Modified Clostridium neurotoxin as a vaccine and conjugate vaccine platform

Abstract

This invention provides a means to inactivate the inherent toxicity of tetanus toxin and produce a safe and effective vaccine. [Solution] This specification provides engineered non-catalytic, non-toxic tetanus toxin variants and methods for using such engineered tetanus toxin variants as low-dose prophylactic vaccines that are more effective than non-toxic and their respective chemically inactivated toxoids. In addition, this specification provides conjugate vaccine carriers containing engineered tetanus toxin variants and methods for using such conjugate vaccines to induce a T-cell-dependent immunomemory response that can target a broad range of microbial pathogens as a single vaccine.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 599,444, filed December 15, 2017, which is incorporated herein by reference as if its entire contents were fully disclosed herein.

[0002] Description of federally funded research This invention was made with government support under authorization numbers R01 AI030162 and AI118389 granted by 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 150kDa double-chain protein consisting of a 100kDa heavy chain (HC) and a 50kDa light chain (LC) linked by disulfide bonds. The HC further contains an N-terminal domain (H) that assists in the translocation of the LC into the cytosol. N ) and the C-terminal domain (H) that recognizes and binds to cell surface receptors of nerve cells. CIt is divided into (Montal, 2010). Once inside the cell, LC specifically cleaves the soluble N-ethylmaleimide sensitive-factor attachment protein receptor (SNARE) portion, thereby inactivating neurotransmitter release (Montecucco and Schiavo, 1993, Trends in biochemical sciences 18, 324-327; Schiavo et al., 1995). To date, experimental vaccines have been used to protect "at-risk" populations from botulism, but the use of this chemically inactivated BoNT toxoid vaccine has been discontinued due to reduced efficacy. Furthermore, conventional tetanus toxin fragment vaccines are not ideal due to problems associated with low antigenicity and immunogenicity. Therefore, there is still a need in this field for non-catalytic, non-toxic variants of tetanus and botulinum toxins for use as adjuvant and conjugate vaccines. [Overview of the project]

[0004] Summary of Disclosure This specification provides recombinant non-catalytic, non-toxic mutant forms of tetanus toxin and the use of such mutant toxins. The data described herein show a significant reduction in toxicity compared to natural tetanus toxin and compared to previously described tetanus mutants. The inventors envision several independent, engineered mutations, including, but not limited to, elimination of catalytic activity by elimination of substrate affinity or reduction of reaction rate, elimination of receptor binding, inhibition of translocation, or interference with intradomain cleavage or disulfide bond breakdown of the toxin, which can be combined to inactivate the intrinsic toxicity of tetanus toxin and produce a safe and effective vaccine. This specification describes experiments in which the inventors have engineered toxins having mutations that reduce host receptor binding along with reduced catalytic activity. These data demonstrate the potential of recombinant toxins, including selected independent mutations, that make them suitable as non-toxic and conjugate vaccines that do not require chemical crosslinking to mitigate toxicity.

[0005] In a first embodiment, this specification provides a modified tetanus toxin polypeptide having at least 95% identity with SEQ ID NO: 1 and comprising a sequence having mutations at the R372 and Y375 positions, and further including mutations at two or more positions selected from E334, K768, R1126, and W1289, each position being numbered relative to SEQ ID NO: 1, wherein the polypeptide exhibits reduced catalytic activity, transfer, and receptor binding compared to the toxicity and receptor binding of SEQ ID NO: 1. The amino acid R at the R372 position can be substituted with amino acid A, and the amino acid Y at the Y375 position can be substituted with amino acid F. Mutations may include R372A, Y375F, E334Q, R1226L, and W1289A. The modified polypeptide may further comprise a covalently linked glycan, thereby becoming a polypeptide-glycan conjugate. The modified polypeptide may be encoded by SEQ ID NO: 2.

[0006] In some cases, mutations may include R372A, Y375F, E334Q, 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 positions of L231 and Y26, each position being numbered relative to SEQ ID NO: 1. Mutations at one or both positions of 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 embodiment, this specification provides compositions comprising a modified polypeptide as described herein and a pharmaceutically acceptable carrier.

[0008] In a further embodiment, this specification provides a method for reducing the risk of a subject 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] The aforementioned and other aspects and advantages of the present invention will become apparent from the following description. The description refers to the accompanying drawings, which, in part, illustrate preferred embodiments of the present invention as examples. Such embodiments do not necessarily represent the entire scope of the present invention, and therefore, the claims and this specification should be referenced in order to interpret the scope of the present invention. [Brief explanation of the drawing]

[0010] [Figure 1]Figure 1 shows recombinant proteins used to evaluate the host immune response to vaccination. (Upper 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 cell studies. Domain junctions were defined using the crystal structure of BoNT / A1 (PDB:3BTA). The single-letter amino acid notation above each schematic diagram indicates the introduction of amino acid substitutions introduced to reduce catalytic activity (LC) or receptor binding (HCC). Note that single-stranded BoNT and LCHCN were used for vaccination. (Lower 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 nicked and reduced with trypsin; 3, M-LCHCN / A1; 4, M-LCHCN / A1 nicked and reduced with trypsin; 5, LC / A1RY; 6, HCC / A1W; and 7, TeNTRY. The migration of molecular weight marker proteins (kDa) is shown in the left lane. Note that in lane 2, the nicked HC migrates to approximately 80 kDa, which has been shown in other experiments to be due to trypsin-induced cleavage of the HC belt region. [Figure 2]Figure 2 shows ELISA results for serum from mice inoculated with M-BoNT / A1 or M-BoNT / A1W and stimulated with the heterologous subtype, natural BoNT / A2. Mice were inoculated with M-BoNT / A1 (upper panel) or M-BoNT / A1W (lower panel), and serum was collected before antigen stimulation with natural BoNT / A2 with an LD50 of 106. Mice that survived antigen stimulation (A) or mice that did not survive (D) are shown. Antibody titers were measured by ELISA for serum (1:20,000 dilution) of M-BoNT / A1; M-LCHCN / A1; LC / A1RY; HCC / A1W; TeNTRY; and a protein-free control (Con). Bounded mouse antibodies were detected using TMB reagent with goat α-mouse IgG-HRP (1:20,000 dilution). The reaction was stopped with dilute H2SO4 and read at 450 nm. The data are shown as the mean of 5 mice (A) and 4 mice (D) after M-BoNT / A1 inoculation, and 3 mice (A) and 6 mice (D) after M-BoNT / A1W inoculation, from two independent experiments with two replicates, with the standard deviations shown. Statistical analysis was performed as described herein: P, .05 = *. [Figure 3]Figure 3 shows the ELISA results for serum from mice inoculated with BoNT derivatives and stimulated with natural BoNT / A1 antigens. 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 antigen stimulation. The ELISA determined the antibody titers of serum (1:30,000 dilution) of M-BoNT / A1; M-LCHCN / A1; LC / A1RY; HCC / A1W; TeNTRY; and a protein-free control (Con). Bounded mouse antibodies were detected using TMB reagent with goat α-mouse IgG-HRP (1:20,000 dilution). The reaction was stopped with dilute H2SO4 and read at 450 nm. Except for HCC / A1W-inoculated mice, where data are from surviving (A)7 or non-surviving (D)3 mice stimulated with the natural BoNT / A1 antigen, data are expressed as the mean of 10 independent serum samples from mice surviving BoNT / A1 antigen stimulation in Experiment 3 of Table 1, analyzed in two independent experiments with two replicates, with the standard deviations shown. Variations in titer ranges were due to differences in antibody titers between individual mice, not due to ELISA replication. 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 from individual mice inoculated with M-BoNT derivatives and individual mice that survived antigen stimulation with natural BoNT / A1. Serum obtained before BoNT antigen stimulation from individual mice that survived natural BoNT / A1 antigen stimulation and were inoculated with BoNT / A1W (#7 and #3), HCC / A1W (#78), and LCHCN / A1 (#21, #24, and #25) was analyzed by ELISA using M-BoNT / A1;M-LCHCN / A1;LC / A1RY;HCC / A1W as antigens (1:30,000 dilution). Bounded mouse antibodies were detected using TMB reagent with goat α-mouse IgG-HRP (1:20,000 dilution). The reaction was stopped with dilute H2SO4 and read at 450 nm. The data shown are the average of two independent experiments with two replicates, with the standard deviation shown. [Figure 5] Figure 5 shows the serum neutralization of native BoNT / A1 cleavage of SNAP25 in human induced pluripotent stem cells (hiPSCs). Serum obtained before BoNT antigen stimulation from individual mice inoculated with M-BoNT / A1W (#7 and #3), HCC / A1W (#78), and M-LCHCN / A1 (#21, #24, and #25) that survived BoNT / A1 antigen stimulation was 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 in 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 a serial dilution of filtered and decontaminated serum in culture medium and incubated at 37°C for 1 hour. An "antibody-free" buffer was used as a control. 50 μl of each antibody-toxin mixture was added to each well of hiPSC-derived neurons in at least two replicates, and the cells were incubated at 37°C and 5% CO2 for 24 hours. Toxins / antibodies were aspirated from these cells, and after PAGE of the cell lysates, analysis for SNAP-25 cleavage was performed by Western blotting (Pellett et al., 2007; Pellett et al., 2010). Cleaved SNAP-25 was quantified relative to uncleaved SNAP-25 using a concentration meter, and the protection percentage was determined by comparison with the "antibody-free" control. IC50 values ​​were evaluated using GraphPad Prism 6 software and nonlinear regression, gradient variability, and four parameters. [Figure 6] Figure 6 shows a representative ELISA of serum from M-BoNT / A1W-vaccinated mice that survived antigen stimulation with BoNT / A1. Serum from BoNT / A1W-vaccinated mice that survived BoNT / A1 antigen stimulation was analyzed by ELISA using the indicated antigen, as described in the Methods section. The values ​​shown are the standard deviations and represent the average of representative measurements performed in two replicates. [Figure 7]Figure 7 shows that M-BoNT / A1W is more immunogenic than M-LCHCN / A1 or LCHCN / A1+HCC / A1W. Serum from individual mice that survived antigen stimulation with BoNT / A1 and were 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) were analyzed by ELISA for antibodies against M-BoNT / A1, M-LCHCN / A1, LC / A1RY, HCC / A1W, TeNTRY, or protein-free (Con). Data are expressed as the mean of 10 independent serum samples from mice that survived BoNT / A1 antigen stimulation in Experiment 3 of Table 1, analyzed in two independent experiments with two replicates, with the standard deviation shown. Statistical analysis was performed as described herein: P<0.05=*, 0.01=**, 0.001=***, and 0.0001=****. [Figure 8] Figure 8 shows the amino acid sequence encoding the toxin of wild-type tetanus bacteria (Clostridium tetani) (SEQ ID NO: 1). [Figure 9] Figure 9 shows the amino acid sequence encoding 2M-TT (SEQ ID NO: 2). [Figure 10] Figure 10 shows the amino acid sequence encoding 5M-TeNT (SEQ ID NO: 4). [Figure 11] Figure 11 shows the amino acid sequence encoding 6M-TeNT (SEQ ID NO: 5). [Figure 12] Figure 12 shows the amino acid sequence encoding 7M-TeNT (SEQ ID NO: 6). [Figure 13] Figure 13 shows 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) between toxins. [Figure 15] Figure 15 shows the crystal structure of TeNTRY PDB:5n0b. Four TT functions were inactivated: light chain E234Q, R373A, Y376F (Zn++ binding), L231K (VAMP-2 cleavage), and Y26A (VAMP-2 binding), K768A (LC transition), and R1226L and W1289A (receptor binding). [Modes for carrying out the invention]

