intradomain cleavage or disulfide bond disruption of the toxin, among other steps of toxin intoxication, that can be combined to inactivate the inherent toxicity of tetanus toxin and produce a safe and effective vaccine. Herein, we describe experiments in which the inventors engineered toxins with mutations that reduce host receptor binding in addition to reduced catalytic activity. These data demonstrate the potential of recombinant toxins containing selected independent mutations that render them non-toxic and suitable for use in vaccines and conjugate vaccines without the need for chemical cross-linking to reduce toxicity.

[0005] In a first aspect, there is provided herein a polypeptide having at least 95% identity to SEQ ID NO: 1 and having mutations at positions R372 and Y375, and E234 , K768, R1226 and W1289, each position numbered relative to SEQ ID NO: 1, wherein 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 substituted with the amino acid A, and the amino acid Y at position Y375 can be substituted with the amino acid F. Mutations include R372A, Y375F, E234 The modified polypeptide may include the residues Q, R1226L, and W1289A. The modified polypeptide may further include a covalently attached carbohydrate moiety, thereby making the polypeptide a polypeptide-carbohydrate conjugate. The modified polypeptide may be encoded by SEQ ID NO:2.

[0006] In some cases, the mutations are R372A, Y375F, E234 The modified polypeptide may include the following mutations: Q, K768A, R1226L, and W1289A. The modified polypeptide may be encoded by SEQ ID NO:5. In some cases, the modified polypeptide may further include mutations at one or both of positions L231 and Y26, each position being numbered relative 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.

[0007] In another aspect, provided herein are compositions comprising a modified polypeptide as described herein and a pharmaceutically acceptable carrier.

[0008] In a further aspect, provided herein are methods of reducing a subject's risk of developing tetanus by inducing an immune response by administering to the subject a therapeutically effective amount of a modified polypeptide as described herein. In some cases, the modified polypeptide is used as an adjuvant. In some cases, the modified polypeptide is used as a vaccine.

[0009] These and other aspects and advantages of the present invention will become apparent from the following description. In the description, reference is made to the accompanying drawings, which form a part hereof, and in which there is shown by way of example preferred embodiments of the invention. Such embodiments do not necessarily represent the full scope of the invention, and therefore, reference is made to the claims and this specification for interpreting the scope of the invention. [Brief explanation of the drawings]

[0010] [Figure 1]Figure 1 shows the recombinant proteins used to evaluate the host immune response to vaccination. (Top panel) Schematic diagrams of the BoNT derivatives used in this study are shown. Where indicated, two epitopes (His6 and Strep) were used for protein purification. 3XFLAG (3XF) and two consecutive hemagglutinin (2HA) epitopes were included for cellular studies. Domain junctions were defined using the crystal structure of BoNT / A1 (PDB: 3BTA). The single-letter amino acid designations above each diagram indicate the introduction of amino acid substitutions introduced to reduce catalytic activity (LC) or receptor binding (HCC). Note that single-chain BoNT and LCHCN were used for vaccination. (Bottom panel) 4 μg of the indicated proteins were subjected to SDS-PAGE and Coomassie blue staining. Lanes: 1, M-BoNT / A1; 2, M-BoNT / A1 trypsin nicked and reduced; 3, M-LCHCN / A1; 4, M-LCHCN / A1 trypsin nicked and reduced; 5, LC / A1RY; 6, HCC / A1W; and 7, TeNTRY. The migration of molecular weight marker proteins (kDa) is indicated in the left lanes. Note that in lane 2, the nicked HC migrates at approximately 80 kDa, which has been shown in other experiments to be due to cleavage of the belt region of the HC by trypsin. [Figure 2]Figure 2 shows ELISA results for sera from mice inoculated with M-BoNT / A1 or M-BoNT / A1W and challenged with heterosubtypic native BoNT / A2. Mice were inoculated with M-BoNT / A1 (upper panel) or M-BoNT / A1W (lower panel), and serum was collected before challenge with 10 LD50 of native BoNT / A2. Mice that survived challenge (A) or did not survive challenge (D) are shown. Antibody titers were measured by ELISA in sera from M-BoNT / A1; M-LCHCN / A1; LC / A1RY; HCC / A1W; TeNTRY; and a no-protein control (Con) (1:20,000 dilution). Bound mouse antibodies were detected with goat α-mouse IgG-HRP (1:20,000 dilution) using TMB reagent. The reaction was stopped with diluted H2SO4 and read at 450 nm. Data are shown as the mean of five (A) and four (D) mice after M-BoNT / A1 inoculation and three (A) and six (D) mice after M-BoNT / A1W inoculation from two independent experiments performed in duplicate with standard deviations indicated. Statistical analysis was performed as described herein; P = .05 = *. [Figure 3]Figure 3 shows ELISA results for sera from mice inoculated with BoNT derivatives and challenged with native BoNT / A1. Mice were inoculated with M-BoNT / A1W (0.3 μg), M-LCHCN / A1 (0.2 μg), M-LCHCN / A1 (0.2 μg) + HCC / A1W (0.1 μg), or HCC / A1W (0.3 μg). Serum was obtained before BoNT challenge. ELISA determined antibody titers for sera (1:30,000 dilution) from M-BoNT / A1; M-LCHCN / A1; LC / A1RY; HCC / A1W; TeNTRY; and a no-protein control (Con). Bound mouse antibodies were detected with goat α-mouse IgG-HRP (1:20,000 dilution) using TMB reagent. The reaction was stopped with dilute H2SO4 and read at 450 nm. Except for HCC / A1W-inoculated mice, where data were from 7 survivors (A) or 3 non-survivors (D) of natural BoNT / A1 challenge, data are presented as the mean of 10 independent sera from mice surviving BoNT / A1 challenge in Experiment 3 in Table 1, analyzed in two independent experiments performed in duplicate, with the standard deviations shown. Variation in titer range was due to variation in antibody titers between individual mice, not due to ELISA replicates. Statistical analysis was performed as described in the Methods section: P < 0.05 = *, 0.01 = **, 0.001 = ***, and 0.0001 = ****. [Figure 4] Figure 4 shows ELISA results for sera from individual mice inoculated with M-BoNT derivatives and from individual mice that survived challenge with native BoNT / A1. Sera obtained before BoNT challenge from individual mice inoculated with BoNT / A1W (#7 and #3), HCC / A1W (#78), and LCHCN / A1 (#21, #24, and #25), which survived challenge with native BoNT / A1, were analyzed by ELISA using M-BoNT / A1; M-LCHCN / A1; LC / A1RY; and HCC / A1W as antigens (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 diluted H2SO4 and read at 450 nm. Data shown are the mean of two independent experiments, each performed in duplicate, with the standard deviation indicated. [Figure 5] Figure 5 shows serum neutralization of native BoNT / A1 cleavage of SNAP25 in human induced pluripotent stem cells (hiPSCs). Sera obtained before BoNT challenge from individual mice inoculated with M-BoNT / A1W (#7 and #3), HCC / A1W (#78), and M-LCHCN / A1 (#21, #24, and #25), which survived BoNT / A1 challenge, were analyzed for their ability to neutralize native BoNT / A1. Human induced pluripotent stem cell (hiPSC)-derived neurons were seeded at a density of 35,000–40,000 cells per well on poly-L-ornithine and Matrigel-coated plates and maintained in iCell Neurons culture medium for 7 days prior to the neutralization assay. To detect neutralizing antibodies in mouse serum, 2 pM native BoNT / A1 was combined with serial dilutions of filter-cleaned serum in culture medium and incubated for 1 h at 37°C. "No antibody" buffer was used as a control. 50 μl of each antibody-toxin mixture was added in at least duplicate per well of hiPSC-derived neurons, and the cells were incubated at 37°C and 5% CO for 24 hours. The toxin / antibody was aspirated from the cells, and cell lysates were subjected to PAGE and then analyzed by Western blot for SNAP-25 cleavage (Pellett et al., 2007; Pellett et al., 2010). Cleaved versus uncleaved SNAP-25 was quantified by densitometry, and percent protection was determined by comparison with the "no antibody" control. IC50 values ​​were estimated using GraphPad Prism 6 software and nonlinear regression, slope of variation, four parameters. [Figure 6] Figure 6 shows a representative ELISA of sera from M-BoNT / A1W-vaccinated mice that survived challenge with BoNT / A1. Sera from BoNT / A1W-vaccinated mice that survived challenge with BoNT / A1 were analyzed by ELISA using the indicated antigens as described in the Methods section. Values ​​are the means of representative measurements performed in duplicate, with the standard deviations shown. [Figure 7]Figure 7 shows that M-BoNT / A1W is more immunogenic than M-LCHCN / A1 or LCHCN / A1 + HCC / A1W. Sera from individual mice inoculated with M-BoNT / A1 (0.3 μg), M-LCHCN / A1W (0.2 μg), or M-LCHCN / A1 (0.2 μg) + HCC / A1W (0.1 μg) that survived challenge with 10 LD BoNT / A1 were analyzed by ELISA for antibodies to M-BoNT / A1, M-LCHCN / A1, LC / A1RY, HCC / A1W, TeNTRY, or no protein (Con). Data are presented as the mean of 10 independent sera from mice surviving challenge with BoNT / A1 in Experiment 3 of Table 1, analyzed in two independent experiments performed in duplicate, with standard deviations shown. Statistical analysis was performed as described herein: P<0.05=*, 0.01=**, 0.001=***, and 0.0001=****. [Figure 8] FIG. 8 is the amino acid sequence encoding wild-type Clostridium tetani toxin (SEQ ID NO: 1). [Figure 9] FIG. 9 is the amino acid sequence encoding 2M-TT (SEQ ID NO: 2). [Figure 10] FIG. 10 is the amino acid sequence encoding 5M-TeNT (SEQ ID NO:4). [Figure 11] FIG. 11 is the amino acid sequence encoding 6M-TeNT (SEQ ID NO:5). [Figure 12] FIG. 12 is the amino acid sequence encoding 7M-TeNT (SEQ ID NO: 6). [Figure 13] FIG. 13 is the amino acid sequence encoding 8M-TeNT (SEQ ID NO:7). [Figure 14]Figure 14 shows that K768 mediates light chain translocation in tetanus toxin. (Left panel) TT767DKE (WT), TT767AAA, TT767RKK, or TT767DAE were incubated with neurons and assayed for translocation as reporter (β-lactamase) CCF2 cleavage. (Right panel) Alignment of TT and BT in 767DKE. Note the conserved lysine (K) among the toxins. [Figure 15] Figure 15 is the crystal structure of TeNTRY PDB:5n0b. Four TT functions were inactivated: light chain E234Q, R372A, Y375F (Zn++ binding), L231K (VAMP-2 cleavage) and Y26A (VAMP-2 binding), K768A (LC translocation), and R1226L and W1289A (receptor binding). DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description of Disclosure While the invention has been described in terms of one or more preferred embodiments, it is to be understood that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and fall within the scope of the invention.

