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JP-2026076161-A - Modified Clostridium neurotoxin as a vaccine and conjugate vaccine platform

JP2026076161AJP 2026076161 AJP2026076161 AJP 2026076161AJP-2026076161-A

Abstract

[Problem] To provide 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. [Selection Diagram] Figure 1

Inventors

  • バルビエリ ジョセフ ティー
  • ジョンソン エリック エイ
  • ペレット サビーヌ
  • テップ ウィリアム エイチ
  • プルゼドペルスキ アマンダ

Assignees

  • ザ メディカル カレッジ オブ ウィスコンシン インク
  • ウィスコンシン アラムニ リサーチ ファンデーション

Dates

Publication Date
20260511
Application Date
20251224
Priority Date
20171215

Claims (19)

  1. A modified tetanus toxin polypeptide having at least 95% identity with SEQ ID NO: 1, and containing mutations at positions R372 and Y375, and further containing mutations at two or more positions selected from E334, K768, R1226, and W1289, with each position numbered relative to SEQ ID NO: 1, wherein the polypeptide exhibits reduced toxicity and receptor binding compared to 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 ID No. 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, encoded by sequence number 6 or sequence number 7.
  10. A modified polypeptide according to any one of claims 1 to 9, further comprising covalently bonded sugar chains, 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 a therapeutically effective amount of the modified polypeptide described in any one of claims 1 to 10 to the subject.
  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 native amino acid residues at selected positions with non-native amino acid residues, thereby inactivating one or more protein functions related to the domain; and expressing a nucleic acid sequence encoding a full-length bacterial protein toxin containing the substituted non-native amino acid residues in a host cell, thereby causing the expressed protein to exhibit 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 native 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, computerized 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.

Description

This application, which cross-references related applications , claims priority to U.S. Provisional Patent Application No. 62/599,444, filed December 15, 2017, and is incorporated herein by reference as if its entire contents were fully disclosed herein. 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. Botulinum neurotoxin (BoNT), the most toxic substance to humans, is a protein toxin produced by selected strains of Clostridium botulinum, Clostridium butyricum, and Clostridium baratii (Hill and Smith, 2013; Johnson and Montecucco, 2008). BoNT is synthesized as a 150 kDa double-chain protein consisting of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC) linked by disulfide bonds. The HC is further divided into an N-terminal domain ( H₁N₂ ) that assists in the translocation of LC into the cytosol and a C-terminal domain ( H₁C₂ ) that recognizes and binds to cell surface receptors of nerve cells (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). While experimental vaccines have been used to protect "at-risk" populations from botulism, 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. 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. 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, translocation, 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 covalently linked glycans, thereby becoming a polypeptide-glycan conjugate. The modified polypeptide may be encoded by SEQ ID NO: 2. In some cases, mutations may include R372A, Y375F, E334Q, K768A, R1226L, and W1289A. The modified polypeptide may be coded 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 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 coded by SEQ ID NO: 6 or SEQ ID NO: 7. In another embodiment, this specification provides compositions comprising a modified polypeptide as described herein and a pharmaceutically acceptable carrier. 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. The aforementioned and ot