[0011] Detailed explanation of disclosure Although the present invention has been described with respect to one or more preferred embodiments, it goes without saying that many other equivalents, substitutions, variations, and modifications are possible and fall within the scope of the present invention.

[0012] The methods and compositions described herein are at least in part based on the inventors' development of genetically engineered toxins that are non-catalytic and unable to bind to neuronal cells. Tetanus toxin and botulinum toxin (BoNT) having engineered defects that interfere with catalytic activity and receptor binding, as described in the following paragraphs and examples, provide a platform for the development of vaccines for toxin-mediated diseases. For example, modified tetanus toxin and BoNT made non-catalytic and unable to bind to receptors by engineered mutations showed no detectable toxicity. Furthermore, such modified toxins were suitable as vaccines for protection against botulinum neurotoxicity.

[0013] composition Preferably, the genetically modified toxins of this disclosure include genetic modifications at multiple targets relative to the wild-type toxin, such modifications eliminate residual attributes and enhance safety, as detailed below. Although BoNT and tetanus toxin act on different substrates and receptors, the inventors have found that immune cells act on a non-catalytic, non-receptor-bound form of BoNT (referred to herein as "M-BoNT"). W We examined a variant (referred to as "M-BoNT") and demonstrated that it elicits a neutralizing immune response similar to that seen with non-catalytic BoNT ("M-BoNT"). Based on these findings, further independent mutation sites were determined to manipulate other non-catalytic, non-receptor-type toxin variants. Modified as described herein, the resulting engineered non-catalytic, non-receptor-binding toxins are suitable for use as platforms for vaccines and conjugate vaccines.

[0014] While not bound by a specific theory or mode of action, mutations manipulated at independent sites in tetanus toxin and botulinum toxin render the toxin proteins toxic through independent mechanisms, thus providing a fail-safe against accidental 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] Accordingly, this specification provides recombinantly inactivated bacterial toxins that are irreversibly nontoxic, more effective than existing chemically inactivated toxoids, easier to produce and handle, and thus provide improved vaccines and conjugate vaccine carriers. In a first embodiment, this specification provides isolated preparations of recombinant, non-catalytic, non-toxic modified forms of bacterial protein toxins (e.g., tetanus toxin and botulinum neurotoxin), where the modified toxin is a full-length toxin containing at least four amino acid substitutions that render the protein toxic by an independent mechanism. “Preparation” means any concentration of the toxin polypeptide concentrated or purified relative to its natural presence. Preferably, the preparation is substantially pure or combined with other components to form a pharmaceutical formulation. In some cases, the preparation of the present invention may contain one or more adjuvants or carriers that may be bound to a toxin polypeptide sequence that helps stimulate the immune system. In other cases, the preparation itself has adjuvant activity and is effective in enhancing the immune response to a bound antigen or co-administered antigen.

[0016] As used herein, “toxin” refers to a toxic substance or poison (e.g., cytotoxin) that is formed or synthesized during the metabolism and growth of certain microorganisms as an integrated part of a cell or tissue (endotoxin), as an intracellular or extracellular product (exotoxin), or as a combination thereof. As used herein, the term “modified toxin” refers to a non-catalytic, non-toxic variant form of a toxin, where the toxin is made non-catalytic and non-toxic by genetically engineered (e.g., unnatural, artificial) modification of the amino acid sequence of a polypeptide toxin. In exemplary embodiments, modified toxins are genetically engineered or otherwise modified variants of toxins produced by Clostridium bacteria (e.g., C. difficile, C. novyi, C. sordellii, Clostridium perfringens, Clostridium tetani, and Clostridium botulinum). These toxins may be recombinants, synthetics, or part of fusion proteins covalently bound to and / or chemically crosslinked with an antigen (e.g., an antigen or polypeptide (e.g., His6) that facilitates the purification of the fusion protein). 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 a second composition containing a modified form of a particular protein active ingredient can be administered to a mammal at the same or a higher dose level as a first composition containing a particular protein active ingredient (e.g., wild-type TT), without causing death to that mammal, compared to a first composition containing a particular protein active ingredient (e.g., wild-type TT). Reduced toxicity includes the partial or complete elimination of toxicity that is detectable by methods known to those skilled in the art. In addition, reduced toxicity includes reduced systemic toxicity (i.e., in the case of intravenous administration) or reduced toxicity in the case of intramuscular administration.

[0018] In certain embodiments, the preparation contains a modified tetanus toxin. Generally, the modified tetanus toxins described herein exhibit one or more property changes compared to the wild-type tetanus toxin polypeptide shown in SEQ ID NO: 1, such as a significant decrease in catalytic activity and receptor binding activity. In some embodiments, the modified tetanus toxins described herein are at most one-millionth of the toxicity of the wild-type tetanus toxin. [Table 1]

[0019] In certain embodiments, the preparation comprises a modified tetanus toxin having mutations in amino acid residues 372 and 375, and further mutations in one or more of residues 334, 1226, and 1289, where these residue positions are numbered relative to the full-length wild-type tetanus neurotoxin (Tetanus CN3911; GenBank accession number X06214), indicated as Sequence ID No. 1. In certain embodiments, the amino acid mutations in residues 372 and 375 are R372A and Y375F, and the modified toxin further comprises at least one mutation selected from E334Q, R1226L, and W1289A. In some cases, the modified toxin comprises five mutations (R372A, Y375F, E334Q, R1226L, and W1289A) numbered relative to Sequence ID No. 1, 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 shown as Sequence ID No. 4.

