Mutants of cholesterol-dependent cytolysins and uses thereof
View Patent ↗Mutants of cholesterol-dependent cytolysins comprising at least one amino acid substitution in at least one of Loop 1, Loop 2, or Loop 3 of Domain 4, nucleic acids encoding such polypeptide mutants, and compositions and vaccines comprising such polypeptide mutants.
1. A purified mutant pneumolysin polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:1 and comprises a substitution in at least one of amino acid positions 458, 459, and 460; and wherein said mutant pneumolysin polypeptide has reduced hemolytic activity and reduced pore-forming activity as compared to a wild type pneumolysin polypeptide.
2. The purified mutant pneumolysin polypeptide of claim 1 wherein the amino acid sequence is at least 95% identical to SEQ ID NO:1.
3. The purified mutant pneumolysin polypeptide of claim 1 wherein the amino acid sequence is at least 96% identical to SEQ ID NO:1.
4. The purified mutant pneumolysin polypeptide of claim 1 wherein the amino acid sequence is at least 97% identical to SEQ ID NO:1.
5. The purified mutant pneumolysin polypeptide of claim 1 wherein the amino acid sequence is at least 98% identical to SEQ ID NO:1.
6. The purified mutant pneumolysin polypeptide of claim 1 wherein the amino acid sequence is at least 99% identical to SEQ ID NO:1.
7. The purified mutant pneumolysin polypeptide of claim 1 comprising a substitution in amino acid position 458.
8. The purified mutant pneumolysin polypeptide of claim 1 comprising a substitution in amino acid position 459.
9. The purified mutant pneumolysin polypeptide of claim 1 comprising a substitution in amino acid position 460.
10. The purified mutant pneumolysin polypeptide of claim 1 wherein the mutant pneumolysin polypeptide is substantially non-hemolytic, non-pore forming, and non-binding to cell membranes.
11. The purified mutant pneumolysin polypeptide of claim 1 wherein the hemolytic activity is less than 30% of that of the wild type pneumolysin polypeptide.
12. The purified mutant pneumolysin polypeptide of claim 1 wherein the hemolytic activity is less than 20% of that of the wild type pneumolysin polypeptide.
13. The purified mutant pneumolysin polypeptide of claim 1 wherein the hemolytic activity is less than 10% of that of the wild type pneumolysin polypeptide.
14. The purified mutant pneumolysin polypeptide of claim 1 wherein the hemolytic activity is less than 5% of that of the wild type pneumolysin polypeptide.
15. The purified mutant pneumolysin polypeptide of claim 1 wherein the hemolytic activity is less than 1% of that of the wild type pneumolysin polypeptide.
16. A composition comprising one or more of the mutant pneumolysin polypeptides of claim 1 disposed in a pharmaceutically-acceptable carrier, vehicle, adjuvent or diluent.
17. A vaccine comprising the purified mutant pneumolysin polypeptide of claim 1 .
18. The vaccine of claim 12 further comprising capsular polysaccharide serotypes, or immunogenic proteins or protein subunits or fragments.