[0012] The methods and compositions described herein are based, at least in part, on the inventors' development of genetically engineered toxins that are non-catalytic and incapable of neuronal binding. As described in the following paragraphs and examples, tetanus toxin and botulinum toxin (BoNT) with engineered defects that prevent catalysis and receptor binding provide a platform for the development of vaccines against toxin-mediated diseases. For example, modified tetanus toxin and BoNT that have been rendered non-catalytic and incapable of receptor binding by engineered mutations have not demonstrated detectable toxicity. Furthermore, such modified toxins have been suitable as vaccines to protect against botulinum neurotoxic challenge.

[0013] composition Preferably, the genetically modified toxins of the present disclosure contain genetic modifications at multiple targets relative to the wild-type toxin, such modifications eliminating residual attributes and enhancing safety, as described in more detail below. While BoNT and tetanus toxin act on different substrates and receptors, the inventors have demonstrated that immune cells can bind to a non-catalytic, non-receptor-binding form of BoNT (referred to herein as "M-BoNT"). W ") and showed that it elicited a neutralizing immune response similar to that seen with noncatalytic BoNT ("M-BoNT"). Based on this observation, additional independent mutation sites were identified for engineering other noncatalytic, nonreceptor-binding toxin variants. When modified as described herein, the resulting engineered noncatalytic, nonreceptor-binding toxins are suitable for use as vaccine and conjugate vaccine platforms.

[0014] Without being bound by any particular theory or mode of action, engineered mutations at separate sites in tetanus toxin and botulinum toxin render the toxin proteins incapable of expressing toxicity through independent mechanisms, thus providing a fail-safe against inadvertent genetic reversion to toxicity. Such properties are advantageous for the use of mutant toxins as vaccines against tetanus and botulism, and as platforms for conjugate vaccines.

[0015] Thus, provided herein are recombinantly inactivated bacterial toxins that are irreversibly non-toxic and more effective and easier to produce and manipulate than current chemically inactivated toxoids, thus providing improved vaccines and conjugate vaccine carriers. In a first aspect, provided herein are isolated preparations of recombinant, non-catalytic, non-toxic modified forms of bacterial protein toxins (e.g., tetanus toxin and botulinum neurotoxin), where the modified toxins are full-length toxins containing at least four amino acid substitutions that render the proteins incapable of expressing toxicity through an independent mechanism. A "preparation" refers to a toxin polypeptide at any concentration, enriched or purified relative to its natural occurrence. Preferably, the preparation is substantially pure or is combined with other components to form a pharmaceutical formulation. In some cases, the preparations of the invention may include one or more adjuvants or carriers that may be coupled to the toxin polypeptide sequence to help stimulate the immune system. In other cases, the preparation itself possesses adjuvant activity and is effective for enhancing the immune response to the conjugated or co-administered antigen.

[0016] As used herein, "toxin" refers to a poisonous or toxic substance (e.g., a cytotoxin) that is formed or synthesized during the metabolism and growth of a particular microorganism as an integral part of the cell or tissue (endotoxin), as an intracellular or extracellular product (exotoxin), or a combination thereof. As used herein, the term "modified toxin" refers to a non-catalytic, non-toxic mutant form of a toxin, where the toxin is rendered non-catalytic and non-toxic by genetically engineered (e.g., non-natural, artificial) modifications to the amino acid sequence of the polypeptide toxin. In exemplary embodiments, the modified toxins are genetically engineered or otherwise modified variants of toxins produced by Clostridium bacteria (e.g., C. difficile, C. novyi, C. sordellii, C. perfringens, C. tetani, and C. botulinum). These toxins can be recombinant, synthetic, part of a fusion protein (e.g., containing an antigen or a polypeptide (e.g., His6) that facilitates purification of the fusion protein) covalently linked to an antigen and / or chemically crosslinked to the antigen. In some cases, non-catalytic, non-toxic forms of toxins are called toxoids. Toxoids lack toxicity but retain their antigenicity and their immunopotency.

[0017] As used herein, the term "reduced toxicity" means that, compared to a first composition containing a particular active protein ingredient (e.g., wild-type TT), a second composition containing a modified form of a particular active protein ingredient can be administered to a mammal at a dose level equal to or greater than the lethal dose level of the first composition without causing death to the mammal. Reduced toxicity includes partial or complete elimination of toxicity as detectable by methods known to those skilled in the art. Additionally, reduced toxicity includes reduced systemic toxicity (i.e., when administered intravenously) or reduced toxicity upon intramuscular administration.

[0018] In certain embodiments, the preparation comprises a modified tetanus toxin. Generally, a tetanus toxin modified as described herein exhibits one or more altered properties when compared to the wild-type tetanus toxin polypeptide set forth in SEQ ID NO: 1, such as, for example, a significant reduction in catalytic activity and receptor binding activity. In some embodiments, a modified tetanus toxin described herein is at least 1,000,000-fold less toxic than wild-type tetanus toxin. [Table 1]

[0019] In certain embodiments, the preparation has mutations at amino acid residues 372 and 375 and further comprises 234 , 1226, and 1289, where these residue positions are numbered relative to the full-length wild-type tetanus neurotoxin (Clostridium tetani CN3911; GenBank Accession No. X06214) shown as SEQ ID NO: 1. In certain embodiments, the amino acid mutations at residues 372 and 375 are R372A and Y375F, and the modified toxin comprises E234 In some cases, the modified toxin further comprises at least one mutation selected from the group consisting of Q, R1226L, and W1289A. In some cases, the modified toxin further comprises at least one mutation selected from the group consisting of the five mutations numbered relative to SEQ ID NO: 1 (R372A, Y375F, E234 Q, R1226L, and W1289A), and is referred to herein as "5M-TeNT" or "5M-TT." See Table 1. In some cases, 5M-TeNT is encoded by the amino acid sequence set forth as SEQ ID NO:4.

[0020] In some embodiments, the tetanus toxin has an altered translocation domain. For example, the lysine (K) residue at position 768 is located within the loop connecting two long alpha helices. Mutation of this single amino acid to alanine (A) inactivates or blocks light chain translocation. In some cases, the K768A mutation is added to a 5M-TT modified toxin to create a 6M-TT, whereby the resulting modified tetanus toxin contains independent mutations at six sites (see Tables 2 and 3). In some cases, the modified toxin contains six mutations numbered relative to SEQ ID NO: 1 (R372A, Y375F, E234 6M-TeNT includes the following mutations: Q, K768A, R1226L, and W1289A, and is referred to herein as "6M-TeNT" or "6M-TT." In some cases, 6M-TeNT is encoded by the amino acid sequence set forth as SEQ ID NO: 5. Without being bound to a particular mechanism or theory, 6M-TT is likely to have a higher vaccine efficacy than 5M-TT, but should have a lower reversion rate than 5M-TT. The addition of one or more of the mutations D767, K768, or E769A to 5M-TT results in more complete inactivation of the genetically engineered vaccine by inactivating the function of the translocation domain in addition to disrupting the function of the catalytic and receptor-binding domains. [Table 2]

[0021] In some embodiments, tetanus toxin was modified to inhibit VAMP-2 cleavage. For example, mutation of the leucine residue at position 231 (e.g., mutation of leucine to lysine (K)) inactivates the toxin's catalytic activity for VAMP-2 cleavage. In some cases, a L231K mutation was added to a 6M-TT modified toxin to create 7M-TT, whereby the resulting modified tetanus toxin contains independent mutations at seven sites (Table 3). In some cases, the modified toxin contains seven independent mutations numbered relative to SEQ ID NO: 1 (R372A, Y375F, E234Q, R1226L, W1289A, K768A, and L231K), and is referred to herein as "7M-TeNT" or "7M-TT." In some cases, 7M-TeNT is encoded by the amino acid sequence set forth as SEQ ID NO:6.

[0022] In some embodiments, tetanus toxin has been modified to inhibit VAMP-2 binding. For example, mutation of the tyrosine (Y) residue at position 26 (e.g., mutation of tyrosine to alanine (A)) inactivates the toxin's ability to bind VAMP-2. In some cases, a Y26A mutation is added to a 7M-TT modified toxin to create 8M-TT, whereby the resulting modified tetanus toxin contains independent mutations at eight sites (Table 3). In some cases, the modified toxin contains eight independent mutations numbered relative to SEQ ID NO: 1 (R372A, Y375F, E234 Q, R1226L, W1289A, K768A, L231K, and Y26A), and is referred to herein as "8M-TeNT" or "8M-TT." In some cases, 8M-TeNT is encoded by the amino acid sequence set forth as SEQ ID NO:7. [Table 3]

[0023] In some cases, modified tetanus toxins have been used, 372, 275, 234 , 768, 1226, 1289, 231, and / or 26. For example, amino acids that can be substituted for the listed amino acids include substitutions that result in the opposite charge or hydrophobicity of the original residue, conservative amino acid substitutions, and substitutions that result in the deletion of the original residue.

[0024] As is well known to those skilled in the art, alterations to the primary structure of a polypeptide by conservative amino acid substitutions do not significantly alter the activity of the polypeptide, even when the substitutions are in regions critical for determining the conformation of the polypeptide, because the side chain of the amino acid introduced into the sequence can form similar bonds and contacts with the side chain of the substituted amino acid.

