Immunologically active peptides with altered toxicity useful for the preparation of antipertussis vaccine

Abstract

Immunologically active polypeptides with no or reduced toxicity useful for the preparation of an antipertussis vaccine. Method for the preparation of said polypeptides which comprises, cultivating a microorganisms transformed with a hybrid plasmid including the gene/s which codes for at least one of said polypeptides in a suitable medium and recovering the desired polypeptide from the cells or from the culture medium.

1 - 19 . (canceled)  
     
     
         20 . A polypeptide comprising the S1 subunit of pertussis toxin wherein the S1 subunit is modified by the substitution in one or more sites selected from the group consisting of tyrosine in position 8, arginine in position 9, phenylalanine in position 50, threonine in position 53, glutamic acid in position 129, glycine in position 121, alanine in position 124, aspartic acid in position 109, glycine in position 99, arginine in position 135, threonine in position 159 and tyrosine in position 111 with another amino acid capable of destroying or reducing the toxicity of the S1 subunit.  
     
     
         21 . The polypeptide of  claim 20  wherein at least glutamic acid in position 129 of the S1 subunit is substituted with another amino acid.  
     
     
         22 . The polypeptide of  claim 21  wherein said polypeptide is further modified by the substitution in one or more sites selected from the group consisting of tyrosine in position 8, arginine in position 9, phenylalanine in position 50, threonine in position 53, glycine in position 121, alanine in position 124, aspartic acid in position 109, glycine in position 99, arginine in position 135, threonine in position 159, and tyrosine in position 111 with another amino acid capable of destroying or reducing the toxicity of the S1 subunit.  
     
     
         23 . The polypeptide of  claim 22  wherein arginine in position 9 of the S1 subunit is substituted with another amino acid.  
     
     
         24 . The polypeptide of  claim 20  wherein said polypeptide further comprises at least one of the S2, S3, S4, and S5 subunits of the  pertussis  toxin.  
     
     
         25 . The polypeptide of  claim 24  wherein said polypeptide comprises the S2, S3, S4, and S5 subunits and said S2, S3, S4, and S5 subunits have the same arrangement as that present in natural  pertussis  toxin.  
     
     
         26 . The polypeptide of  claim 20  wherein tyrosine in position 8 of the S1 subunit is substituted with aspartic acid and wherein arginine in position 9 of the S1 subunit is substituted with glycine.  
     
     
         27 . The polypeptide of  claim 20  wherein phenylalanine in position 50 of the S1 subunit is substituted with glutamic acid and wherein threonine in position 53 of the S1 subunit is substituted with glycine.  
     
     
         28 . The polypeptide of  claim 20  wherein glycine in position 99 of the S1 subunit is substituted with glutamic acid.  
     
     
         29 . The polypeptide of  claim 20  wherein the glycine in position 121 of the S1 subunit is substituted with glutamic acid.  
     
     
         30 . The polypeptide of  claim 20  wherein the alanine in position 124 of the S1 subunit is substituted with aspartic acid.  
     
     
         31 . A method for the preparation of a polypeptide of  claim 20 , comprising: 
 a) modifying by direct mutagenesis a DNA molecule coding for an S1 subunit of  pertussis  toxin a base sequence of which codes for an amino acid selected from the group consisting of (1) tyrosine in position 8, (2) arginine in position 9, (3) phenylalanine in position 50, (4) threonine in position 53, (5) glutamic acid in position 129, (6) glycine in position 121, (7) alanine in position 124, (8) aspartic acid in position 109, (9) glycine in position 99, (10) arginine in position 135, (11) threonine in position 159, and (12) tyrosine in position 111 of the S1 subunit with a base sequence that codes for an amino acid of interest;    b) constructing a hybrid plasmid linking a cloning vector with the DNA molecule;    c) transforming a host microorganism with the hybrid plasmid;    d) cultivating the transformed host microorganism in a suitable culture medium; and    e) recovering the polypeptide from the culture medium or from the host microorganism.    
     
     
         32 . The method of  claim 31  wherein the DNA molecule is the gene coding for the S1 subunit is contained in the  pertussis  toxin operon.  
     
     
         33 . The method of  claim 32  wherein the DNA molecule further encodes at least one of the S2, S3, S4, or S5 subunits of the  pertussis  toxin.  
     