[0020] In some embodiments, the tetanus toxin has a modified transition domain. For example, the lysine (K) residue at position 768 is located within a loop connecting two long α-helices. Mutation of this single amino acid to alanine (A) inactivates or blocks light chain transition. In some cases, the K768A mutation is added to the 5M-TT modified toxin to create 6M-TT, thereby the resulting modified tetanus toxin contains six independent mutations at six sites (see Tables 2 and 3). In some cases, the modified toxin contains six mutations (R372A, Y375F, E334Q, K768A, R1226L, and W1289A) numbered to Sequence ID No. 1, and is referred to herein as "6M-TeNT" or "6M-TT". In some cases, 6M-TeNT is encoded by the amino acid sequence shown as Sequence ID No. 5. While not bound by any specific mechanism or theory, the vaccine efficacy of 6M-TT appears to be higher than that of 5M-TT, but it must have a lower reversion rate. Adding one or more mutations from D767, K768, or E769A to 5M-TT results in more complete inactivation of the genetically engineered vaccine by disrupting the function of the catalytic and receptor-binding domains, as well as inactivating the function of the transition domain. [Table 2]

[0021] In some embodiments, the tetanus toxin was modified to inhibit VAMP-2 cleavage. For example, a mutation in the leucine residue at position 231 (e.g., a mutation from leucine to lysine (K)) inactivates the catalytic activity of the toxin with respect to VAMP-2 cleavage. In some cases, the 6M-TT modified toxin was modified with the L231K mutation to create 7M-TT, thereby resulting in a modified tetanus toxin containing seven independent mutations (Table 3). In some cases, the modified toxin contains eight independent mutations (R372A, Y375F, E334Q, R1226L, W1289A, K768A, and L231K) numbered relative to SEQ ID NO: 1, and is referred to herein as "7M-TeNT" or "7M-TT". In some cases, 7M-TeNT is encoded by the amino acid sequence shown as SEQ ID NO: 6.

[0022] In some embodiments, tetanus toxin has been modified to inhibit VAMP-2 binding. For example, a mutation in the tyrosine (Y) residue at position 26 (e.g., a mutation from tyrosine to alanine (A)) inactivates the toxin's VAMP-2 binding ability. In some cases, the Y26A mutation is added to the 7M-TT modified toxin to create 8M-TT, thereby resulting in a modified tetanus toxin containing eight independent mutations (Table 3). In some cases, the modified toxin contains eight independent mutations (R372A, Y375F, E334Q, R1226L, W1289A, K768A, L231K, and Y26A) numbered relative to Sequence ID No. 1, and is referred to herein as "8M-TeNT" or "8M-TT". In some cases, 8M-TeNT is encoded by the amino acid sequence shown as Sequence ID No. 7. [Table 3]

[0023] In some cases, the modified tetanus toxin contains other amino acid substitutions at the residues at positions 372, 275, 334, 768, 1226, 1289, 231, and / or 26. For example, the amino acids that can substitute 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, altering the primary structure of a polypeptide by conservative amino acid substitution does not significantly alter the polypeptide's activity, because the side chain of the amino acid introduced into its sequence can form similar bonds and contacts to the side chain of the substituted amino acid. This is true even when the substitution is in a region important for determining the polypeptide's conformation.

[0025] Conservative amino acid substitution is understood in the art as the substitution of one amino acid with another amino acid having similar characteristics. Conservative amino acid substitution 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 their 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 permissible, as long as these substitutions do not disrupt the three-dimensional structure of the epitope within the polypeptide, for example, they do not interfere with the polypeptide's immunogenicity (e.g., antigenicity) and do not restore toxicity.

[0027] In certain embodiments, the modified tetanus toxin comprises mutations at amino acid residues 372 and 375, and further mutations at one or more of residues 334, 1226, and 1289, where the modified toxin is conjugated or coupled to another peptide as described below for a suitable therapeutic method. Advantageously, the modified tetanus toxin of this 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 degradation inhibitor, but such amounts are less (e.g., an order of magnitude less) than the amounts commonly used to detoxify wild-type tetanus toxin (or tetanus toxin not modified as described herein) to form a “tetanus toxoid” (about 0.4%).

[0028] In certain embodiments, the modified toxins described herein further comprise molecules such as glycans, proteins or peptides (e.g., antigens), and chemical moieties. In particular, herein provides recombinant non-catalytic, non-toxic mutant toxin forms (e.g., modified tetanus toxin and modified botulinum neurotoxin) that are further modified to include conjugated or chemically linked (e.g., crosslinked) glycans. Thus, modified toxins conjugated to glycans 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 crosslinked moiety as “cargo.”

[0029] In some cases, the modified glycan-conjugated tetanus toxin contains five mutations (R372A, Y375F, E334Q, R1226L, and W1289A) numbered relative to Sequence ID No. 1, and is referred to herein as "TeNT(CB)". In other cases, the modified glycan-conjugated BoNT toxin contains four mutations (E224A, R363A, Y366F, and W1266A) numbered relative to UniProtKB / Swiss-Prot:P10845.4, and is referred to herein as "BoNT(CB)". In some cases, the glycans are conjugated to the modified toxin by chemical crosslinking. Common chemical reactions for covalently attaching polysaccharides to polypeptides such as toxins include, but are not limited to, reductive amination, cyanation conjugation, and carbodiimide reactions. In other cases, glycan-toxin conjugates are prepared by other synthetic schemes, such as the scheme described by Chu et al., 1983. Infect and Immun. 40(1):245-256.

[0030] As used herein, the term "glycan" is intended to include monomeric sugars, polysaccharides, oligosaccharides, and other glycan polymers. Generally, polysaccharides have about 10 to a maximum of 2,000 or more repeating units, preferably about 100 to 1,900 repeating units. Oligosaccharides generally have about 2 to 10 repeating units to about 15, 20, 25, 30, or 35 to about 40 or 45 repeating units. In some cases, suitable glycans 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] As used herein, the terms “polypeptide,” “peptide,” and “protein” refer to polymers comprising amino acid residues linked to each other primarily by covalent amide bonds. The term “protein” is used by the inventors 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 non-natural amino acid polymers. As used herein, these terms may encompass amino acid chains of any length, including full-length proteins, in which amino acids are linked by covalent peptide bonds. Proteins or peptides may be isolated from natural organisms, produced by recombinant techniques, or produced by synthetic manufacturing techniques known to those skilled in the art.

[0032] In some cases, the spacer portion is used as a spacer arm bridge between the modified toxin and the linked molecule. The spacer portion may 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 their alignments. If equal positions in the sequences being compared are occupied by the same amino acids, then those molecules are identical at those positions. The alignment score, as an identity percentage, is a function of the number of identical amino acids at positions shared by the sequences being compared. When comparing sequences, the optimal alignment may require the introduction of one or more gaps in the sequences to account for possible insertions and deletions. The sequence comparison method can use a gap penalty; therefore, for the same number of identical molecules in the sequences being compared, a sequence alignment with as few gaps as possible (reflecting a higher association between the two comparison sequences) will score higher than one with more gaps. Calculating the maximum identity percentage involves creating the optimal alignment, taking the gap penalty into account. As mentioned above, the sequence identity percentage can be determined using the Needleman-Wunsch global sequence alignment tool, available at blast.ncbi.nlm.nih.gov / Blast.cgi, with 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 present invention can be produced by synthesis using conventional synthesis equipment. Alternatively, they can be produced using recombinant DNA technology and incorporated into a suitable expression vector, which is then used to transform suitable host cells such as prokaryotic cells like Escherichia coli (E. coli). The transformed host cells are cultured, and polypeptides are isolated from them.

[0035] In another embodiment, the present invention provides nucleic acid sequences encoding a modified toxin preparation and other nucleic acid sequences that hybridize with the above-mentioned nucleotide sequence as a nucleic molecule under high stringency conditions. In certain embodiments herein, the present invention provides DNA sequences encoding a modified tetanus toxin having mutations at amino acid residues 372 and 375, and further mutations in one or more of residues 334, 1226, and 1289, or comprising a modified catalytic domain as described herein. In some cases, the nucleic acid sequence encoding the modified tetanus toxin is shown as Sequence ID No. 3.

[0036] The term "stringent conditions," as used herein, means parameters well known in the art. For example, nucleic acid hybridization parameters can be found in reference literature 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 FM Ausubel et al., John Wiley & Sons, Inc., New Yorks. More specifically, high stringency conditions, as used herein, refer to hybridization at 65°C in a hybridization buffer (3.5 × SSC, 0.02% Ficol, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 25 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). 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 DNA-transferred membrane is washed with 2 × SSC at room temperature, and then with 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 sequences in expression vectors for transfecting host cells and cell lines (these being prokaryotes (e.g., Escherichia coli) or eukaryotes (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 described above, be functionally linked to a promoter.

[0038] In another aspect of this specification, an immunogenic composition is provided comprising a modified toxin, as described herein, which, upon introduction into a host, confers immunity to the host if the host that produced the protein is subsequently attacked 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 excipients and / or diluents suitable for administration to a subject requiring vaccination against the development of a disease caused by Clostridium tetani, Clostridium botulinum, or their purified toxins.

[0039] As used herein, the term “vaccine” refers to a composition containing an antigen. Vaccines may also include biological preparations that enhance immunity against a particular disease. Vaccines generally contain a drug called an antigen, which resembles a disease-causing microorganism. This drug can often be made from one of the attenuated or dead forms of the microorganism, its toxin, or its surface proteins. The antigen prompts the body’s immune system to recognize the drug 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 “enhance” the immune response against the target pathogen by acting as vaccine adjuvants themselves. Adjuvants can be classified according to their physicochemical properties or mechanism of action. Two main classes of adjuvants include compounds that act directly on the immune system, such as bacterial toxins that stimulate the immune response, and molecules that can assist in antigen presentation in a controlled manner and behavior as carriers.

