Mutant botulinum neurotoxin serotype a polypeptide and uses thereof
AssigneeThomas Jefferson University
InventorsLance L. Simpson, Mohammed Elias
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Abstract
Modified polypeptides based on the botulinum neurotoxin A heavy chain containing the double mutation Trp-Tyr->Leu-Ser in the ganglioside binding motif Ser-X-Trp-Tyr do not bind polysialogangliosides and nerve endings. The polypeptides are useful in the preparation of nontoxic vaccines against the effects of C. botulinum infection. The modified polypeptides are also useful as vehicles for the transepithelial delivery of diagnostic and therapeutic entities, through formation of conjugates between the polypeptides and the diagnostic or therapeutic entities.
Claims54
A modified BoNT/A polypeptide comprising:
- an isolated BoNT/A heavy chain polypeptide,
- a fragment of a BoNT/A heavy chain polypeptide comprising at least about 20% of the molecular mass of said heavy chain polypeptide and containing the ganglioside binding motif Ser-X-Trp-Tyr, or
- a fusion polypeptide comprising said BoNT/A heavy chain polypeptide fragment,
- wherein the modified polypeptide contains the double mutation Trp-Tyr→Leu-Ser in said ganglioside binding motif.
A nucleic acid encoding a modified BoNT/A polypeptide comprising:
- an isolated BoNT/A heavy chain polypeptide,
- a fragment of a BoNT/A heavy chain polypeptide comprising at least about 20% of the molecular mass of said heavy chain polypeptide and containing the ganglioside binding motif Ser-X-Trp-Tyr, or
- a fusion polypeptide comprising said BoNT/A heavy chain polypeptide fragment,
- wherein the modified polypeptide contains the double mutation Trp-Tyr→Leu-Ser in said ganglioside binding motif.
A method of preparing a modified BoNT/A polypeptide comprising:
- an isolated BoNT/A heavy chain polypeptide,
- a fragment of a BoNT/A heavy chain polypeptide comprising at least about 20% of the molecular mass of said heavy chain polypeptide and containing the ganglioside binding motif Ser-X-Trp-Tyr, or
- a fusion polypeptide comprising said BoNT/A heavy chain polypeptide fragment,
- wherein the modified polypeptide contains the double mutation Trp-Tyr→Leu-Ser in said ganglioside binding motif;
- the method comprising culturing a transfected host cell with an expression vector comprising a nucleic acid sequence that encodes said modified BoNT/A polypeptide, under conditions wherein said modified BoNT/A polypeptide is expressed.
A method for delivery of a therapeutic or diagnostic entity across a non-keratinized epithelium of an animal comprising:
- contacting an epithelium of said animal with a conjugate comprising said therapeutic or diagnostic entity linked to a modified BoNT/A polypeptide, the modified BoNT/A polypeptide comprising:
- an isolated BoNT/A heavy chain polypeptide, or
- a fragment of a BoNT/A heavy chain polypeptide comprising at least about 20% of the molecular mass of said heavy chain polypeptide and containing the ganglioside binding motif Ser-X-Trp-Tyr,
- wherein the modified polypeptide contains the double mutation Trp-Tyr→Leu-Ser in said ganglioside binding motif;
- whereby the therapeutic or diagnostic is transported across the non-keratinized epithelium.
A method of inducing an immune response in a vertebrate against a selected antigen comprising:
- contacting an epithelium of said vertebrate with an effective amount of a conjugate comprising said antigen linked to a modified BoNT/A polypeptide, the modified BoNT/A polypeptide comprising:
- an isolated BoNT/A heavy chain polypeptide, or
- a fragment of a BoNT/A heavy chain polypeptide comprising at least about 20% of the molecular mass of said heavy chain polypeptide and containing the ganglioside binding motif Ser-X-Trp-Tyr,
- wherein the modified polypeptide contains the double mutation Trp-Tyr→Leu-Ser in said ganglioside binding motif;
- whereby an immune response to the antigen is induced in the vertebrate.
Dependencies are inferred from the claim text.