Determining and reducing immunoresistance to botulinum toxin therapy using botulinum toxin a peptides

Abstract

The present invention provides BoNT/A peptide compositions, tolerogizing compositions, BoNT/A immune response inducing compositions and antibody compositions, as well as methods of determining immunoresistance to botulinum toxin therapy in an individual, methods of preventing or reducing immunoresistance to botulinum toxin therapy in an individual, methods of vaccinating an individual against botulinum toxin, methods of preparing anti-BoNT/A antibodies, methods of treating botulinum toxicity in an individual and methods of reducing anti-botulinum toxin antibodies in an individual.

1 . A method of determining immunoresistance to botulinum toxin therapy in an individual, the method comprising the steps of:
 a) combining a BoNT/A peptide and a test sample under conditions suitable for the selective binding of the BoNT/A peptide to an anti-BoNT antibody, the BoNT/A peptide having a length of at least 5 amino acids and at most 60 amino acids; and
 wherein the BoNT/A peptide has a length of at least 5 amino acids and at most 60 amino acids and comprises an amino acid sequence selected from the group consisting of amino acids 519-537 of SEQ ID NO: 1, amino acids 533-551 of SEQ ID NO: 1, amino acids 547-565 of SEQ ID NO: 1, amino acids 1177-1195 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, and an immunoreactive BoNT/A amino acid sequence fragment thereof; 
 wherein said conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 519-537 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 533-551 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 547-565 of SEQ ID NO: 1, or 1-4 conservative amino acid substitutions to amino acids 1177-1195 of SEQ ID NO: 1; 
 wherein said immunoreactive BoNT/A amino acid sequence fragment comprises at least six consecutive amino acids of 519-537 of SEQ ID NO: 1, at least six consecutive amino acids of 533-551 of SEQ ID NO: 1, at least six consecutive amino acids of 547-565 of SEQ ID NO: 1, at least six consecutive amino acids of 1177-1195 of SEQ ID NO: 1; and 
   b) determining the presence of an anti-BoNT antibody-BoNT/A peptide complex, the antibody-peptide complex formed by the selective binding of an anti-BoNT antibody and the BoNT/A peptide; where the presence of the anti-BoNT antibody-BoNT/A peptide complex is indicative of immunoresistance to a BoNT therapy.   
     
     
         2 . The immunoresistance method of  claim 1 , wherein the BoNT/A peptide is at most 60 amino acids and comprises an amino acid sequence selected from the group consisting of amino acids 519-537 of SEQ ID NO: 1, amino acids 533-551 of SEQ ID NO: 1, amino acids 547-565 of SEQ ID NO: 1, and amino acids 1177-1195 of SEQ ID NO: 1. 
     
     
         3 . The immunoresistance method of  claim 1 , wherein the BoNT/A peptide comprises amino acids 519-537 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, or an immunoreactive BoNT/A amino acid sequence fragment thereof;
 wherein the conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 519-537 of SEQ ID NO: 1; and   wherein the immunoreactive BoNT/A amino acid sequence fragment comprises at least six consecutive amino acids of 519-537 of SEQ ID NO: 1.   
     
     
         4 . The immunoresistance method of  claim 1 , wherein the BoNT/A peptide comprises amino acids 533-551 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, or an immunoreactive BoNT/A amino acid sequence fragment thereof;
 wherein the conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 533-551 of SEQ ID NO: 1; and   wherein the immunoreactive BoNT/A amino acid sequence fragment comprises at least six consecutive amino acids of 533-551 of SEQ ID NO: 1.   
     
     
         5 . The immunoresistance method of  claim 1 , wherein the BoNT/A peptide comprises amino acids 547-565 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, or an immunoreactive BoNT/A amino acid sequence fragment thereof;
 wherein the conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 547-565 of SEQ ID NO: 1; and   wherein the immunoreactive BoNT/A amino acid sequence fragment comprises at least six consecutive amino acids of 547-565 of SEQ ID NO: 1.   
     
     
         6 . The immunoresistance method of  claim 1 , wherein the BoNT/A peptide comprises amino acids 1177-1195 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, or an immunoreactive BoNT/A amino acid sequence fragment thereof;
 wherein the conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 1177-1195 of SEQ ID NO: 1; and   wherein the immunoreactive BoNT/A amino acid sequence fragment comprises at least six consecutive amino acids of 1177-1195 of SEQ ID NO: 1.   
     