[0025] Conservative amino acid substitutions are recognized in the art as substituting one amino acid for another with similar characteristics. Conservative amino acid substitutions can be achieved by modifying a nucleotide sequence to introduce a nucleotide change that encodes the conservative substitution. For example, each amino acid can be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic, and hydrophilic. Conservative substitutions include substitutions between amino acids within each group. Acidic amino acids include aspartic acid and glutamic acid. 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 aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, and tyrosine. Hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tryptophan. Amino acids can also be described in terms of relative size, with alanine, cysteine, aspartic acid, glycine, asparagine, proline, threonine, serine, and valine being considered small.

[0026] In some cases, non-conservative substitutions are also possible as long as they do not disrupt the three-dimensional structure of the epitope within the polypeptide, e.g., do not interfere with the immunogenicity (e.g., antigenicity) of the polypeptide, and do not restore toxicity.

[0027] In a particular embodiment, the modified tetanus toxin comprises mutations at amino acid residues 372 and 375, and further comprises 234, 1226, and 1289, wherein the modified toxin is conjugated or coupled to another peptide as described below for appropriate therapeutic methods. Advantageously, the modified tetanus toxin of the present disclosure does not need to be detoxified with formalin for use as a vaccine or adjuvant. In some cases, a small amount of formalin (about 0.04%) or another fixative or stabilizing reagent (e.g., formalin, glutaraldehyde, β-propiolactone, etc.) is applied to the modified tetanus toxin as a stabilizing agent, although such amount is less (e.g., an order of magnitude less) than the amount (about 0.4%) typically used to detoxify wild-type tetanus toxin (or tetanus toxin that has not been modified as described herein) to form a "tetanus toxoid."

[0028] In certain embodiments, the modified toxins described herein further comprise molecules such as glycosylation, proteins or peptides (e.g., antigens), and chemical moieties. In particular, provided herein are recombinant non-catalytic, non-toxic mutant toxin forms (e.g., modified tetanus toxin and modified botulinum neurotoxin) that have been further modified to include conjugated or chemically linked (e.g., cross-linked) glycosylation. In this manner, modified toxins conjugated to glycosylation provide a platform for use as T-cell-dependent immunogens. In some cases, other molecules or moieties (e.g., antigens) can be further linked to the cross-linking moiety as "cargo."

[0029] In some cases, the modified glycoconjugated tetanus toxin contains five mutations numbered relative to SEQ ID NO: 1 (R372A, Y375F, E234 In other cases, modified carbohydrate-conjugated BoNT toxins contain four mutations numbered relative to UniProtKB / Swiss-Prot:P10845.4 ( E224Q, R363A, Y366F, and W1266A), referred to herein as "BoNT(CB)." In some cases, the glycan is conjugated to the modified toxin by chemical crosslinking. Common chemical reactions for covalently attaching a polysaccharide to a polypeptide such as a toxin include, but are not limited to, reductive amination, cyanation conjugation, and carbodiimide reaction. In other cases, the glycan-toxin conjugate is prepared by other synthetic schemes, such as the scheme described by Chu et al., 1983. Infect and Immun. 40(1):245-256.

[0030] The term "glycan," as used herein, is intended to include polysaccharides, oligosaccharides, and other glycopolymers, including monomeric sugars. Polysaccharides generally have from about 10 up to 2,000 or more repeating units, preferably about 100-1900 repeating units. Oligosaccharides generally have from about 2-10 repeating units to about 15, 20, 25, 30, or 35 to about 40 or 45 repeating units. In some cases, suitable glycosyl groups for conjugation to the modified toxins provided herein include, but are not limited to, polysaccharides having carboxyl groups. In such cases, polysaccharides having carboxyl groups can be conjugated to the modified toxins via thiol derivatives of the carboxyl groups.

[0031] The terms "polypeptide," "peptide," and "protein," as used herein, refer to polymers comprising amino acid residues linked together primarily by covalent amide bonds. By the term "protein," we mean to encompass all of the above definitions. These terms apply to amino acid polymers in which one or more amino acid residues may be artificial chemical mimics of natural amino acids, as well as to natural and unnatural amino acid polymers. As used herein, these terms can encompass amino acid chains of any length, including full-length proteins, in which the amino acids are linked by covalent peptide bonds. Proteins or peptides may be isolated from natural organisms, produced by recombinant technology, or produced by synthetic manufacturing techniques known to those of skill in the art.

[0032] In some cases, a spacer moiety is used as a spacer arm bridge between the modified toxin and the linked molecule. The spacer moiety can be any of a variety of molecular structures, including, but not limited to, dextran, polyglutamic acid, and oligopeptides.

[0033] Sequence identity between amino acid sequences can be determined by comparing sequence alignments. If an equal position in the compared sequences is occupied by the same amino acid, the molecules are identical at that position. Scoring an alignment as a percentage identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignment may require the introduction of gaps into one or more of the sequences to account for possible insertions and deletions within the sequences. Gap penalties can be used in sequence comparison methods; therefore, for the same number of identical molecules in the compared sequences, a sequence alignment with as few gaps as possible (reflecting a higher relatedness between the two compared sequences) will score higher than one with more gaps. Calculating the maximum percent identity involves creating an optimal alignment that takes gap penalties into account. As mentioned above, sequence identity percentages can be determined using the Needleman-Wunsch global sequence alignment tool, publicly available at blast.ncbi.nlm.nih.gov / Blast.cgi, using default parameter settings. The Needleman-Wunsch algorithm is published in J. Mol. Biol. (1970) vol. 48:443-53.

[0034] The polypeptides and nucleic acids of the invention can be made synthetically using conventional synthesizers. Alternatively, they can be produced using recombinant DNA technology and incorporated into suitable expression vectors, which are then used to transform suitable host cells, such as prokaryotic cells such as E. coli. The transformed host cells are cultured and the polypeptides isolated therefrom.

[0035] In another embodiment, the present invention is directed to nucleic acid sequences encoding modified toxin preparations and other nucleic acid sequences that hybridize under high stringency conditions to a nucleic acid molecule consisting of the above-described nucleotide sequence. In a specific embodiment herein, the nucleic acid molecule has mutations at amino acid residues 372 and 375, and further comprises the amino acid sequence 376.234 , 1226, and 1289. In some cases, the modified tetanus toxin is provided in a DNA sequence encoding the modified tetanus toxin, which comprises a mutation or modified catalytic domain as described herein in one or more of: The amino acid sequence of is the sequence number 4 is shown as:

[0036] The term "stringent conditions" as used herein refers to parameters well known in the art. For example, nucleic acid hybridization parameters can be found in references compiling such methods, such as Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, edited by F. M. Ausubel et al., John Wiley & Sons, Inc., New York. More specifically, high stringency conditions as used herein refer to hybridization 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) at 65°C. SSC is 0.15 M sodium chloride / 0.015 M sodium citrate, pH 7; SDS is sodium dodecyl sulfate; and EDTA is ethylenediaminetetraacetic acid. After hybridization, the membrane onto which the DNA has been transferred is washed with 2×SSC at room temperature, followed by 0.1-0.5×SSC / 0.1×SDS at temperatures up to 68°C, e.g., 55°C, 60°C, 65°C, or 68°C. Alternatively, high stringency hybridization can be performed using commercially available hybridization buffers, such as ExpressHyb™ buffer (Clontech), using the hybridization and washing conditions described by the manufacturer.

[0037] It will also be understood that the present invention encompasses the use of the sequences in expression vectors and for transfecting host cells and cell lines, whether prokaryotic (e.g., E. coli), or eukaryotic (e.g., dendritic cells, CHO cells, COS cells, yeast expression systems, recombinant baculovirus expression in insect cells). These expression vectors require that the appropriate sequences, i.e., those operably linked to a promoter, be present.

[0038] In another aspect herein, there is provided an immunogenic composition comprising a modified toxin as described herein, which, when introduced into a host, confers immunity to the host when the host that produced the protein is subsequently challenged by the same microorganism (e.g., Clostridium tetani). In a preferred embodiment, the immunogenic composition is a vaccine comprising a modified toxin as described herein, and further comprising a suitable excipient and / or diluent when the composition is administered to a subject in need of vaccination against the development of a disease caused by Clostridium tetani or Clostridium botulinum, or a purified toxin thereof.

[0039] The term "vaccine" as used herein refers to a composition containing an antigen. Vaccines can also include biological preparations that enhance immunity against a specific disease. Vaccines generally contain an agent called an antigen that resembles a disease-causing microorganism; this agent can often be made from an attenuated or killed form of the microorganism, its toxin, or one of its surface proteins. The antigen prompts the body's immune system to recognize the agent as non-self, destroy it, and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms it encounters later. Similarly, modified toxin preparations, when combined with vaccines against other pathogens, can themselves act as vaccine adjuvants to "boost" the immune response against the targeted pathogen. Adjuvants can be classified according to their physicochemical properties or mechanism of action. Two major classes of adjuvants include compounds that act directly on the immune system, such as bacterial toxins that stimulate an immune response, and molecules that can aid in antigen presentation in a controlled manner and behave as carriers.

[0040] Selection of appropriate vaccine components is within the ordinary skill of those skilled in the art. For example, the vaccine compositions of the present invention can be conveniently formulated using pharmaceutically acceptable excipients or diluents, such as aqueous solvents, non-aqueous solvents, and non-toxic excipients, e.g., salts, preservatives, buffers, and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, e.g., ethyl oleate. Aqueous solvents include water, alcoholic / aqueous solutions, saline, and parenteral vehicles, e.g., sodium chloride, Ringer's dextrose, and the like. Preservatives include antimicrobial agents, antioxidants, chelating agents, and inert gases. The pH and precise concentrations of the various components in the vaccine composition are adjusted according to routine methods.

[0041] In some cases, the preparations described herein may contain purified modified toxins, including preparations containing partial toxin complexes. In some embodiments, the preparations may further contain stabilizers known to stabilize modified BoNT and tetanus toxin proteins. Suitable stabilizers are known in the art and include, but are not limited to, human or bovine serum albumin, gelatin, and recombinant albumin, such as those described in U.S. Patent Application Publication No. 2005 / 0238663, the entire contents of which are incorporated herein by reference.