[0040] The selection of appropriate vaccine components is within the ordinary capabilities of those skilled in the art. For example, the vaccine compositions of the present invention can conveniently be prepared using, for example, aqueous solvents, non-aqueous solvents, non-toxic excipients, and pharmaceutically acceptable excipients or diluents such as salts, preservatives, and buffers. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, and organic esters for injection, such as ethyl oleate. Aqueous solvents include water, alcohol / aqueous solutions, physiological saline, and parenteral vehicles, such as sodium chloride and glucose-added Ringer's solution. Preservatives include antimicrobial agents, antioxidants, chelating agents, and inert gases. The pH and the exact concentrations of the various components in the vaccine composition are adjusted according to conventional methods.

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

[0042] The terms “subject” and “patient” are used interchangeably and refer to any animal (e.g., mammals), including but not limited to humans, non-human primates, and rodents, that is a recipient of a particular procedure. Generally, the terms “subject” and “patient” are used interchangeably in this specification in reference to human subjects.

[0043] The preparations of the present invention can be administered in therapeutically effective amounts. The term "effective amount" or "therapeutically effective amount" refers to the amount of antigen or vaccine that induces a sufficient immune response in a subject receiving the antigen or vaccine 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. Humoral immunity, cell-mediated immunity, or both humoral and cell-mediated immunity may be induced. The immunogenic response of an animal to the vaccine can be evaluated indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by observing the course of signs and symptoms after administration of the wild-type strain. The protective immunity conferred by the vaccine can be evaluated, for example, by measuring reductions 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 vaccine varies depending on the specific 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 suitable doses or dose ranges for individual treatments are known to those skilled in the art. With respect to the methods provided herein, the preparations of the present invention may be administered by any means that achieves the intended purpose or is deemed appropriate by those skilled in the art. In exemplary embodiments, the modified toxin preparation is administered as a single dose or, where appropriate, as a series of doses, for example, using a minipump system. In some cases, the modified toxin preparation is provided as a liquid dosage form or as a lyophilized dosage form that is reconstituted, for example, before administration.

[0045] As used herein, the term "protected" refers to the induction of immunity in a patient against a disease or condition. Immune induction can occur by administering a vaccine containing an antigen. In particular, in this invention, an immune patient is protected from tetanus disease or its symptoms. In one embodiment, a suitable dose is approximately 1 μg to 20 μg. Preferred 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 drug in this technology are, for example, LD 50 (A lethal dose in 50% of the population) and ED 50 The therapeutic dose (the dose effective in 50% of the population) can be determined by cell culture or standard pharmaceutical methods in test animals. The dose ratio between toxic effect and therapeutic effect is the therapeutic factor, which is the LD50. 50 / ED 50 It can be expressed as a ratio.

[0047] As used herein, “therapeutically effective amount” means an amount of the compound sufficient to treat a disease when administered to a subject for that purpose. “Therapeutically effective amount” varies depending on the compound, the condition being treated, the severity of the disease being treated, the age and relative health status of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors. For the purposes of this invention, “to treat” or “to cure” means the management and care of a patient to combat a disease, condition, or disorder. These terms encompass both preventive therapies, i.e., prophylactic and reactive therapies.

[0048] The conjugate delivery platforms described herein can be adapted to target specific pathological conditions or diseases by adjusting which conjugate modified Tet toxin platform is used.

[0049] method In another aspect of this specification, methods are provided for manipulating vaccines and conjugate vaccines with reduced toxicity and enhanced efficacy. These methods involve genetically modifying specific amino acid residues within the domain of a multi-domain protein toxin so that the toxin is unable to express toxicity either through catalytic activity or receptor binding activity against target cells (e.g., neurons), is defective in its transfer, and has a reduced likelihood of reverting to a toxic form. By modifying multiple independent protein functions, the methods provided herein advantageously provide full-length toxins that are ideal candidates for potent vaccines and conjugate vaccines, with substantially no possibility of reverting the protein to toxicity. As used herein, the term “potency” refers to the specific protective immune performance or performance of a vaccine as indicated by appropriate clinical tests or well-controlled clinical data. In other words, potency is a measure of the strength of a vaccine.

[0050] In some cases, a method for obtaining an engineered bacterial protein toxoid with enhanced potency as a vaccine comprises the following steps: selecting one or more amino acids in each domain of an amino acid sequence encoding a multi-domain bacterial protein toxin, wherein each position is selected to inactivate the protein function associated with each domain, and these domains include two or more of a catalytic domain, a translocation domain, a receptor binding domain, and a substrate binding domain; substituting the 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 a nucleic acid sequence encoding a full-length bacterial protein toxin containing the substituted non-natural amino acid residues in a host cell, 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 containing natural amino acids, or consists essentially of or is derived from them.

[0051] The selection of amino acid residues for modification involves analysis of protein sequence (e.g., primary amino acid sequence) or structural information for identifying individual functional amino acid residues in each domain of the bacterial protein toxin. Preferably, the residues selected are one or more individual amino acid residues of 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. The stability of the protein may be evaluated by any suitable method. In some cases, the modified toxin is tested for stability by measuring trypsin sensitivity 3 and the modified toxin is tested for immunogenicity by measuring the immune response to inoculation with the modified toxin in a mouse model 4 .

[0052] Protein structure information may be obtained by any suitable method, such as X-ray crystallography, electron microscopy, nuclear magnetic resonance imaging, computerized protein structure modeling, or a combination thereof. In some cases, the crystal structure of the bacterial protein toxin of interest can be used to identify, for example, specific sites of interaction between the functional domains of the toxin and functionally conserved residues 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 the alignment of amino acid sequences in 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 E. coli, the protein is recombinantly produced, and tested for loss of toxicity, maintenance of stability, and retention of immunogenicity.

[0053] For example, interdomain and intradomain molecular interactions can be determined from the analysis of the crystal structure of wild-type bacterial protein toxins. Substitutional mutations can be achieved using well-known methods such as site-directed mutagenesis, PCR-mediated mutagenesis, whole-gene synthesis, and other methods known in the art. Exemplary methods of mutagenesis protocols are shown, for example, in the following examples. In some cases, site-directed mutagenesis is used to induce mutations at a single amino acid residue. 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. The 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, the polynucleotide encoding the modified protein toxin is located within the expression vector. In some cases, this vector is located within a host cell (e.g., bacterial cell, yeast cell, eukaryotic cell). Many expression vectors and systems are known in both prokaryotes and eukaryotes, and the selection of an appropriate system depends on the intention. Expression and purification of the modified protein product of the present invention can be easily performed by those skilled 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 exotoxins, which are multi-domain proteins secreted by bacteria that are often similar to enzymes in that they act catalytically and exhibit substrate specificity. Examples of bacterial protein toxins include, but are not limited to, botulinum toxin, tetanus toxin, Shiga toxin, diphtheria toxin, Bordetella pertussis toxin, Escherichia coli thermolabile toxin LT, Bacillus anthracis toxin, Pseudomonas exotoxin A, lethal anthrax factor (LF), cholera enterotoxin, and Staphylococcus aureus exfoliatin B. Table 4 shows exemplary bacterial protein toxins for which crystalline structure information is available for use in the design of genetically engineered recombinant, non-toxic, high-potency toxoids that are particularly advantageous for vaccines and conjugate vaccines.

[0055] [Table 4]

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

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

[0058] In certain embodiments, genetic modifications are made to the C-terminal portion of the heavy chain (HC) to inhibit the host receptor activity.C To inhibit catalytic activity, genetic modifications are introduced to amino acid residues in the light chain that are necessary for or important for the release of neurotransmitters via the SNARE complex.

[0059] The techniques and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art, for example, by conventional methods such as the widely used molecular cloning methods described in Ausubel et al., *Current Protocols in Molecular Biology*, Wiley Interscience Publishers, (1995). Procedures, including the use of commercially available kits and reagents as needed, are generally carried out according to the protocols and / or parameters specified by the manufacturer, unless otherwise noted.

[0060] kit In another aspect of this specification, a kit is provided for administering to a subject a vaccine or adjuvant containing a modified toxin vaccine as described herein. In one embodiment, the kit includes a form of the modified toxin (e.g., a modified tetanus toxin as described herein). The kit may further include instructions enabling a user to carry out a method of vaccinating a subject against the development of diseases caused by tetanus, in particular diseases caused by tetanus toxin. In one embodiment, the modified toxin of the present invention is prepared, delivered, and stored for use under physiological conditions. Suitable pharmaceutical carriers include, but are not limited to, physiological saline (e.g., 0.9% sodium chloride), phosphate-buffered saline, and lactated Ringer's solution.