     
         7 . A method of determining immunoresistance to botulinum toxin therapy in an individual, the method comprising the steps of:
 a) combining at least one BoNT/A peptide and a test sample under conditions suitable for the selective binding of the BoNT/A peptide to an anti-BoNT antibody, the BoNT/A peptide having a length of at least 5 amino acids and at most 60 amino acids; and
 wherein the BoNT/A peptide has a length of at least 5 amino acids and at most 60 amino acids and comprises an amino acid sequence selected from the group consisting of amino acids 519-537 of SEQ ID NO: 1, amino acids 533-551 of SEQ ID NO: 1, amino acids 547-565 of SEQ ID NO: 1, amino acids 1177-1195 of SEQ ID NO: 1, a conservative BoNT/A amino acid sequence variant thereof, and an immunoreactive BoNT/A amino acid sequence fragment thereof; 
 wherein said conservative BoNT/A amino acid sequence variant comprises 1-4 conservative amino acid substitutions to amino acids 519-537 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 533-551 of SEQ ID NO: 1, 1-4 conservative amino acid substitutions to amino acids 547-565 of SEQ ID NO: 1, or 1-4 conservative amino acid substitutions to amino acids 1177-1195 of SEQ ID NO: 1; 
 wherein said immunoreactive BoNT/A amino acid sequence fragment comprises at least six consecutive amino acids of 519-537 of SEQ ID NO: 1, at least six consecutive amino acids of 533-551 of SEQ ID NO: 1, at least six consecutive amino acids of 547-565 of SEQ ID NO: 1, at least six consecutive amino acids of 1177-1195 of SEQ ID NO: 1; and 
   b) determining the presence of an anti-BoNT antibody-BoNT/A peptide complex, the antibody-peptide complex formed by the selective binding of an anti-BoNT antibody and the BoNT/A peptide; where the presence of the anti-BoNT antibody-BoNT/A peptide complex is indicative of immunoresistance to a BoNT therapy.   
     
     
         8 . The immunoresistance method of  claim 7 , wherein in said step (a), said test sample is contacted with two or more BoNT/A peptides. 
     
     
         9 . The immunoresistance method of  claim 8 , wherein in said step (b), said test sample is contacted with two BoNT/A peptides. 
     
     
         10 . The immunoresistance method of  claim 8 , wherein in said step (b), said test sample is contacted with five BoNT/A peptides. 
     
     
         11 . The immunoresistance method of  claim 8 , wherein in said step (b), said test sample is contacted with ten BoNT/A peptides. 
     
     
         12 . The method according to either  claim 1  or  claim 7 , wherein the method comprises a further step of correlating the amount of an antibody-peptide complex formed from the test sample relative to the amount of an antibody-peptide complex formed by the BoNT/A peptide combined to a control sample. 
     
     
         13 . The method according to either  claim 1  or  claim 7 , wherein the test sample comprises blood. 
     
     
         14 . The method according to  claim 13 , wherein the test sample comprises serum. 
     
     
         15 . The method according to  claim 14 , wherein the test sample comprises an IgG antibody component separated from the serum. 
     
     
         16 . The method according to either  claim 1  or  claim 7 , wherein the immunoresistance to botulinum toxin therapy is selected from the group consisting of a BoNT/A immunoresistance, a BoNT/B immunoresistance, a BoNT/C1 immunoresistance, a BoNT/D immunoresistance, a BoNT/E immunoresistance, a BoNT/F immunoresistance and a BoNT/G immunoresistance. 
     
     
         17 . The method according to  claim 16 , wherein the immunoresistance to botulinum toxin therapy is a BoNT/A immunoresistance. 
     
     
         18 . The method according to either  claim 1  or  claim 7 , wherein the individual is a human. 
     
     
         19 . The method according to either  claim 1  or  claim 7 , wherein the presence of an anti-BoNT antibody-BoNT/A peptide complex is determined qualitatively or quantitatively. 
     
     
         20 . The method according to either  claim 1  or  claim 7 , wherein the presence of an anti-BoNT antibody-BoNT/A peptide complex is determined using a competitive assay or a non-competitive assay. 
     
     
         21 . The method according to either  claim 1  or  claim 7 , wherein the presence of an anti-BoNT antibody-BoNT/A peptide complex is determined by an assay format selected from the group consisting of a radioimmunoassay, an enzyme-linked immunosorbent assay, an enzyme immunoassay, a fluorescence immunoassay, and a luminescent immunoassay. 
     
     
         22 . The method according to either  claim 1  or  claim 7 , wherein the presence of at least 10% complex formation of an anti-BoNT antibody to a BoNT/A peptide is indicative of a BoNT immunoresistance. 
     
     
         23 . The method according to  claim 22 , wherein the presence of at least 30% complex formation of an anti-BoNT antibody to a BoNT/A peptide is indicative of a BoNT immunoresistance. 
     
     
         24 . The method according to  claim 23 , wherein the presence of at least 50% complex formation of an anti-BoNT antibody to a BoNT/A peptide is indicative of a BoNT immunoresistance. 
     
     
         25 . The method according to either  claim 1  or  claim 7 , wherein the presence of at most 50% complex formation of an anti-BoNT antibody to a BoNT/A peptide is indicative of a BoNT immunoresistance. 
     
     
         26 . The method according to  claim 25 , wherein the presence of at most 30% complex formation of an anti-BoNT antibody to a BoNT/A peptide is indicative of a BoNT immunoresistance. 
     
     
         27 . The method according to  claim 26  wherein the presence of at most 10% complex formation of an anti-BoNT antibody to a BoNT/A peptide is indicative of a BoNT immunoresistance.