[0042] The terms "subject" and "patient" are used interchangeably and refer to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that will be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0043] The preparations of the present invention can be administered in therapeutically effective amounts. The terms "effective amount" or "therapeutically effective amount" refer to the amount of an antigen or vaccine that induces an immune response sufficient to prevent signs or symptoms of disease, including adverse health effects or complications, caused by infection with a pathogen, such as a virus or bacteria, in a subject receiving the antigen or vaccine. Humoral immunity, cell-mediated immunity, or both humoral and cell-mediated immunity may be induced. The immunogenic response of an animal to a vaccine can be assessed indirectly, for example, by measuring antibody titers or lymphocyte proliferation assays, or directly by monitoring signs and symptoms after administration of a wild-type strain. Protective immunity conferred by a vaccine can be assessed, for example, by measuring the reduction in clinical signs, such as mortality, morbidity, body temperature, and overall physical condition, as well as the overall health and performance of the subject. The therapeutically effective amount of a vaccine will vary depending on the particular preparation used or the condition of the subject and can be determined by a physician.

[0044] The preparations of the present invention can be administered in therapeutically effective amounts depending on the type of treatment required. Methods for determining the appropriate dose or dose range for a particular treatment are known to those of skill in the art. With respect to the methods provided herein, the preparations of the present invention can be administered by any means that achieves the intended purpose or is deemed appropriate by the skilled artisan. In exemplary embodiments, the modified toxin preparation is administered as a single dose or, if appropriate, as a continuous administration using, for example, a minipump system. In some cases, the modified toxin preparation is provided as a liquid dosage form or, for example, as a lyophilized dosage form that is reconstituted prior to administration.

[0045] The term "protected," as used herein, refers to the induction of immunity in a patient against a disease or condition. The induction of immunity can occur by administering a vaccine containing an antigen. In particular, in the present invention, an immunized patient is protected from tetanus disease or its symptoms. In one embodiment, a suitable dose is about 1 μg to 20 μg. Suitable routes of administration for the modified toxin preparations described herein include, but are not limited to, direct injection. In certain embodiments, each dose is administered intramuscularly.

[0046] The dosage, toxicity, and therapeutic efficacy of the drugs of the present technology can be determined, for example, by the LD 50 (lethal dose for 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of a population can be determined by standard pharmaceutical procedures in cell cultures or test animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 It can be expressed as a ratio.

[0047] As used herein, a "therapeutically effective amount" refers to the amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" will vary depending on the compound, the condition being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors. For purposes of this invention, "treating" or "treatment" refers to the management and care of a patient for the purpose of combating a disease, condition, or disorder. These terms include both preventative, i.e., prophylactic, and palliative, therapy.

[0048] The conjugated delivery platforms described herein can be tailored to target specific conditions or diseases by tailoring which conjugated modified Tet toxin platform is used.

[0049] method In another aspect, provided herein are methods for engineering vaccines and conjugate vaccines with reduced toxicity and enhanced efficacy. These methods involve genetically modifying specific amino acid residues within domains of multidomain protein toxins, thereby rendering the toxins unable to express toxicity through either catalytic or receptor-binding activity against target cells (e.g., neurons), defective in translocation, and reduced likelihood of reverting to a toxic form. By modifying multiple independent protein functions, the methods provided herein advantageously provide full-length toxins with substantially no potential for protein reversion to toxicity, making them ideal candidates for potent vaccines and conjugate vaccines. As used herein, the term "efficacy" refers to the specific protective immune efficacy or performance of a vaccine as demonstrated by appropriate clinical testing or well-controlled clinical data. In other words, efficacy is a measure of the strength of a vaccine.

[0050] In some cases, a method for obtaining an engineered bacterial protein toxoid with enhanced efficacy as a vaccine comprises, or consists essentially of, the following steps: selecting one or more amino acids in each domain of an amino acid sequence encoding a multidomain bacterial protein toxin, where each position is selected to inactivate a protein function associated with each domain, these domains including two or more of a catalytic domain, a translocation domain, a receptor-binding domain, and a substrate-binding domain; substituting a natural amino acid residue at each selected position with a non-natural amino acid residue, whereby the substitution inactivates one or more protein functions associated with that domain; and expressing in a host cell a nucleic acid sequence encoding a full-length bacterial protein toxin comprising the substituted non-natural amino acid residue, whereby the expressed protein exhibits a partial or complete loss of catalytic activity, receptor-binding activity, translocation activity, or substrate-binding activity compared to the full-length bacterial protein toxin comprising the natural amino acid.

[0051] Selection of amino acid residues for modification involves analysis of the protein sequence (e.g., primary amino acid sequence) or structural information to identify individual functional amino acid residues in each domain of the bacterial protein toxin. Preferably, the selected residues are one or more individual amino acid residues in each functional domain (e.g., catalytic domain, translocation domain, receptor-binding domain, substrate-binding domain) that can be modified without destabilizing the full-length protein and without loss of immunogenicity. Protein stability may be assessed by any suitable method. In some cases, the modified toxin exhibits stability characterized by its trypsin sensitivity. 3 The modified toxins were tested for immunogenicity by measuring the 4 The test is carried out by measuring the immune response to inoculation with a modified toxin.

[0052] Protein structural information can be obtained by any suitable method, such as, for example, X-ray crystallography, electron microscopy, nuclear magnetic resonance, computerized protein structure modeling, or a combination thereof. In some cases, the crystal structure of a bacterial protein toxin of interest can be used to identify, for example, specific sites of interaction between functional domains of the toxin and residues that are functionally conserved among related protein toxins. For example, using diphtheria toxin as an example, amino acid residues involved in catalysis, substrate binding, translocation, and receptor binding are identified in the literature from previous studies, and based on alignment of amino acid sequences within the crystal structure of diphtheria toxin, manipulations are performed in the gene encoding diphtheria toxin to create mutant diphtheria toxin genes encoding multiple independent mutations in each of the toxin's four functional domains. The nucleic acid sequence encoding the mutant diphtheria toxin is transformed into Escherichia coli, and the protein is recombinantly produced and tested for loss of toxicity, maintenance of stability, and retention of immunogenicity.

[0053] For example, inter- and intra-domain molecular interactions can be determined from analysis of the crystal structure of a wild-type bacterial protein toxin. Substitution mutations can be achieved using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art. Exemplary mutagenesis protocols are provided, for example, in the Examples below. In some cases, site-directed mutagenesis is used to create mutations at single amino acid residues. In some cases, software programs such as PrimerX (available on the World Wide Web at bioinformatics.org / primerx / ) can be used to design oligonucleotide primers for site-directed mutagenesis. A wild-type bacterial protein toxin gene can be cloned into an expression vector to serve as a template for mutagenesis by any suitable method, such as polymerase chain reaction. Mutagenesis can be confirmed by nucleic acid sequencing. In some cases, a polynucleotide encoding a modified protein toxin is located within an expression vector. In some cases, the vector is located within a host cell (e.g., a bacterial cell, a yeast cell, or a eukaryotic cell). Many expression vectors and expression systems are known for both prokaryotes and eukaryotes, and the selection of an appropriate system is a matter of intent. Expression and purification of the modified protein products of the present invention can be readily accomplished by one of ordinary skill in the art. See Sambrook et al., "Molecular cloning - A Laboratory Manual, 2nd Edition."

[0054] These methods are applicable to virtually any bacterial protein toxin, also known as exotoxin, which are secreted by bacteria and are often multidomain proteins resembling enzymes in that they function catalytically and exhibit substrate specificity. Bacterial protein toxins include, but are not limited to, botulinum toxin, tetanus toxin, Shiga 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 exfoliatin B. Table 4 lists exemplary bacterial protein toxins for which crystal structure information is available for use in designing genetically engineered recombinant, non-toxic, highly potent toxoids that are particularly advantageous for vaccines and conjugate vaccines.

[0055] [Table 4]

[0056] In some cases, the independent inactivation modifications are selected to disrupt one or more of the catalytic domain, substrate binding domain, translocation domain, and receptor binding domain of a bacterial protein toxin. For example, diphtheria toxin is a bacterial protein toxin 535 amino acids in length. Inactivation of amino acid residues involved in catalytic function (E149S), substrate binding function (H21A), translocation function (E349K, E362K), and receptor binding function (K516A, K526A, H391A) results in a non-toxic yet effective diphtheria vaccine.

[0057] However, it will be understood that these methods can be applied to virtually any protein with multiple functional domains (e.g., effector domains) for which amino acid sequence and / or crystal structure information is available and for which inactivating modifications can be determined based on known structure-function properties.

[0058] In certain embodiments, the genetic modification to disrupt host receptor activity is in the C-terminal portion of the heavy chain (HCC To disrupt catalytic activity, genetic modifications are introduced into the light chain at amino acid residues necessary or important for neurotransmitter release via the SNARE complex.

[0059] The techniques and procedures described or referenced herein are generally well understood and commonly used by those of skill in the art using conventional methods, such as, for example, the widely used molecular cloning methods described in Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed in accordance with manufacturer-defined protocols and / or parameters unless otherwise specified.

[0060] kit In another aspect herein, there is provided a kit for administering to a subject a vaccine or adjuvant comprising a modified toxin vaccine as described herein. In one embodiment, the kit includes a form of modified toxin (e.g., a modified tetanus toxin as described herein). The kit may further include instructions that enable a user to carry out a method of vaccinating a subject against the development of a disease caused by Clostridium tetani, particularly a disease caused by a 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, saline (e.g., 0.9% sodium chloride), phosphate-buffered saline, lactated Ringer's solution, and the like.

[0061] By "instructions for use," we mean a publication, record, drawing, or any other medium of expression used to communicate the utility of the present invention for one of the purposes set forth herein. The kit's instructional materials may, for example, be affixed to a container containing the present invention or shipped together with a container containing the present invention. Alternatively, the instructional materials may be shipped separately from the container or provided in electronically accessible form on an internet website with the intention that the instructional materials and the biocompatible hydrogel be used cooperatively by the recipient.