[0061] By “Instructions for Use,” the inventors mean any publication, record, figure, or other medium of expression used to convey the usefulness of the present invention for one of the purposes set forth herein. The kit's explanatory materials may, for example, be attached to the container containing the present invention, or be transported together with the container containing the present invention. Alternatively, the explanatory materials may be transported separately from the container, or may be provided in an electronically accessible form on an internet website with the intention that the explanatory materials and the biocompatible hydrogel will be used collaboratively by the recipient.

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

[0063] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural nouns unless otherwise explicitly stated. Similarly, the terms “a” (or “an”), “one or more,” and “at least one” are interchangeable herein. It should also be noted that the terms “comprising,” “including,” “characterized by,” and “having” are interchangeable.

[0064] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. All publications and patents specifically mentioned herein, including descriptions and disclosures of chemicals, apparatus, statistical analyses, and methodologies reported in those publications, which may be used in connection with this invention, are incorporated herein by reference in their entirety as part of this specification. All references cited herein are considered to represent the state of the art in the art. This specification should not be construed as acknowledging that this invention has no prior rights to such disclosures due to prior art.

[0065] The present invention will be better understood by considering the following non-limiting examples. [Examples]

[0066] Example 1: M-BoNT / A1 and M-BoNT / A1 W Isolation and characterization of This embodiment involves 10 full-length BoNTs that have been manipulated to have defects in catalytic activity and acceptor binding. 6 LD 50 This explains the protection from antigenic stimulation by natural 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 institute's Biosafety Committee. In addition, the experiments were conducted in a laboratory approved by the Federal Select Agent Program for this research, by researchers who had undergone suitability assessments, and in compliance with institute policies and procedures. Animal experiments were approved and conducted in accordance with the guidelines of the University of Wisconsin-Madison's Animal Experimentation Committee. The U.S. Department of Health and Human Services states that the BoNT / A gene and protein products encoding three LC mutations (E224A / R363A / Y366F, designated as M) do not meet the regulatory definition of a Select Agent, and that production of M-BoNT / A is possible without Select Agent registration (§73.3 HHS Select 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 using standard toxin purification protocols (Jacobson et al., 2011; Lin et al., 2010; Malizio et al., 2000; Tepp et al., 2012). BoNT / A6 was obtained using CDC41370 B2tox -The toxin was purified using a previously described method (Pellett et al., 2016) from a modified strain of CDC41370 to produce only BoNT / A6. Toxin purity was confirmed by spectroscopy and SDS-PAGE analysis (Whitemarsh et al., 2013). The purified toxin was stored at -20°C in phosphate-buffered saline containing 40% glycerol until use. The activity of the five subtype preparations was determined using standard intraperitoneal mouse bioassay (MBA) methods as previously described (Hatheway, 1988; Schantz, 1978). The median lethal dose of each toxin was defined as 50 units (U) of 1 mouse LD50. 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 derivatives: HC C / A1(W1266A)(HC C / A1 W ), LC / A1(R363A / Y366F)(LC / A1 RY ), LCHC N / A1(E224A / R363A / Y366F)(M-LCHC N / A1), BoNT / A1(E224A / R363A / Y366F)(M-BoNT / A1), BoNT / A1(E224A / R363A / Y366F / W1266A)(M-BoNT / A1 W ) and non-catalytic tetanus toxin (R372A / Y375F) (TeNT RYThe production of ) was carried out 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 cultured at 37°C with shaking for 3-6 hours until the OD600 was approximately 0.6 when 1.0 mM IPTG was added, and then incubated overnight at 16°C with shaking. The 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). The cells were disrupted with a French press, clarified by centrifugation, and filtered through a 0.45 μm surfactant-free cellulose acetate membrane (Thermo Fischer). The lysate was Ni 2+ The protein was further purified by tandem gravity flow chromatography using NTA resin (Qiagen), p-aminobenzamidine-agarose (Sigma), and Strep-tactin Superflow high-capacity resin (IBA-LifeSciences). The purified protein was dialyzed against 10 mM Tris (pH 7.9), 200 mM NaCl, and 40% glycerol, and stored at -20°C. The recombinant protein used in this study is shown (Figure 1). M-BoNT / A1 W The nucleotide sequence encoding this is shown as Sequence ID 4 (see Figure 9).

[0070] Vaccination: Female ICR mice (18-22g) were given the indicated concentration of HCl mixed with an equal amount of HCl hydrogel as an adjuvant. C / A1 W M-LCHC N / A1, M-BoNT / A1, or M-BoNT / A1 W Intraperitoneal immunization was performed using non-trypsin-treated M-BoNT / A1 and M-BoNT / A1. WThe vaccine was used. The vaccine was administered on days 1 and 14, blood was collected by maxillary blood sampling on day 21, and mice were stimulated with antigens using BoNT / A1, BoNT / A2, or BoNT- / A2, / A3, / A5,A6 cocktails on day 26 as shown. As shown, at least 8 mice were used in each group for each experiment. The statistical appropriateness of the results was evaluated by paired Student's two-tailed t-test at p=0.05.

[0071] ELISA: BoNT derivative 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. Next, the plate was washed three times with 0.3 ml of phosphate-buffered saline (PBS) containing 0.05% Tween20, and blocked at room temperature (RT) for 30 minutes with 0.2 ml of PBS containing 1% (wt / vol) bovine serum albumin (BSA). The plate was incubated at RT for 1 hour in PBS containing 1% (wt / vol) BSA (0.1 ml) with serum from individually vaccinated mice at either the indicated dilution ratio of 1:20,000 or 1:30,000. After washing three times with 0.3 ml of PBS containing 0.05% Tween20, the plates were incubated at RT for 1 hour in PBS containing 1% (wt / vol) BSA with goat α-mouse IgG-horseradish peroxidase (IgG-HRP diluted 1:20,000; Thermo). After washing three times with 0.3 ml of PBS containing 0.05% Tween20, 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. A control ELISA measuring antigens bound to α-HA and α-FLAG antibodies showed that the presence of appropriate epitopes within each antigen was within 15% (data not shown). For ELISA, P<0.05 was observed for serogroups (N=10) individually analyzed based on immune induction and / or antigen stimulation conditions.* , 0.01 = ** , 0.001 = *** , and 0.0001 = **** Statistical analysis was performed using unpaired Student's two-tailed t-tests in GraphPad Prism 7. Individual serum samples were analyzed by at least two independent ELISAs with two replicates. Analysis of mouse serum at 1:20,000–1:30,000 dilutions was based on the evaluation of several individual serum samples. Serum dilutions were determined from dose-response ELISAs of several M-BoNT / A1-inoculated mice that survived BoNT / A1 antigen stimulation. Representative ELISAs are shown in Figure 6.

[0072] Cell-based assay for detection of neutralizing antibodies: A cell-based neutralization assay was 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 in 96-well TPP plates coated with poly-L-ornithine and Matrigel® (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 in culture medium with a serial dilution of filtered and decontaminated serum and incubated at 37°C for 1 hour. Serum-free BoNT / A1 was used as an "antibody-free" reference, and serum from naive mice was used as a control. Toxin-free serum was used as a negative control. 50 μl of each antibody-toxin mixture was added to each well of hiPSC-derived neurons in at least two replicates, 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). The cell lysates were analyzed by Western blotting for SNAP-25 cleavage as previously described (Pellett et al., 2007; Pellett et al., 2010). Images were obtained using PhosphaGlo reagent (KPL, Gaithersburg, MD) and the Fotodyne / FOTO / Analyst FX imaging system (Harland, WI). Cleavage (24 kDa) and uncleavage (25 kDa) SNAP-25 signals were analyzed by concentration meter using TotalLab Quant software (Fotodyne, Harland, WI). The percentage of protection was determined by comparing it to an "antibody-free" control, and IC 50 The values ​​were evaluated using GraphPad Prism 6 software, nonlinear regression, variation gradient, and four parameters.

[0073] result M-BoNT / A1 is non-toxic to uninbred mice or cultured cells. Intraperitoneal injection of 10 μg of trypsin-treated or non-trypsin-treated M-BoNT / A1 / mice (ICR) did not produce observable signs of botulism, indicating that M-BoNT / A1 was at most toxic to one part in a million of natural BoNT / A1. In addition, incubation of human iPSC-derived neurons with 80 nM M-BoNT / A1 did not result in detectable SNAP-25 cleavage, while incubation with 50 fM natural BoNT / A1 did cleave SNAP-25, which, according to cell-based assays, is at most toxic to one part in a million (data not shown).