     
         34 . The method of  claim 33  wherein the DNA molecule is the operon that codes for the  pertussis  toxin.  
     
     
         35 . The method of  claim 32  wherein the host microorganism is selected from the group consisting of  E. coli,  a  bacillus,  and a yeast.  
     
     
         36 . The method of  claim 35  wherein the microorganism is  E. coli.    
     
     
         37 . An antipertussis vaccine comprising a therapeutically effective quantity of at least one polypeptide of  claim 21 .  
     
     
         38 . An antipertussis vaccine comprising a therapeutically effective quantity of at least one polypeptide of  claim 22 .  
     
     
         39 . An isolated DNA molecule comprising a nucleotide sequence coding for a polypeptide comprising the S1 subunit of pertussis toxin wherein bases coding for one or more sites of the S1 subunit selected from the group consisting of (1) tyrosine in position 8, (2) arginine in position 9, (3) phenylalanine in position 50, (4) threonine in position 53, (5) glutamic acid in position 129, (6) glycine in position 121, (7) alanine in position 124, (8) aspartic acid in position 109, (9) glycine in position 99, (10) arginine in position 135, (11) threonine in position 159, and (12) tyrosine in position 111 are substituted with bases coding for another amino acid capable of destroying or reducing toxicity of the S1 subunit.  
     
     
         40 . The DNA molecule of  claim 39  wherein at least the bases of the DNA molecule coding for glutamic acid in position 129 of said S1 subunit are substituted with bases coding for another amino acid.  
     
     
         41 . The DNA molecule of  claim 40  which is further modified by the substitution of bases coding for one or more amino acids of the S1 subunit selected from the group consisting of (1) tyrosine in position 8, (2) arginine in position 9, (3) phenylalanine in position 50, (4) threonine in position 53, (5) glycine in position 121, (6) alanine in position 124, (7) aspartic acid in position 109, (10) glycine in position 99, (11) arginine in position 135, (12) threonine in position 159, and (13) tyrosine in position 111 are substituted with bases coding for another amino acid capable of destroying or reducing toxicity of the S1 subunit.  
     
     
         42 . The DNA molecule of  claim 41  wherein bases coding for arginine in position 9 of the S1 subunit are substituted with bases coding for another amino acid.  
     
     
         43 . The DNA molecule of  claim 41  wherein the polypeptide contains at least one of the S2, S3, S4, or S5 subunits of the  pertussis  toxin.  
     
     
         44 . The DNA molecule of  claim 43  wherein the polypeptide comprises the S2, S3, S4, and S5 subunits in the same arrangement as that present in natural  pertussis  toxin.  
     
     
         45 . The DNA molecule of  claim 39  wherein bases coding for tyrosine in position 9 are substituted with bases coding for aspartic acid and wherein bases coded for arginine in position 9 of the S1 subunit are substituted with bases coding for glycine.  
     
     
         46 . The DNA molecule of  claim 39  wherein bases coding for phenylalanine in position 50 are substituted with bases coding for glutamic acid and wherein bases coding for threonine in position 53 of the S1 subunit are substituted with bases coding for isoleucine.  
     
     
         47 . The DNA molecule of  claim 39  wherein bases coding for glycine in position 99 of the S1 subunit are substituted with bases coding for glutamic acid.  
     
     
         48 . The DNA molecule of  claim 39  wherein bases coding for glycine in position 121 of the S1 subunit are substituted with bases coding for glutamic acid.  
     
     
         49 . The DNA molecule of  claim 39  wherein bases coding for alanine in position 124 of the S1 subunit are substituted with bases coding for aspartic acid.  
     
     
         50 . A method for immunizing a human against  pertussis  comprising administering an effective amount of a vaccine selected from the group consisting of the vaccine of  claim 37  and the vaccine of  claim 38 .  
     
     
         51 . A method of preparing an antipertussis vaccine comprising formulating a therapeutically effective amount of a polypeptide in vaccine form, wherein the polypeptide is selected from the group consisting of the polypeptides of claims  21 - 25 .  
     
     
         52 . An isolated preparation of  E. coli  selected from the group consisting of PTE 255-22 deposited as ATCC Accession No. 67542, PTE 255-28 deposited as ATCC Accession No. 67543, and PTE 255-41 deposited as ATCC Accession No. 67544.