[0062] As used in this specification and claims, the terms "including" and "comprising" are open-ended and should be interpreted to mean "including, but not limited to." These terms encompass the more restrictive terms "consisting essentially of" and "consisting of."

[0063] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly stated otherwise. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," "characterized by," and "having" can be used interchangeably.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes, including the description and disclosure of chemicals, apparatus, statistical analyses, and methodologies reported in the publications that may be used in connection with the present invention. All references cited herein are believed to indicate the state of the art. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0065] The present invention will be better understood in light of the following non-limiting examples. [Example]

[0066] Example 1: M-BoNT / A1 and M-BoNT / A1 W Isolation and characterization of This example demonstrates the use of 10 full-length BoNTs engineered to be defective in catalytic activity and receptor binding. 6 LD 50 We describe that a single-stranded toxin protected against challenge with native BoNT / A1. These data demonstrate the potential of genetically engineered toxins with multiple mutations that reduce toxicity through independent mechanisms as a platform strategy for the development of vaccines against botulism and other toxin-mediated diseases.

[0067] Materials and Methods: Biosafety and Biosecurity: Experiments conducted at the University of Wisconsin-Madison were approved by the Institutional Biosafety Committee. Additionally, experiments were conducted in a laboratory approved by the Federal Select Agent Program for this study, by accredited investigators, and in compliance with institutional policies and procedures. Animal experiments were approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison. The U.S. Department of Health and Human Services has approved the use of three LC mutants ( E224Q The gene and protein product encoding BoNT / A (M-BoNT / A) (M-BoNT / A, designated M-BoNT / A) does not meet the regulatory definition of a designated biological agent and allows for the production of M-BoNT / A without designated biological agent registration (§ 73.3 HHS Designated Biological Agents and Toxins 42 CFR 73.3(e)(1)).

[0068] Botulinum neurotoxins: BoNT / A1, / A2, / A3, and / A5 were purified from Clostridium botulinum strains Hall 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 protocols (Jacobson et al., 2011; Lin et al., 2010; Malizio et al., 2000; Tepp et al., 2012). BoNT / A6 was purified from CDC41370 B2tox. - The toxin (modified from the CDC41370 strain to produce only BoNT / A6) was purified using a previously described method (Pellett et al., 2016). Toxin purity was confirmed by spectroscopy and SDS-PAGE analysis (Whitemarsh et al., 2013). Purified toxin was 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 1 mouse LD50 unit (U). 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).

[0069] Recombinant BoNT derivative: 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 Production of α-glucanase (α-glucanase) was performed as previously described (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 grown with shaking at 37°C for 3–6 hours until an OD600 of approximately 0.6 was reached when 1.0 mM IPTG was added, followed by overnight incubation at 16°C with shaking. Cells were harvested, and the pellet was suspended in lysis buffer (20 mM Tris (pH 7.9), 500 mM NaCl, 5 mM imidazole, RNase, DNase, and protease inhibitors) (Sigma). Cells were disrupted using a French press, clarified by centrifugation, and filtered through a 0.45 μm detergent-free cellulose acetate membrane (Thermo Fischer). The lysate was then filtered using a Ni 2+ The recombinant proteins were further purified by tandem gravity-flow chromatography using β-NTA resin (Qiagen), p-aminobenzamidine-agarose (Sigma), and Strep-tactin Superflow high-capacity resin (IBA-LifeSciences). The purified proteins were dialyzed against 10 mM Tris (pH 7.9), 200 mM NaCl, and 40% glycerol and stored at -20°C. The recombinant proteins used in this study are shown in Figure 1. M-BoNT / A1 W The nucleotide sequence encoding this is shown as SEQ ID NO: 4 (see Figure 9).

[0070] Vaccination: Groups of female ICR mice (18–22 g) were vaccinated with the indicated concentrations of HC mixed with an equal volume of Alhydrogel as an adjuvant. C / A1 W , M-LCHC N / A1, M-BoNT / A1, or M-BoNT / A1W The mice were intraperitoneally immunized with non-trypsinized M-BoNT / A1 and M-BoNT / A1. W was used as the vaccine. Vaccines were administered on days 1 and 14, blood was collected by maxillary bleed on day 21, and mice were challenged with BoNT / A1, BoNT / A2, or a cocktail of BoNT / A2, / A3, / A5, or A6 as indicated on day 26. At least eight mice per group were used for each experiment, as indicated. Statistical validity of the results was assessed by a paired two-tailed Student's t-test with p = 0.05.

[0071] ELISA: BoNT derivatives or TeNT RY(250 ng / well) was added to 0.1 ml of coating buffer, 50 mM Na2CO3 (pH 9.6), in a high-protein-binding 96-well plate (Corning) and incubated overnight at 4°C. Plates were then washed three times with 0.3 ml of phosphate-buffered saline (PBS) containing 0.05% Tween 20 and blocked with 0.2 ml of PBS containing 1% (wt / vol) bovine serum albumin (BSA) for 30 min at room temperature (RT). Plates were incubated for 1 h at RT with the indicated dilutions of serum from individually vaccinated mice at either 1:20,000 or 1:30,000 in PBS containing 1% (wt / vol) BSA (0.1 ml). After washing three times with 0.3 ml of PBS containing 0.05% Tween 20, the plates were incubated with goat α-mouse IgG-horseradish peroxidase (IgG-HRP; Thermo) at a 1:20,000 dilution in PBS containing 1% (wt / vol) BSA for 1 hour at room temperature. After washing three times with 0.3 ml of PBS containing 0.05% Tween 20, the plates were incubated with 0.1 ml / well of tetramethylbenzidine (TMB; Thermo Ultra TMB) as substrate. The reaction was terminated after 10 minutes with 0.1 ml of 0.1 M H2SO4, and absorbance was read at 450 nm. Control ELISAs measuring antigen binding with α-HA and α-FLAG antibodies demonstrated that the presence of the appropriate epitope within each antigen was within 15% (data not shown). For ELISA, P < 0.05 = for serogroups (N = 10) analyzed individually based on the time of immune induction and / or antigen challenge conditions. * , 0.01= ** , 0.001= *** , and 0.0001 = ****Statistical analysis was performed by unpaired Student's two-tailed t-test (GraphPad Prism 7). Individual sera were analyzed by at least two independent ELISAs performed in duplicate. Analysis of mouse sera at dilutions of 1:20,000 to 1:30,000 was based on evaluation of several individual sera. Serum dilutions were determined from dose-response ELISAs of several M-BoNT / A1-inoculated mice that survived BoNT / A1 challenge. A representative ELISA is shown in Figure 6.

[0072] 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, WI) were seeded at a density of approximately 35,000–40,000 cells / well onto poly-L-ornithine and Matrigel™-coated 96-well TPP plates (Midwest Scientific, MO) and maintained in iCell Neurons culture medium (Cellular Dynamics International, WI) according to the manufacturer's instructions for 7 days prior to the neutralization assay. To detect neutralizing antibodies in mouse serum, 2 pM BoNT / A1 was combined with serial dilutions of filter-sterilized serum in culture medium and incubated at 37°C for 1 hour. Serum-free BoNT / A1 was used as a "no antibody" reference, and serum from naive mice was used as a control. Toxin-free serum was used as a negative control. Fifty microliters of each antibody-toxin mixture was added per well of hiPSC-derived neurons in at least duplicate, and the cells were incubated at 37°C and 5% CO2 for 24 hours. The toxin / antibody was aspirated from the cells, and cell lysates were prepared in 50 μl of lithium dodecyl sulfate (LDS) sample buffer (Life Technologies). Cell lysates were analyzed by Western blot for SNAP-25 cleavage as previously described (Pellett et al., 2007; Pellett et al., 2010). Images were acquired using PhosphaGlo reagent (KPL, Gaithersburg, MD) and a Fotodyne / FOTO / Analyst FX imaging system (Harland, WI). Cleaved (24 kDa) and uncleaved (25 kDa) SNAP-25 signals were analyzed by densitometry using TotalLab Quant software (Fotodyne, Harland, WI). The percentage of protection was determined relative to a "no antibody" control and expressed as IC 50 Values ​​were evaluated using GraphPad Prism 6 software and nonlinear regression, variance slope, 4 parameters.

[0073] result M-BoNT / A1 is not toxic to outbred mice or cultured cells Intraperitoneal injection of 10 μg of trypsinized or nontrypsinized M-BoNT / A1 / ICR mice (ICR) did not result in observable signs of botulism, indicating that M-BoNT / A1 was at least one millionth less toxic than native BoNT / A1. In addition, incubation of human iPSC-derived neurons with 80 nM M-BoNT / A1 did not result in detectable SNAP-25 cleavage, whereas incubation with 50 fM native BoNT / A1 cleaved SNAP-25, indicating at least one millionth less toxicity in cell-based assays (data not shown).