[0074] M-BoNT / A1 and M-BoNT / A1 W is HCc / A1 W It is a more protective vaccine. Vaccine administration was performed on non-inbred ICR mice (n=8-10) using primary immunization and a subsequent booster immunization to represent innate immune changes within the host (Rai et al., 2009). Previous studies have shown that HC C / A1(W1266A)(HC C / A1 W ) is a mouse model of botulism in HC C Since it showed vaccine efficacy equivalent to / A1 (Przedpelski et al., 2013), M-BoNT / A1 W The following was also performed: 0.3 μg / mouse single-stranded M-BoNT / A1 W Alternatively, 0.2 μg / mouse M-LCHC N Mice inoculated with / A1 showed 10 6 LD 50 Natural BoNT / A1 or 10 5 LD 50 BoNT / A subtype cocktails (A2, A3, A5, A6, 2.5 each x 10) 4 LD 50 ) is protected from antigen stimulation, 10 6 LD 50It was partially protected from antigenic stimulation by the heterologous subtype, native BoNT / A2 (Table 4). 0.1 μg / mouse HC C / A1 W Mice that were inoculated with 10 3 LD 50 These data were partially protected from antigenic stimulation by natural BoNT / A1 or natural BoNT / A2. W Vaccines and M-LCHC N / A1 vaccine is HC C / A1 W This shows that it provided 1,000 times more protection against toxins than the vaccine. In a comparison of vaccines using body weight equivalent doses, 0.3 μg / mouse HC C / A1 W Mice that were inoculated with 10 5 LD 50 Natural BoNT / A1 or 10 5 LD 50 It was partially protected from antigenic stimulation by the natural BoNT / A subtype cocktail. This was due to the use of an equivalent concentration (and a 3x molar excess of HC). C Even if it is M-BoNT / A1 W Vaccines and M-LCHC N The M-BoNT / A1 vaccine demonstrated better protection against allogeneic and heterogeneous BoNT / A antigen stimulation. W No difference was observed in protection between the vaccine and the M-BoNT / A1 vaccine, indicating that additional "receptor-binding" mutations do not affect vaccine efficacy. Overall, M-BoNT / A1, M-BoNT / A1 W and M-LCHC N / A1 is HC C / A1 W It was a more potent vaccine. The duration of action in cultured neurons was further investigated in human iPSC-derived neurons by exposing the neurons to either BoNT / A1 or BoNT / A6 serial dilution for 72 hours, completely removing the extracellular toxin, and then further incubating them in culture medium. Cells were collected in three replicates with each dilution system on days 3, 39, and 70, and EC against SNAP-25 cleavage at each time point was measured. 50This decision was made. In 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). In BoNT / A1, EC 50 The values ​​were approximately 0.7, 6.3, and 28 U / 50 μl / well (313, 2940, and 12,880 fM, respectively) at days 3, 39, and 70 (Figure 2). The half-lives of BoNT / A1 and / A6 in these hiPSC-derived neurons were EC 50 When determined over time from the values, the duration of action was similar for both BoNT / A1 and BoNT / A6, approximately 12 and 14 days, respectively (Figure 2). Considering these results together, BoNT / A6 has a longer duration of action, similar to other BoNT / A subtypes.

[0075] Antibody responses to BoNT inoculation varied qualitatively and quantitatively in uninbred mice. M-BoNT / A1 W And M-BoNT / A1 vaccination is 10 5 LD 50 Complete protection against antigen stimulation by BoNT / A2, and 10 6 LD 50 This provided partial protection against antigen stimulation by the heterologous subtype, natural BoNT / A2 (Table 5). Antibody responses of vaccinated mice, analyzed by ELISA, showed that both mice that survived natural BoNT / A2 antigen stimulation and those that did not survive were resistant to BoNT and LCHC. N They showed equivalent dominant antibody titers against each other, and there was no statistically significant difference between them (Figure 2). Therefore, partial protection from natural BoNT / A2 antigen stimulation is likely due to specific differences in the composition of neutralizing epitopes between BoNT / A subtypes, rather than the ability of vaccinated mice to elicit an immune response to the delivered vaccine. [Table 5]

[0076] Antibody response in vaccinated mice is LCA1RY , HC C / A1 W , M-LCHC N / A1, or M-BoNT / A1 holotoxin was used as a binding substrate and analyzed by ELISA for each individual serum. The antibody responses within each group of vaccinated mice varied quantitatively and qualitatively within the group. M-BoNT / A1 W -vaccinated mice (Figure 3, lower left) showed dominant antibody titers against BoNT (mean titer 2.2 (range 1.3 - 2.6)) and LCHC N (mean titer 1.7 (range 0.8 - 2.4)). The titers against HC C varied among mice (mean titer 0.41 (range 0.07 - 1.83)). The titers against LC did not exceed the control, indicating that most of the antibody responses were against HC. The variation in the titer range was due to the variation in antibody titers among individual mice, not due to the variation in ELISA replicates. A similar immune response was seen against M-BoNT / A1 inoculation (data not shown). M-LCHC N / A1-vaccinated mice (Figure 3, upper left) also showed dominant antibody titers against BoNT and LCHC N , but had lower titers on average than M-BoNT / A1 W -vaccinated mice. M-LCHC N / A1 + HC C / A1 W -vaccinated mice showed qualitatively similar antibody titer characteristics to M-BoNT / A1 W -vaccinated mice, but quantitatively, they were comparable to mice vaccinated with M-LCHC N / A1 alone (Figure 7). HC C / A1 W -vaccinated mice showed antibody titers correlated with survival against BoNT / A1 antigen stimulation (Figure 3, lower right). M-BoNT / A1 C -vaccinated mice had limited antibody titers against TeNT W (Figure 3), indicating that the observed antibody responses were BoNT-specific.

[0077] ​​Characteristics of serum derived from individual vaccinated mice that survived BoNT antigen stimulation M-BoNT / A1 that survived stimulation with natural BoNT / A1 antigen W M-LCHC N / A1, or HC C / A1 W Analysis of individual serums from inoculated mice revealed several representative immune responses to vaccination (Figure 4). In our earlier research (Przedpelski et al., 2013 Infect Immun. 81(7):2638-44), M-LCHC N Since the antibody response to / A1 inoculation was not characterized, the LCHC of the three individuals was not determined. N Serum from mice inoculated with / A1 was analyzed. ELISA results showed M-BoNT / A1 W Inoculated mice were BoNT and LCHC N (#7) or BoNT, LCHC N , and HC C It demonstrated a dominant antibody titer against (mouse #3). M-LCHC N / A1-vaccinated mice were BoNT and LCHC N They showed a dominant antibody response to (mouse #21, #24, and #25), while HC C / A1 W Mice that were inoculated with and survived BoNT / A1 antigen stimulation were classified as HC. C A dominant antibody response was observed against (mouse #78). Overall, TeNT RY The antibody response to was low, indicating that the immunoreactivity detected by ELISA was BoNT-specific.

[0078] BoNT / A and HC C The vaccine is LCHC N It elicits a stronger neutralizing antibody response than vaccines. Neutralizing antibodies in the serum of vaccinated mice were measured by a cell line assay using hiPSC-derived neurons. Ten serum samples were pooled from each vaccination group, each administered an equimolar dose of the vaccine, and their ability to neutralize BoNT / A1-induced SNAP25 cleavage was tested in the cell line assay. M-BoNT / A1 W The vaccination pool is IC 50 With a value of 0.004, it has the highest neutralizing efficacy, and this is HC C / A1 W Inoculation pool and M-LCHC N / A1+HC C / A1 W In approximately half of the vaccination pool, M-LCHC N This was about one-fifth of the / A1 vaccination pool (data not shown). C The similarity in neutralizing antibody titers between inoculated mice and M-BoNT / A1-inoculated mice was remarkable, considering that the M-BoNT / A1 vaccine protected mice from toxic stimulation more than 1,000 times greater than that of the HCc vaccine. To further investigate this, the ability of each of the six serum samples to neutralize SNAP25 cleavage by BoNT / A1 was also determined using cell line assays (Figure 5). Overall, each of the six serum samples neutralized BoNT / A1 activity, with a difference of approximately 10-fold in serum potency. C HC containing a dominant antibody response to C / A1 W Inoculation (mouse #78) and M-BoNT / A1 W Serum from inoculated mouse #3 (Figure 4) was the most effective inhibitor of SNAP-25 BoNT / A1 cleavage. Detectable HC C Serums without an antibody response (mouse #7, #21, #24, and #25) were less effective in inhibiting SNAP-25 cleavage. Therefore, in this assay, a vaccine containing the HCc epitope was effective against LCHC. N It elicited a stronger "neutralizing / inhibitory antibody" response than the vaccine. Considering these data together, BoNT / A1 HC C The domain is HC N It elicits a stronger neutralizing / blocking antibody response than the domain or LC domain, but this HC NThis demonstrates that the domain, and possibly the LC domain, plays a major role in biological defense.

