Mutants of clostridium difficile toxin B and methods of use

Abstract

An active, or passive vaccine utilizing purified non-toxic mutant TcdB toxins from Clostridium difficile for humans and animals against infections caused by C. difficile and/or C. sordellii. Persons most potentially affected by C. difficile infections include hospitalized patients, infants, and elderly persons. The TcdB toxin mutant of the vaccine preferably lacks the toxicity of a native C. difficile TcdB toxin. A serum comprising antibodies raised to the TcdB toxin mutant is also available for treating humans or animals against C. difficile infections. The serum may be used in a method for conferring passive immunity against C. difficile. Antibodies to the TcdB toxin mutant may be used in diagnostic tests or in treatments to clear TcdB toxin from bodily fluids. The mutant TcdB toxin may be produced by recombinant methods using cDNA encoding the toxin, the cDNA contained for example in a plasmid or host cell.

1 . An isolated mutant of  Clostridium difficile  TcdB toxin polypeptide comprising:
 a modified  Clostridium difficile  TcdB toxin having SEQ ID NO: 3, SEQ ID NO: 5, (SEQ ID NO: 7, or SEQ ID NO: 9, wherein the mutant is effective in inhibiting or modulating the cytotoxic effect of C difficile TcdB toxin and C sordellii TcsL toxin.   
     
     
         2 . A composition comprising the mutant of  claim 1  disposed within a pharmaceutically-acceptable carrier. 
     
     
         3 . A method of inhibiting, modulating or treating a  Clostridium difficile  and/or a  Clostridium sordellii  infection or the symptoms or toxin thereof in a subject, comprising administering an effective amount of the mutant composition of  claim 1  to the subject, wherein the mutant is non-cytotoxic. 
     
     
         4 . The method of  claim 3  wherein the subject is a human. 
     
     
         5 . The method of  claim 3  wherein the subject is an animal. 
     
     
         6 . The method of  claim 3  wherein the method comprises administering at least two mutants. 
     
     
         7 . A vaccine composition comprising at least one mutant of  claim 1  and a pharmaceutically effective carrier, and wherein the mutant is non-toxic. 
     
     
         8 . The vaccine composition of  claim 7  further comprising an adjuvant. 
     
     
         9 . The vaccine composition of  claim 7  further comprising mote than one mutant of  claim 1 . 
     
     
         10 . A vaccine composition comprising the mutant of  claim 1  or an immunogenic fragment thereof which is effective in generating an antibody which is effective against  Clostridium difficile  TcdB toxin. 
     
     
         11 . A method of immunizing a human or animal against a  Clostridium difficile  infection comprising treating the human or animal with an immunogenic amount of the vaccine of  claim 10 . 
     
     
         12 . An antibody raised against the mutant of  claim 1  wherein the antibody binds to  Clostridium difficile  TcdB toxin. 
     
     
         13 . The antibody of  claim 12  wherein the antibody also binds to  Clostridium sordellii  TcsL toxin. 
     
     
         14 . A method of making an antibody against  Clostridium difficile  TcdB toxin comprising:
 immunizing an animal with an immunogenic amount of the mutant of  claim 1 , wherein the mutant is non-cytotoxic; and   obtaining the antibody from the animal.   
     
     
         15 . The method of  claim 14  wherein the antibody is also effective against  Clostridium sordellii  TcsL toxin. 
     
     
         16 . A serum comprising the antibody made by the method of  claim 14 . 
     
     
         17 . A method of making a hybridoma which secretes an antibody against  Clostridium dfficile  TcdB toxin, comprising:
 fusing a lymphocyte from an animal immunized with a mutant of  claim 1  with cells capable of replicating indefinitely in cell culture to produce the hybridoma, wherein the mutant is non-cytotoxic; and   isolating the hybridoma.   
     
     
         18 . A hybridoma produced by the method of  claim 17 , which hybridoma secretes an antibody against  Clostridium difficile  TcdB toxin. 
     
     
         19 . A method of making a monoclonal antibody that recognizes  Clostridium difficile  TcdB toxin, comprising isolating the antibody produced by the hybridoma of  claim 18 . 
     
     
         20 . The method of  claim 19  wherein the monoclonal antibody also recognizes  Clostridium sordellii  TcsL toxin. 
     
     
         21 . The antibody produced by the method of  claim 20  wherein the antibody is humanized. 
     
     
         22 . An immunoassay for  Clostridium difficile  TcdB toxin, comprising:
 contacting a sample to be tested for a Clostridium difficile TcdB toxin or a portion thereof with an antibody of  claim 12  to form an antibody-TcdB toxin complex; and   detecting the antibody-TcdB toxin complex to determine the presence or absence of the  Clostridium difficile  TcdB toxin in the sample.   
     
     
         23 . The immunoassay of  claim 22  wherein the immunoassay is also effective in detecting  Clostridium sordellii  TcsL toxin. 
     
     
         24 . A polynucleotide which encodes the mutant of Clostridium difficile TcdB toxin polypeptide as defined in  claim 1 . 
     
     
         25 . A vector containing the polynucleotide of  claim 24 . 
     
     
         26 . The vector of  claim 25  wherein the polynucleotide is operatively associated with an expression control sequence. 
     
     
         27 . A host cell containing the vector of  claim 25 . 
     
     
         28 . A process for producing a mutant of  Clostridium difficile  TcdB toxin polypeptide, comprising:
 culturing the host cell of  claim 27  thereby expressing the mutant, and wherein the mutant is non-cytotoxic; and   purifying the mutant from the cultured host cell.   
     
     
         29 . The mutant of  Clostridium difficile  TcdB toxin produced by the process of  claim 28 .