[0074] M-BoNT / A1 and M-BoNT / A1 W HCc / A1 W It is a more protective vaccine than Vaccination was performed in outbred ICR mice (n = 8–10) using a primary immunization followed by one booster immunization to represent innate immune variability within the host (Rai et al., 2009). C / A1(W1266A)(HC C / A1 W ) in a mouse model of botulism C / A1 (Przedpelski et al., 2013). W We also engineered 0.3 μg / mouse single-chain M-BoNT / A1 W or 0.2 μg / mouse M-LCHC N Mice inoculated with / A1 were 10 6 LD 50 of natural BoNT / A1 or 10 5 LD 50 BoNT / A subtype cocktail (A2, A3, A5, A6 each 2.5 × 10 4 LD 50 ) and 10 6 LD 50The HC of 0.1 μg / mouse partially protected against challenge with the heterologous subtype of native BoNT / A2 (Table 4). C / A1 W Mice inoculated with 10 3 LD 50 These data support the conclusion that at equimolar doses, M-BoNT / A1 partially protected against challenge with either native BoNT / A1 or native BoNT / A2. W Vaccines and M-LCHC N / A1 vaccine is HC C / A1 W Comparison of vaccines using weight-equivalent doses showed that 0.3 μg / mouse HC provided 1,000-fold greater protection against the toxin than the vaccine. C / A1 W Mice inoculated with 10 5 LD 50 of natural BoNT / A1 or 10 5 LD 50 This was due to the partial protection from challenge with the natural BoNT / A subtype cocktail at an equal concentration (and a 3-fold molar excess of HC C ) even if M-BoNT / A1 W Vaccines and M-LCHC N The results show that the M-BoNT / A1 vaccine provided better protection against homologous and heterologous BoNT / A challenge. W No difference in protection was observed between the M-BoNT / A1 vaccine and the M-BoNT / A1 vaccine, indicating that the additional "receptor-binding" mutations do not affect vaccine efficacy. W and M-LCHC N / A1 is HC C / A1 W The duration of action in cultured neurons was further investigated in human iPSC-derived neurons by exposing neurons to serial dilutions of either BoNT / A1 or BoNT / A6 for 72 hours followed by complete removal of exotoxin and further incubation in culture medium. Cells were harvested in triplicate with each dilution on days 3, 39, and 70, and the EC values ​​for SNAP-25 cleavage at each time point were calculated. 50In BoNT / A6, EC 50 The values ​​were approximately 0.04, 0.7, and 1 U / 50 μl / well (32, 560, and 800 fM) on days 3, 39, and 70, respectively (Figure 2). 50 The values ​​were approximately 0.7, 6.3, and 28 U / 50 μl / well (313, 2940, and 12,880 fM, respectively) on days 3, 39, and 70 (Figure 2). The half-lives of BoNT / A1 and / A6 in these hiPSC-derived neurons were approximately EC 50 The time course of the effects of BoNT / A1 and BoNT / A6 was comparable, approximately 12 and 14 days, respectively (Figure 2). Taken together, these results indicate that BoNT / A6 has a long duration of action similar to other BoNT / A subtypes.

[0075] Antibody responses to BoNT vaccination vary qualitatively and quantitatively in outbred mouse strains. M-BoNT / A1 W and M-BoNT / A1 vaccination was 10 5 LD 50 Complete protection against challenge with BoNT / A2 and 10 6 LD 50 The antibody responses of vaccinated mice analyzed by ELISA showed that both mice that survived the challenge with native BoNT / A2 and mice that did not survive the challenge with native BoNT / A2 were partially protected against BoNT and LCHC. N The results showed that all vaccinated mice had comparable predominant antibody titers against BoNT / A2, which were not statistically different (Fig. 2). Thus, the partial protection from natural BoNT / A2 challenge appears to be due to specific differences in the composition of neutralizing epitopes between BoNT / A subtypes, rather than the ability of vaccinated mice to mount an immune response to the delivered vaccine. [Table 5]

[0076] The antibody response of vaccinated mice wasRY , H.C. C / A1 W , M-LCHC N Individual sera were analyzed by ELISA using either M-BoNT / A1 or M-BoNT / A1 holotoxin as the binding substrate. Antibody responses within each group of vaccinated mice varied quantitatively and qualitatively within the groups. W Inoculated mice (Figure 3, bottom left) were infected with BoNT (mean titer 2.2 (range 1.3-2.6)) and LCHC. N The antibody titer was predominant against HC (mean titer 1.7 (range 0.8-2.4)). C The titers against HC varied among mice (mean titer 0.41 (range 0.07-1.83)). The titers against LC did not exceed those of the control, indicating that most of the antibody response was directed against HC. The variation in the titer range was due to variations in antibody titers between individual mice, not due to variations in ELISA replicates. Similar immune responses were observed against M-BoNT / A1 inoculation (data not shown). M-LCHC N / A1-inoculated mice (Fig. 3, upper left) also expressed BoNT and LCHC N Although the antibody titers against M-BoNT / A1 were predominant, W The titers were lower on average than those of the inoculated mice. N / A1+HC C / A1 W Inoculated mice were qualitatively M-BoNT / A1 W The antibody titer profile was similar to that of the inoculated mice, but quantitatively, they were significantly higher than those of the M-LCHC N The results were comparable to those of mice inoculated with HC / A1 alone (Fig. 7). C / A1 W Inoculated mice were HC C The antibody titers against M-BoNT / A1 correlated with survival after BoNT / A1 antigen stimulation (Fig. 3, bottom right). W Inoculated mice were treated with TeNT RY (Fig. 3), indicating that the antibody response observed was BoNT-specific.

[0077] Characterization of sera from individual vaccinated mice surviving BoNT challenge M-BoNT / A1 survives natural BoNT / A1 antigen stimulation W , M-LCHC N / A1 or HC C / A1 W Analysis of individual sera from inoculated mice revealed several representative immune responses to vaccination (Figure 4). In our earlier study (Przedpelski et al., 2013 Infect Immun. 81(7):2638-44), M-LCHC N The antibody response to / A1 vaccination was not characterized, so the three LCHC N Sera from mice inoculated with M-BoNT / A1 were analyzed. ELISA results showed that M-BoNT / A1 W Inoculated mice were infected with BoNT and LCHC. N (#7) or BoNT, LCHC N , and H.C. C (Mouse #3) showed a predominant antibody titer against M-LCHC. N / A1-inoculated mice were treated with BoNT and LCHC N (mice #21, #24, and #25) showed predominant antibody responses to HC C / A1 W Mice inoculated with and surviving BoNT / A1 challenge were HC C The mouse #78 showed a predominant antibody response to TeNT. RY The antibody response to BoNT was low, indicating that the immunoreactivity detected by ELISA was BoNT-specific.

[0078] BoNT / A and HC C The vaccine is LCHC N Elicits a stronger neutralizing antibody response than vaccines Neutralizing antibodies in the sera of vaccinated mice were measured in a cell-based assay using hiPSC-derived neurons. Ten sera from each vaccination group receiving equimolar doses of the vaccine were pooled and tested for their ability to neutralize BoNT / A1-induced SNAP25 cleavage in a cell-based assay. W The inoculation pool is IC 50 The value of 0.004 indicates the highest neutralization efficacy, which is HC C / A1 W Inoculation pool and M-LCHC N / A1+HC C / A1 W Approximately half of the inoculum pool contained M-LCHC N The HC population was approximately one-fifth of the HC population in the / A1 inoculation pool (data not shown). C The similarity in neutralizing antibody titers between the HCc-vaccinated and M-BoNT / A1-vaccinated mice was striking, given that the M-BoNT / A1 vaccine protected mice from a toxic challenge that was >1,000-fold stronger than the HCc vaccine. To further explore this, we also determined the ability of each of the six individual sera to neutralize BoNT / A1 cleavage of SNAP25 in a cell-based assay (Fig. 5). Overall, each of the six sera neutralized BoNT / A1 action, with a difference in serum potency of approximately 10-fold. C HC containing a predominant antibody response against C / A1 W inoculation (mouse #78) and M-BoNT / A1 W Serum from the inoculated mouse (mouse #3) (Fig. 4) was the most potent inhibitor of BoNT / A1 cleavage of SNAP-25. C Sera without antibody responses (mice #7, #21, #24, and #25) were less effective in inhibiting SNAP-25 cleavage. Thus, in this assay, the vaccine containing the HCc epitope inhibited LCHC N These data suggest that BoNT / A1 HC induces a greater neutralizing / blocking antibody response than the vaccine. C The domain is HC N Although this HC domain elicits a stronger neutralizing / blocking antibody response than the HC domain or LC domain, NThis indicates that the PDK domain and possibly the LC domain play a major role in host defense.

[0079] Consideration In an outbred mouse model of botulism, M-BoNT / A1, M-BoNT / A1 W and M-LCHC N / A1 is HC C / A1 W Evaluation of serum from vaccinated mice that survived BoNT / A1 challenge was performed using LCHC N This is consistent with the presence of neutralizing epitopes within the LCHC N M-BoNT / A1 elicits a similar protective immune response to M-BoNT / A1. W The ability of BoNT to bind to host cells demonstrated that reduced host cell binding did not adversely affect vaccine efficacy. Therefore, full-length BoNT engineered to possess defects in both the catalytic and receptor-binding domains represents a novel platform strategy for the development of vaccines against botulism and other toxin-mediated diseases. Collier and colleagues (Killeen et al., 1992) demonstrated the feasibility of creating second-site mutations that partially reverted the activity of genetically inactivated diphtheria toxin as a vaccine development test. In addition, recent work by Smith and colleagues demonstrated the need for attenuation beyond simple catalytic reduction for some serotypes of BoNT-based vaccines (Webb et al., 2017).

[0080] In early studies, LCHC N was considered a BoNT vaccine candidate (Shone et al., 2009). N It is produced in large quantities by Escherichia coli through fermentation and is effective against low-dose BoNT challenge (10 3 LD 50 In this report, the inventors demonstrated efficacy as a single-dose vaccine against BoNT (BoNT). In this report, the inventors demonstrated efficacy in LCHC by direct comparison with other BoNT vaccine candidates using a prime immunization and one booster immunization. N is an effective vaccine, and LCHCN confirmed the presence of neutralizing epitopes within the LC (Shone et al., 2009). Dolly and coworkers identified a LC-specific monoclonal antibody (MAb) that blocked BoNT / A action (Cenci Di Bello et al., 1994), while Marks and coworkers identified a BoNT / A-neutralizing mAb with LC function that inhibited SNARE cleavage (Cheng et al., 2009) and HC (Cheng et al., 2009). N We identified a mAb that targeted BoNT and neutralized several BoNT serotypes (Garcia-Rodriguez et al., 2011). Taken together, these studies suggest that BoNT vaccination may contribute to the prevention of LCHC infection. N It has been shown that M-BoNT / A1 elicits the production of antibodies against neutralizing epitopes within the domain. W M-LCHC N HC / A1 elicited a greater antibody response than HC / A1 (Fig. 7). C M-BoNT / A1 was also found to produce antibodies with the greatest neutralizing / blocking capacity in cultured cells. W HC etc. C Vaccines containing LCHC N or HC C There appears to be greater protection under "high dose" exposure than with vaccine derivatives.