[0079] Consideration In a non-inbred mouse model of botulism, M-BoNT / A1, M-BoNT / A1 W and M-LCHC N / A1 is HC C / A1 W It was a vaccine that was as effective as possible. Evaluation of serum from vaccinated mice that survived BoNT / A1 antigen stimulation was performed using LCHC. N LCHC consistent with the presence of neutralizing epitopes within it N They showed a common response to M-BoNT / A1. M-BoNT / A1 elicits a similar protective immune response against M-BoNT / A1. W The ability demonstrated that reduced host cell binding did not adversely affect vaccine efficacy. Therefore, full-length BoNTs engineered to have defects in both the catalytic and receptor-binding domains represent a novel platform strategy for the development of vaccines against botulism and other toxin-mediated diseases. Collier and collaborators (Killeen et al., 1992) showed that a mutation in the second site partially restoring genetically inactivated diphtheria toxin could be created as a trial for vaccine development. In addition, recent research by Smith and collaborators has indicated the need for attenuation of BoNT-based vaccines beyond mere reduction of catalytic activity against several serotypes (Webb et al., 2017).

[0080] Early research showed that LCHC N It was considered a candidate for the BoNT vaccine (Shone et al., 2009). LCHC N It is produced in large quantities by E. coli through fermentation, and low-dose BoNT antigen stimulation (10 3 LD 50 It was effective as a single-dose vaccine against LCHC (BoNT). In this report, the inventors directly compared it with other BoNT vaccine candidates using primary immunization and one booster immunization. N It was recognized as an effective vaccine, LCHCN The presence of a neutralizing epitope within the molecule was confirmed (Shone et al., 2009). Dolly and collaborators identified an LC-specific monoclonal antibody (Mab) that inhibited BoNT / A action (Cenci Di Bello et al., 1994), while Marks and collaborators identified a BoNT / A neutralizing mAb with LC function that inhibited SNARE cleavage (Cheng et al., 2009) and HC. N We identified several mAbs that targeted BoNT serotypes and neutralized them (Garcia-Rodriguez et al., 2011). Considering these studies together, BoNT inoculation is linked to LCHC N It has been shown to induce the production of antibodies against neutralizing epitopes within the domain. M-BoNT / A1 W M-LCHC N Since it elicited a greater antibody response than / A1 (Figure 7), HC C This decision was made in conjunction with the finding that M-BoNT / A1 produced the antibody with the greatest neutralizing / blocking ability in cultured cells. W HC C Vaccines containing LCHC N or HC C It appears to offer greater protection under "high-dose" exposure than vaccine derivatives.

[0081] HC C , vaccines against botulism using DNA vectors and viral vectors, and HC C This domain is well-established for developing protein-based vaccines constructed in early studies, demonstrating neutralizing efficacy (Clayton et al., 1995) and ease of production (Baldwin et al., 2008). Smith and collaborators developed HC in the yeast Pichia pastoris. C This expresses the protective immunity induced by HC(c) (Byrne and Smith, 2000), and then recombinant HC C / A and HC CA bivalent vaccine consisting of / B(rBV A / B) has been reported, and this vaccine is currently in clinical trials (Webb and Smith, 2013). Escherichia coli also has seven serotypes (A-G) of HC in response to BoNT antigen stimulation. C It is used as a heterologous host for BoNT vaccine development, including vaccine production (Baldwin et al., 2008). To enhance vaccine efficacy, HC C A mutation is introduced that blocks host receptor binding, and here HC C W It retained vaccine efficacy (Przedpelski et al., 2013). Ease of production is HC C While this makes it an attractive vaccine platform, current research is focused on HC. C / A1 W M-BoNT / A1 W This indicates that the vaccine was more potent. This is related to BoNT's LCHC N Using human serum derived from BoNT-resistant cervical dystonia patients whose immunogenicity is consistent with that of LC and HC, N This is supported by the findings of Atassi and his collaborators who detected immunoepitopes within the cells (Atassi et al., 2011; Dolimbek et al., 2007).

[0082] In a recent study by Smith and collaborators (Webb et al., 2017), catalytically inactive BoNT was found to be effective after 1000 LD after a single inoculation. 50 HC in response to antigen stimulation by toxin administration C They reported that it showed greater efficacy than the individual HC. The antigen stimulation test described by Smith et al. measured threshold toxin stimulation, which differs from current tests that measure protection against endpoint toxin stimulation. This data shows that, in both cases measuring protection against toxin stimulation, whether threshold or endpoint, the full-length BoNT vaccine showed greater efficacy than the individual HC. C It was more potent than the subunit. M-BoNT / A1 has defects in catalytic activity and host receptor binding. WIn endpoint toxin stimulation, it was more effective than the subunit. By inactivating multiple functional sites to reduce potential toxicity due to cell binding or entry, M-BoNT / A1 as a vaccine candidate W Its usefulness addresses the concern that genetic inactivation of catalytic function alone does not provide sufficient safety margin for developing a full-length BoNT vaccine (Webb et al., 2017).

[0083] HC as a vaccine candidate against botulism C Although its usefulness has been established (Baldwin et al., 2008; Henderson, 2006), current research suggests that multi-domain derivatives of BoNT are superior to HC C This indicates that it is a more powerful vaccine than M-BoNT / A1. W In non-inbred mice, LCHC N A common dominant antibody response was induced against HC, C Antibody responses varied. The ability to reduce both catalytic activity and receptor binding suggests that M-BoNT / A1 could be a vaccine platform against botulism. W This supports the use of the BoNT / A subtype cocktail. Protection from the BoNT / A subtype cocktail ensures the broad neutralizing ability of this vaccine. M-BoNT / A1 was used as the vaccine in this study. W Although it was not processed into an activated double-stranded form and no toxicity was detected in mice or cells, this does not mean that single-stranded M-BoNT / A1 is a safe and effective vaccine. W This suggests that...

[0084] Example 2 - Characterization of impaired light chain translocation in the lysine 768 TeNT mutant This section describes the first identification and characterization of a single amino acid point mutation within the transition domain of TT that blocked light chain (LC) translocation. Identifying the role of K768 in LC translocation provides for the first time an opportunity to inactivate the independent activity (see Table 6), catalytic activity, translocation, and receptor binding of TT, and similarly, BT, for recombinant vaccine development.

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

[0086] Independent mutations in botulinum toxin (BT) (see Table 6) and TT vaccine candidates inactivate each of the three functions of the toxin: catalytic action, translocation, and receptor binding, thereby increasing vaccine safety. Multiple independent mutations exponentially reduce toxin potency, enhancing vaccine safety without impairing protein structure and possibly immunogenicity, and reducing the possibility of reversion during large-scale production. [Table 6]

[0087] Example 3 - Modified 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 (transition and receptor-binding heavy chain, HC). Zn of TT ++ Mutations in two amino acids within the binding pocket (R372A, Y375F) are Zn ++ This inhibited binding, resulting in 2M-TT, which reduced toxicity to 1 / 125,000th of that of natural 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 bond inhibition, E234 is used with Zn ++ An additional mutation (E234Q) was added based on stabilizing H233, which directly coordinates to binding. Next, 5M-TT was created by inhibiting neuron binding to dual ganglioside receptor binding by generating two independent mutations (R1226L, W1289A) (see Table 7). As proof of principle, the inventors 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 of approximately 6 mg / liter. Four mice injected with 20 μg of single-stranded or double-stranded 6M-TT, respectively, did not show symptoms of tetanus. Therefore, 6M-TT is a soluble, well-expressed, non-toxic protein. [Table 7]

[0088] 6M-TT was further manipulated to sequentially inhibit VAMP-2 binding and cleavage by introducing mutations at the Y26 and L231 positions, resulting in the creation of 7M-TT and 8M-TT. Based on earlier studies showing that the L231 mutation reduced kcat without affecting VAMP-2 affinity, and that the L231K mutation did not affect the overall structure of LC, the L231K mutation was chosen.9 We selected this because Y26 was located in the S7 pocket of HCR / T and the Y26A mutation reduced the affinity of LC / T to VAMP-2. 9 The selection was based on the following evidence (see Table 7). For 8M-TT and its intermediate products (6M-TT and 7M-TT), circular dichroism was examined to assess the secondary structure, trypsin sensitivity was examined to assess the overall stability of the protein, and mass spectrometry was performed to assess the protein composition. 42 . The inventors have found that 6M-TT is produced as a soluble protein at this stage, is highly expressed, and is non-toxic when injected at 20 μg in mice. Therefore, the toxicity of 6M-TT, 7M-TT, and 8M-TT will be analyzed by examining VAMP-2 cleavage after cell entry and VAMP-2 cleavage in the cell lysate. 64 Evaluation is performed using an assay based on human nerve cells. If no transection or cytotoxicity is detected, the absence of in vivo toxicity is confirmed in a mouse model using non-inbred female ICR mice (18-22g, 5 mice / group) (Table 8). [Table 8]

[0089] In the initial test, 20, 50, 250, or 1000 μg of 8M-TT (1000 μg is approximately 4 × 10) was administered intraperitoneally to the mice. 7 LD 50 Inject (equivalent to wild-type tetanus toxin). 65 Injected mice will be scored for 3 days using mouse bioassay and observed for up to 14 days for symptoms indicating TT pathology, including weight gain, signs of stress, organ damage, and tetanus symptoms. Male mice will also be tested for sex differences.

