Recombinant attenuated clostridium organisms and vaccine
The present invention discloses attenuated Clostridium perfringens organisms that express a substantially nontoxic alpha-toxin. The expressed alpha-toxin is a deletion mutein that relative to the alpha-toxin of the mature alpha-toxin of Clostridium perfringens strain 13, is missing at least nine consecutive amino acid residues including His 68 . The present invention also discloses attenuated organisms that encode the muteins, as well as the use of such attenuated organisms as vaccines.
1. A substantially nontoxic mutein of Clostridium perfringens alpha-toxin; wherein the mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus at least 9 consecutive amino acid residues, with no more than 48 consecutive amino acid residues deleted; and wherein one of the deleted amino acid residues is His 68 .
2. The substantially nontoxic mutein of Clostridium perfringens alpha-toxin of claim 1 wherein the mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 minus at least 12 consecutive amino acid residues; and wherein one of the deleted amino acid residues is His 68 .
3. The substantially nontoxic mutein of Clostridium perfringens alpha-toxin of claim 2 , wherein said mutein comprises the amino acid sequence of SEQ ID NO: 3 minus at least 18 consecutive amino acid residues; and wherein one of the deleted amino acid residues is His 68 .
4. The substantially nontoxic mutein of Clostridium perfringens alpha-toxin of claim 1 , wherein nine consecutive amino acid residues that range from Tyr 62 through Trp 70 , are deleted and replaced by a single Leu residue.
5. The substantially nontoxic mutein alpha-toxin of claim 1 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 36 consecutive amino acid residues deleted.
6. The substantially nontoxic mutein alpha-toxin of claim 5 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 24 consecutive amino acid residues deleted.
7. The substantially nontoxic mutein alpha-toxin of claim 6 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 18 consecutive amino acid residues deleted.
8. The substantially nontoxic mutein alpha-toxin of claim 2 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 36 consecutive amino acid residues deleted.
9. The substantially nontoxic mutein alpha-toxin of claim 8 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 24 consecutive amino acid residues deleted.
10. The substantially nontoxic mutein alpha-toxin of claim 3 wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 with no more than 36 consecutive amino acid residues deleted.
11. A substantially nontoxic mutein of Clostridium perfringens alpha-toxin;
wherein the substantially nontoxic mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 in which His 68 is deleted together with an additional 4 to 60 flanking amino acid residues; and
wherein the attenuated Clostridium perfringens organism is substantially nontoxic due to a lack of a functional wild-type plc gene.
12. A substantially nontoxic mutein of Clostridium perfringens alpha-toxin; wherein the mutein alpha-toxin comprises the amino acid sequence of SEQ ID NO: 3 wherein nine consecutive amino acid residues that range from Tyr 62 through Trp 70 , are deleted and replaced by a single Leu residue.