[0081] HC C , DNA vector and viral vector vaccines against botulism, and HC C This is a well-established domain for developing vaccines based on proteins constructed from HCs, with early studies demonstrating their neutralizing potency (Clayton et al., 1995) and ease of production (Baldwin et al., 2008). Smith and coworkers have demonstrated the use of HCs in the yeast Pichia pastoris. C to express HC(c) and the protective immunity elicited by HC(c) (Byrne and Smith, 2000), and then the recombinant HC C / A and HC CA bivalent vaccine consisting of rBV A / B (rBV A / B) has been reported, and this vaccine is currently undergoing clinical trials (Webb and Smith, 2013). E. coli also has HC of seven serotypes (A to G) in response to BoNT antigen stimulation. C It has been used as a heterologous host for BoNT vaccine development, including vaccine production (Baldwin et al., 2008). C introduce a mutation that blocks host receptor binding into HC C W The vaccine retained its efficacy (Przedpelski et al., 2013). C makes HC an attractive vaccine platform, but current research is focused on C / A1 W than M-BoNT / A1 W This indicates that the LCHC of BoNT was a more potent vaccine. N Using human serum from a patient with cervical dystonia who was resistant to BoNT therapy, which is consistent with the immunogenicity of LC and HC, N This is supported by the findings of Atassi and colleagues (Atassi et al., 2011; Dolimbek et al., 2007), who detected immune epitopes within the

[0082] A recent study by Smith and coworkers (Webb et al., 2017) found that catalytically inactive BoNT can produce 1000 LD after a single inoculation. 50 HC responding to antigen stimulation by toxin administration C reported that the antigen challenge test described by Smith et al. measured a threshold toxic challenge, which differs from current tests that measure protection against end-point toxic challenge. This data indicates that in both cases, whether threshold or end-point protection against toxic challenge is measured, full-length BoNT vaccines exhibited greater efficacy than their respective HCs. C M-BoNT / A1 is defective in catalysis and host receptor binding. WThe subunit was more effective in stimulating end-point toxicity than the subunit. Inactivating multiple functional sites to reduce potential toxicity through cell binding or entry may be useful for the development of M-BoNT / A1 as a vaccine candidate. W The utility of this approach addresses concerns that genetic inactivation of catalytic function alone may not provide sufficient safety margin for full-length BoNT vaccine development (Webb et al., 2017).

[0083] HC as a vaccine candidate against botulism C Although the utility of HCs has been established (Baldwin et al., 2008; Henderson, 2006), current studies have demonstrated that multidomain derivatives of BoNTs are more effective against HCs. C This indicates that M-BoNT / A1 is a more potent vaccine than M-BoNT / A1. W In outbred mice, LCHC N Although the antibody responses against HC C The antibody responses were variable. The ability to reduce both catalytic activity and receptor binding supports the use of M-BoNT / A1 as a vaccine platform against botulism. W The protection from the BoNT / A subtype cocktail confirms the broad neutralizing capacity of this vaccine. W The single-chain M-BoNT / A1 was not processed into its active two-chain form, and no toxicity was detected in mice or cells, indicating the potential of the single-chain M-BoNT / A1 as a safe and effective vaccine. W This suggests that...

[0084] Example 2 - Characterization of impaired light chain translocation in lysine 768 TeNT mutants This section describes the first identification and characterization of a single amino acid point mutation within the translocation domain of TT that blocked light chain (LC) translocation. Identification of the role of K768 in LC translocation provides the first opportunity to inactivate the independent activities of TT, and by analogy, BT (see Table 6), catalysis, translocation, and receptor binding, for the development of recombinant vaccines.

[0085] Using tetanus toxin, we recently identified a rate-limiting step in light chain (LC) translocation that is encoded within the translocation domain. Lysine (K) 768 is located within the loop connecting two long α-helices (helices 12-13 and 16-17). Site-directed mutagenesis identified a point mutation, K768A, located within the loop connecting the two long α-helices of the translocation domain, that inhibited translocation (Figure 12). Cellular studies showed that M-TT (K768A) did not bind to neuronal membranes, supporting the role of the loop in membrane permeabilization. Control experiments showed that the K768A mutation did not inhibit TT binding, entry, transport, or membrane pore formation in host cells, nor did it inhibit the preferred LC-HC cleavage by trypsin, indicating that this mutation did not disrupt the overall M-TT structure and implicating a direct role for this loop in light chain translocation. Other experiments indicated that K768 was not a pH-triggered component. It is a single amino acid within the translocation domain required for light chain translocation, implicating a role for the two long α-helices (helices 12-13 and 16-17) in toxin-membrane interactions. Other experiments showed that the D767A / E769A mutation also resulted in a translocation defect in tetanus toxin, suggesting that D767 / E769 complement and / or add to K768 to inhibit LC translocation in tetanus toxin.

[0086] Creating independent mutations in botulinum toxin (BT) (see Table 6) and TT vaccine candidates inactivates each of the three functions of toxin action: catalysis, translocation, and receptor binding, thus enhancing vaccine safety. Multiple independent mutations exponentially reduce toxin potency, enhancing vaccine safety without compromising protein structure and possibly immunogenicity, and reducing the likelihood of reversion during large-scale production. [Table 6]

[0087] Example 3 - Engineered M-Tetanus Toxin (M-TT) as a Low-Dose Protective Vaccine Tetanus toxin (TT), which has a similar structure to BT, is an AB toxin containing an N-terminal domain (catalytic light chain, LC) and a C-terminal domain (translocation and receptor-binding heavy chain, HC). ++ Mutation of two amino acids in the binding pocket (R372A, Y375F) inhibits Zn ++ This resulted in 2M-TT, which inhibited the binding of tetanus toxin and was 125,000-fold less toxic than native tetanus toxin. The amino acid sequence of 2M-TT is shown as SEQ ID NO:2, and the nucleotide sequence is shown as SEQ ID NO:3. In preliminary experiments to further reduce toxicity, Zn ++ To increase the degree of inhibition of binding, E234 was added to Zn ++ An additional mutation (E234Q) was added based on stabilizing H233, which directly coordinates the binding. Next, neuron binding to dual ganglioside receptor binding was inhibited by creating two independent mutations (R1226L, W1289A) to create 5M-TT (see Table 7). As proof of principle, we engineered 6M-TT to have an additional mutation (K768A) to inhibit LC translocation (Figure 14). 6M-TT contains mutations in each of the tetanus toxin functions: catalysis, translocation, and receptor binding. 6M-TT was purified from E. coli in batch cultures at approximately 6 mg / liter. Four mice injected with 20 μg of either single-chain or double-chain 6M-TT showed no symptoms of tetanus. Thus, 6M-TT is a soluble, well-expressed, nontoxic protein. [Table 7]

[0088] 6M-TT was further engineered to sequentially inhibit VAMP-2 binding and cleavage by introducing mutations at positions Y26 and L231 to create 7M-TT and 8M-TT. L231 was chosen based on earlier studies that showed that this mutation reduced kcat without affecting VAMP-2 affinity, and the L231K mutation did not affect the overall structure of the LC.9 Y26 was selected because it is located within the S7 pocket of HCR / T and the Y26A mutation reduced the affinity of LC / T for VAMP-2. 9 The 8M-TT and intermediate products (6M-TT and 7M-TT) were selected based on evidence that they are soluble in water (see Table 7). Circular dichroism analysis was performed to assess secondary structure, trypsin sensitivity to assess overall protein stability, and mass spectrometry to assess protein composition. 42 . At this stage, we have found that 6M-TT is produced as a soluble protein, is highly expressed, and is nontoxic in mice upon injection of 20 μg. Therefore, we first analyzed the toxicity of 6M-TT, 7M-TT, and 8M-TT by analyzing VAMP-2 cleavage after cell entry and VAMP-2 cleavage in cell lysates. 64 The absence of cleavage or cytotoxicity is assessed in a human neuronal cell-based assay, and the absence of in vivo toxicity is confirmed in a mouse model using outbred female ICR mice (18-22 g, 5 mice / group) (Table 8). [Table 8]

[0089] In the first study, mice were intraperitoneally administered 20, 50, 250, or 1000 μg of 8M-TT per mouse (1000 μg of 8M-TT is approximately 4 × 10 7 LD 50 (corresponding to wild-type tetanus toxin) 65 Injected mice are scored for survival in a mouse bioassay for 3 days and observed for up to 14 days for symptoms indicative of TT pathology, including no weight gain, signs of stress, organ damage, and tetanus symptoms. Male mice are also tested for gender differences.

[0090] Outbred female ICR mice (8 mice / group) were immunized with 0.01–0.1 μg of optimized M-TT or an equivalent amount of chemically inactivated tetanus toxoid, followed by a booster vaccination on day 14 (Table 9). Mice were bled on day 26 and vaccinated 10 times on day 30. 3 ~106 To examine long-term protection, vaccinated mice are maintained for 180 days and challenged with TT to test the duration of the immune response. Mice surviving 3 days are considered protected. Sera obtained before challenge were analyzed as previously described. 6 For anti-TT by ELISA and as an inhibition of VAMP-2 cleavage 71-72 Cultured neurons are tested for their potency in neutralizing tetanus toxin intoxication. Male mice are also tested to ensure there are no gender differences. [Table 9]

[0091] Because each independent LC point mutation introduced inhibits an independent step in catalysis, we expect these LC mutations to show a fold-fold decrease in catalytic activity, reflecting a corresponding decrease in M-TT toxic potency. We do not believe these LC or HC mutations affect protein stability or immunogenicity based on initial studies of their effects as individual mutations in LC-TT or HC-TT. Injection of 1000 μg of 8M-TT does not appear to be toxic in our mouse model, 8M-TT appears to elicit a higher neutralizing immune response than chemically inactivated TT, and 8M-TT allows for immune induction at lower doses.