[0090] Non-inbred female ICR mice (8 individuals / 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 vaccine on day 14 (Table 9). Blood was collected from the mice on day 26, and the mice were vaccinated on day 30. 3 ~106 Antigen stimulation is performed with tetanus toxin U. To examine long-term protection, vaccinated mice are maintained for 180 days and stimulated with antigen TT to test the duration of the immune response. Mice that survive for 3 days are evaluated as protected. Serum obtained before antigen stimulation is used as previously described. 6 Regarding anti-TT properties and inhibition of VAMP-2 cleavage, ELISA was used. 71-72 We will test the neutralizing efficacy of cultured neurons against tetanus toxin poisoning. Male mice will also be tested to confirm that there are no sex differences. [Table 9]

[0091] The inventors expect that, since each introduced independent LC point mutation inhibits an independent step in the catalytic process, these LC mutations will show a ploidy-like decrease in catalytic activity, and reflect this, a decrease in the toxic efficacy of M-TT. Based on earlier studies on their effects as individual mutations in LC-TT or HC-TT, the inventors do not believe that these LC or HC mutations affect the stability or immunogenicity of the protein. Injection of 1000 μg of 8M-TT did not appear toxic in the inventors' mouse model, and 8M-TT appears to have a higher neutralizing immune response than chemically inactivated TT, allowing for low-dose immune induction with 8M-TT.

[0092] Recent reviews have assessed that only a portion of the potential immune-inducing efficacy of current conjugate vaccines has been achieved. 4 In the study of microbial pathogens, further immunogens requiring conjugation with protein toxoids to produce an effective T cell-dependent immune response continue to be identified. These include, but are not limited to, Neisseria meningitidis. 15 ,fungi 16 , and pneumococcus 17-18 It contains the capsule. In addition, synthetic glycans are 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 Based on our knowledge of the structural-functional properties of tetanus toxin (TT), our basic information on tetanus toxin as a chemically inactivated toxoid, and the continuing global demand for tetanus vaccination, tetanus toxin is among the best candidates for the development of these next-generation recombinant conjugate vaccines. The production of a safe, easy-to-produce, and protective recombinant TT vaccine will, for the first time, enable the analysis of full-length, non-toxic recombinant TT as a conjugate vaccine carrier. Currently, there is no knowledge regarding the protective properties of conjugate vaccine carriers, including tetanus toxoids. 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 an antigen when M-TT is conjugated to chemically inactivated TT.

[0093] The protocol for conjugating oligosaccharides to M-TT is a publicly available protocol from the Lees laboratory. 74 The procedure follows this. Briefly, polysaccharides (PS) are reduced to a molecular weight of 100-300 kDa using an LV-1 microfluidizer. PS is prepared in water at 5 mg / ml and activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP, 0.5 mg / mg). 74Add an equal mass of protein (5 mg / ml) and maintain the solution at pH 9. Monitor the reaction by size exclusion chromatography (SEC) HPLC and quench with excess glycine. Purify the conjugate by SEC and determine the molecular weight by SEC multi-angle light scattering. Crosslink the certified polysaccharides and peptides with 8M-TT: (i) Group B Streptococcus (GBS) polysaccharide serotypes Ia, Ib, II, III, IV and IV; (2) Poly-β-(1-6)-N-acetylglucosamine (PNAG) which mediates biofilm formation as a candidate broad-spectrum vaccine for Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomonas maltophilia, and Burkholderia cepacia complex (BCC). 45 (3) A peptide currently being tested in influenza vaccines.

[0094] Non-inbred female ICR mice (8 individuals / 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 vaccine on day 14 (Table 10). Blood was collected from the mice on day 26, and the mice were vaccinated on day 30. 3 ~10 6 Antigen stimulation is performed with tetanus toxin U. To examine long-term protection, vaccinated mice are maintained for 180 days and stimulated with antigen TT to test the duration of the immune response. Mice that survive for 3 days are evaluated as protected. Serum obtained before antigen stimulation is used as previously described. 6 74 ELISA will be used to test for anti-conjugate and anti-TT properties. Male mice will also be tested for sex differences. [Table 10]

[0095] PNAG oligosaccharide, GBS polysaccharide, and influenza peptide are individually conjugated to the 8M-TT vaccine. In subsequent experiments, a multi-polysaccharide conjugated 8M-TT vaccine is created by combining GBS and PNAG. Next, a multi-peptide-polysaccharide conjugated 8M-TT vaccine is created by combining influenza peptide and PNAG. 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 evoke equivalent immune responses to their respective conjugates and stronger immune responses to TT than each conjugate chemically inactivated TT vaccine. The inventors also anticipate that the immune responses to the polysaccharides and peptides within the 8M-TT vaccine will evoke equivalent immune responses to those when the individual antigens are conjugated to 8M-TT. The inventors anticipate that the immune response to 8M-TT will correlate with protection against innate TT antigen stimulation.

[0097] References JPEG2026076161000012.jpg84155 JPEG2026076161000013.jpg235160 JPEG2026076161000014.jpg230158 JPEG2026076161000015.jpg231157 JPEG2026076161000016.jpg194153

[0098] It should be noted that the above description, accompanying drawings, and their descriptions are illustrative and not intended to limit the invention. Many subject matter and variations of the invention will be apparent to those skilled in the art and in view of this disclosure. All such subject matter and variations are within the scope of its intent. For example, while the invention has been described in relation to various exemplary embodiments outlined above, various alternatives, modifications, variations, improvements, and / or substantial equivalents, whether known, rare, or not currently anticipated, will be apparent to at least those with ordinary art. Various modifications can be made without departing from the spirit and scope of the invention. Thus, the invention encompasses all known or future-developed alternatives, modifications, variations, improvements, and / or substantial equivalents of these exemplary embodiments.

Claims

1. A modified tetanus toxin polypeptide having at least 95% identity with SEQ ID NO: 1, and comprising a sequence having mutations at positions R372 and Y375, and further comprising mutations at two or more positions selected from E334, K768, R1226, and W1289, with each position being numbered relative to SEQ ID NO: 1, wherein the toxicity and receptor binding are reduced compared to the toxicity and receptor binding of SEQ ID NO:

1.

2. The modified polypeptide according to claim 1, wherein amino acid R at position R372 is substituted with amino acid A, and amino acid Y at position Y375 is substituted with amino acid F.

3. The modified polypeptide according to claim 1, wherein the mutations include R372A, Y375F, E334Q, R1226L, and W1289A.

4. The modified polypeptide according to claim 3, coded by sequence number 4.

5. The modified polypeptide according to claim 1, wherein the mutations include R372A, Y375F, E334Q, K768A, R1226L, and W1289A.

6. The modified polypeptide according to claim 5, coded by sequence number 5.

7. The modified polypeptide according to claim 1, further comprising mutations at one or both positions L231 and Y26, each position being numbered relative to SEQ ID NO:

1.

8. The modified polypeptide according to claim 7, wherein mutations at one or both positions of L231 and Y26 include L231K and Y26A.

9. A modified polypeptide according to claim 7, coded by sequence number 6 or sequence number 7.

10. A modified polypeptide according to any one of claims 1 to 9, further comprising a covalently bonded sugar chain, wherein the polypeptide is a polypeptide-sugar chain conjugate.

11. A composition comprising a modified polypeptide according to any one of claims 1 to 10 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 according to any one of claims 1 to 10.

13. Use of the modified polypeptide as an adjuvant according to any one of claims 1 to 10.

14. Use of the modified polypeptide as a vaccine according to any one of claims 1 to 10.

15. A method for obtaining a modified bacterial protein toxoid with enhanced efficacy as a vaccine, Selecting one or more amino acid positions in each domain of an amino acid sequence encoding a multi-domain bacterial protein toxin, where each position is selected to inactivate the protein function associated with each domain, and the domain comprises two or more of the following: a catalytic domain, a transition domain, a receptor-binding domain, and a substrate-binding domain; Substituting a native amino acid residue at each selected position with a non-native amino acid residue, thereby inactivating one or more protein functions related to the domain in which the substitution occurs; and In a host cell, a nucleic acid sequence encoding a full-length bacterial protein toxin containing the substituted non-natural amino acid residue is expressed, thereby 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 bacterial protein toxin containing natural amino acids. Methods that include...

16. The method according to claim 15, wherein the selection includes identifying individual functional amino acid residues based on the primary sequence or structure of a bacterial protein toxin.

17. The method according to claim 16, wherein the structure is obtained using X-ray crystallography, electron microscopy, nuclear magnetic resonance spectroscopy, computer protein structure modeling, or a combination thereof.

18. The method according to claim 15, wherein the selection includes identifying individual amino acid residues that can be modified without destabilizing the full-length protein or without loss of immunogenicity.

19. The method according to claim 15, wherein the substitution includes site-directed mutagenesis.

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