[0092] A recent review assessed that current conjugate vaccines have only achieved a portion of their potential immune-inducing efficacy. 4 Research into microbial pathogens continues to identify additional immunogens that require conjugation with protein toxoids to generate an effective T-cell dependent immune response. These include, but are not limited to, Neisseria meningitidis. 15 ,fungi 16 , and Streptococcus pneumoniae 17-18 In addition, synthetic glycans represent a promising future alternative to vaccines based on natural polysaccharides, which vary in purity and content. 20-22Tetanus toxoid is an immunogenic carrier protein for polysaccharides. 23 Tetanus toxin is among the best candidates for developing these next-generation recombinant conjugate vaccines, based on our knowledge of TT structure-function properties and the basic information on tetanus toxin as a chemically inactivated toxoid, as well as the continuing global demand for tetanus vaccination. The production of a safe, easy-to-produce, and protective recombinant TT vaccine will, for the first time, allow the analysis of full-length, avirulent (non-toxic) recombinant TT as a conjugate vaccine carrier. Currently, there is no knowledge of the protective properties of conjugate vaccine carriers, including tetanus toxoid. 73 Non-toxic recombinant M-TT can be used as a carrier for several conventional antigens to measure the enhancement of the immune response to the antigen when conjugated to M-TT relative to chemically inactivated TT.

[0093] The protocol for conjugating oligosaccharides to M-TT is based on the published protocol from the Lees laboratory. 74 Briefly, polysaccharides (PS) are reduced to molecular weights of 100–300 kDa using an LV-1 microfluidizer. PS is prepared at 5 mg / ml in water and activated with 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP, 0.5 mg / ml). 74An equal mass of protein (5 mg / ml) is added, and the solution is maintained at pH 9. The reaction is monitored by size-exclusion chromatography (SEC) HPLC and quenched with excess glycine. The conjugate is purified by SEC, and the molecular weight is determined by SEC multi-angle light scattering. Authenticated polysaccharides and peptides are crosslinked with 8M-TT: (i) Group B Streptococcus (GBS) polysaccharide serotypes Ia, Ib, II, III, IV, and IV; (2) poly-β-(1-6)-N-acetyl-glucosamine (PNAG) mediating biofilm formation as a candidate broad-spectrum vaccine against Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomonas maltophilia, and Burkholderia cepacia complex (BCC). 45 ;(3) Peptides currently being tested in influenza vaccines.

[0094] Outbred female ICR mice (8 mice / group) were immunized with 0.01-0.1 μg of conjugated optimized M-TT or an equivalent amount of chemically inactivated tetanus toxoid, followed by a booster vaccination on day 14 (Table 10). Mice were bled on day 26 and vaccinated 10 times on day 30. 3 ~10 6 To examine long-term protection, vaccinated mice are maintained for 180 days and challenged with TT to test the duration of the immune response. Mice surviving 3 days are considered protected. Sera obtained before challenge were analyzed as previously described. 6 74 Anti-conjugate and anti-TT are tested by ELISA. Male mice are also tested for gender differences. [Table 10]

[0095] PNAG oligosaccharide, GBS polysaccharide, and influenza peptide are individually conjugated to the 8M-TT vaccine. In subsequent experiments, GBS and PNAG are combined to create a multi-polysaccharide conjugate 8M-TT vaccine. Next, influenza peptide and PNAG are combined to create a multi-peptide-polysaccharide conjugate 8M-TT vaccine. These experiments test the potential of 8M-TT as a vaccine carrier.

[0096] The individual conjugate vaccines, PNAG-8M-TT, GBS-8M-TT, and peptide-8M-TT, are expected to elicit comparable immune responses to the conjugates and stronger immune responses to TT than the individual conjugate chemically inactivated TT vaccines. We also predict that immune responses to the polysaccharides and peptides in the 8M-TT vaccine will be comparable to those when the individual antigens are conjugated to 8M-TT. We predict that the immune response to 8M-TT will correlate with protection against native TT antigen challenge.

[0097] References TIFF0007797441000011.tif84155 TIFF0007797441000012.tif235160 TIFF0007797441000013.tif230158 TIFF0007797441000014.tif231157 TIFF0007797441000015.tif194153

[0098] It should be noted that the above description, the accompanying drawings, and their descriptions are intended to be illustrative, not limiting, of the present invention. Many themes and variations of the present invention will suggest themselves to those skilled in the art and in light of this disclosure. All such themes and variations are within the contemplation of the present invention. For example, while the present invention has been described in terms of various exemplary embodiments outlined above, various alternatives, modifications, variations, improvements, and / or substantial equivalents, whether known, rare, or presently unanticipated, will become apparent to at least those of ordinary skill in the art. Various changes can be made without departing from the spirit and scope of the present invention. Accordingly, the present invention is intended to embrace all known or later-developed alternatives, modifications, variations, improvements, and / or substantial equivalents of these exemplary embodiments. Another aspect of the present invention may be as follows. [1] A modified tetanus toxin polypeptide having at least 95% identity to SEQ ID NO: 1 and comprising an array containing mutations at positions R372 and Y375, and further mutations at two or more positions selected from E334, K768, R1226, and W1289, each position being numbered relative to SEQ ID NO: 1, wherein the polypeptide has reduced toxicity and receptor binding compared to the toxicity and receptor binding of SEQ ID NO: 1. [2] The modified polypeptide according to [1], wherein the amino acid R at position R372 is replaced with the amino acid A, and the amino acid Y at position Y375 is replaced with the amino acid F. [3] The modified polypeptide according to [1], wherein the mutations include R372A, Y375F, E334Q, R1226L, and W1289A. [4] The modified polypeptide described in [3] above, which is encoded by SEQ ID NO: 4. [5] The modified polypeptide of [1], wherein the mutations include R372A, Y375F, E334Q, K768A, R1226L, and W1289A. [6] The modified polypeptide described in [5] above, which is encoded by SEQ ID NO: 5. [7] The modified polypeptide described in [1], further comprising a mutation at one or both of positions L231 and Y26, each position being numbered relative to SEQ ID NO: 1. [8] The modified polypeptide according to [7], wherein the mutations at one or both of the L231 and Y26 positions include L231K and Y26A. [9] The modified polypeptide according to [7], which is encoded by SEQ ID NO: 6 or SEQ ID NO: 7.

[10] The modified polypeptide according to any one of [1] to [9] above, further comprising a covalently bound sugar chain, thereby making the polypeptide a polypeptide-sugar chain conjugate.

[11] A composition comprising the modified polypeptide according to any one of [1] to

[10] above and a pharmaceutically acceptable carrier.

[12] A method for reducing the risk of a subject developing tetanus, comprising inducing an immune response by administering to the subject a therapeutically effective amount of a modified polypeptide described in any one of [1] to

[10] .

[13] Use of the modified polypeptide according to any one of [1] to

[10] above as an adjuvant.

[14] Use of the modified polypeptide described in any one of [1] to

[10] above as a vaccine.

[15] A method for obtaining an engineered bacterial protein toxoid with enhanced efficacy as a vaccine, comprising: selecting one or more amino acid positions in each domain of an amino acid sequence encoding a multidomain bacterial protein toxin, wherein each position is selected to inactivate a protein function associated with each domain, said domains including two or more of a catalytic domain, a translocation domain, a receptor-binding domain, and a substrate-binding domain; substituting a non-natural amino acid residue for the natural amino acid residue at each selected position, whereby said substitution inactivates one or more protein functions associated with said domain; and expressing in a host cell a nucleic acid sequence encoding a full-length bacterial protein toxin comprising the substituted unnatural amino acid residue, whereby the expressed protein exhibits a partial or complete loss of catalytic activity, receptor binding activity, translocation activity, or substrate binding activity compared to a full-length bacterial protein toxin comprising a natural amino acid; A method comprising:

[16] The method according to

[15] , wherein the selection comprises identifying individual functional amino acid residues based on the primary sequence or structure of the bacterial protein toxin.

[17] The method according to

[16] , wherein the structure is obtained using X-ray crystallography, electron microscopy, nuclear magnetic resonance, computer protein structure modeling, or a combination thereof.

[18] The method of

[15] , wherein the selection comprises identifying individual amino acid residues that can be modified without destabilizing the full-length protein or losing immunogenicity.

[19] The method of

[15] above, wherein the substitution comprises site-directed mutagenesis.

Claims

1. 1. A method for obtaining an engineered tetanus protein toxoid with enhanced vaccine efficacy, comprising: selecting one or more amino acid positions in each domain of an amino acid sequence encoding a multi-domain tetanus protein toxin, wherein each position is selected to inactivate a protein function associated with each domain, said domains including two or more of a catalytic domain, a translocation domain, a receptor-binding domain, and a substrate-binding domain; substituting a non-naturally occurring amino acid residue for the natural amino acid residue at each selected position, whereby the substitution inactivates one or more protein functions associated with the domain, wherein the non-naturally occurring amino acid substitutions include: (a) R372A and Y375F; (b) at least one of D767A, K768A, and E769A; and (c) R1226L and W1289A, wherein each of the positions is numbered relative to SEQ ID NO: 1; and A method comprising expressing in a host cell a nucleic acid sequence encoding a full-length tetanus toxin protein comprising the substituted unnatural amino acid residue, whereby the expressed protein exhibits partial or complete loss of catalytic activity, receptor binding activity, translocation activity, or substrate binding activity compared to a full-length tetanus toxin protein comprising natural amino acids, wherein the expressed protein comprises a sequence having at least 95% identity to SEQ ID NO:

1.

2. 2. The method of claim 1, wherein the selection comprises identifying individual functional amino acid residues based on the primary sequence or structure of the tetanus toxin protein.

3. 3. The method of claim 2, wherein the structure is obtained using X-ray crystallography, electron microscopy, nuclear magnetic resonance spectroscopy, computer protein structure modeling, or a combination thereof.

4. 2. The method of claim 1, wherein the selection comprises identifying individual amino acid residues that can be modified without destabilizing the full-length protein or losing immunogenicity.

5. The method of claim 1 , wherein the substitution comprises site-directed mutagenesis.

6. The method described in claim 1, wherein the expressed protein further comprises the mutation E234Q, said position being numbered relative to sequence number 1.

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