Method of detoxification
The present invention relates to a method of detoxifying bacterial cytolysins such as pneumococcal pneumolysin, utilizing chemical cross-linking compounds.
1. A process for reducing the haemolytic activity of pneumolysin, the process comprising treating said pneumolysin with a chemical cross-linking compound, where said chemical cross-linking compound is a heterobifunctional cross-linking agent that is reactive with primary amine and sulfhydryl groups, whereby the haemolytic activity of the pneumolysin is reduced following said treatment.
2. The process of claim 1 wherein said chemical cross-linking compound cross-links the sulfhydryl group of a c-terminal cysteine residue with the primary amine group of a lysine residue.
3. The process of claim 2 wherein the cysteine and lysine residues are more than twenty amino acids apart in the primary structure of the pneumolysin.
4. The process of claim 2 wherein the cysteine and lysine residues are more than forty amino acids apart in the primary structure of the pneumolysin.
5. The process of claim 1 wherein the haemolytic activity of the pneumolysin is reduced by at least 90%.
6. The process of claim 1 wherein the pneumolysin retains a monomeric quaternary structure.
7. The process of claim 1 wherein said chemical cross-linking compound contains a group selected from (a) an N-hydroxysuccinimide ester group that reacts preferentially with primary amine groups, and (b) a maleimide group that reacts preferentially with sulfhydryl groups.
8. The process of claim 1 wherein said chemical cross-linking compound is not cleavable using a reducing agent.
9. The process of claim 1 wherein the distance between the reactive groups on said chemical cross-linking compound is between 1.5 and 20 Angstroms.
10. The process of claim 1 wherein at least five amino acid residues of said pneumolysin are covalently bound by molecules of said chemical cross-linking compound, said amino acid residues selected from lysine and arginine.
11. The process of claim 1 wherein said chemical cross-linking compound is selected from the group consisting of SMPT, Sulfo-LC-SMPT, Sulfo-KMUS, LC-SMCC, KMUA, Sulfo-LC-SPDP, LC-SPDP, SMPB, Sulfo-SMPB, SMPH, Sulfo-SMCC, SMCC, SIAB, Sulfo-SIAB, GMBS (N-(gamma-maleimidobutyryloxy)succinimide ester), Sulfo-GMBS, MBS, Sulfo-MBS, Sulfo-EMCS, EMCA, EMCS, BMPS, SPDP, SBAP, BMPA, AMAS, SATP and SIA.
12. The process of claim 1 wherein treatment with said chemical cross-linking compound takes place under pH conditions of between 5.0 and 9.0.
13. The process of claim 1 wherein treatment with said chemical cross-linking compound takes place in a salt concentration of between 100 mM and 1 M.
14. The process of claim 1 wherein during the treatment, said chemical cross-linking compound is present at a molar excess of between 50 to 500, compared to the pneumolysin.
15. The process of claim 1 wherein during the treatment, said chemical cross-linking compound is present at a molar excess of between 2 to 20, compared to lysine residues in the pneumolysin.
16. The process of claim 1 wherein said chemical cross-linking compound is a heterobifunctional cross-linking reagent having groups that are reactive with lysine and cysteine residues, and in which the reactive groups are spaced 10 to 20 Angstroms apart.
17. The process of claim 10 wherein said chemical cross-linking compound covalently binds to between 5 and 30 amino acid residues of the pneumolysin, said amino acid residues selected from lysine and arginine, and wherein the non-bound end of the cross-linking compound is quenched.
18. The process of claim 1 wherein a c-terminal cysteine residue is cross-linked to another amino acid of the pneumolysin which, in the primary sequence of the pneumolysin, is more than 2 amino acids from said c-terminal cysteine residue.
19. The process of claim 18 wherein said c-terminal cysteine is cross-linked to another amino acid of the pneumolysin which, in the primary sequence of the pneumolysin, is more than 20 amino acids from said c-terminal cysteine residue.
20. The process of claim 18 where said c-terminal cysteine residue is cross-linked to an amino acid selected from lysine and arginine.
21. Pneumolysin produced by the process of claim 1 wherein the haemolytic activity of the pneumolysin is reduced by at least 90% due to the process.
22. A conjugate comprising a pneumolysin according to claim 21 bonded to a saccharide.
23. A process of making a vaccine comprising the steps of: (a) preparing pneumolysin using the method of claim 1 , and (b) combining said pneumolysin produced by step (a) with a pharmaceutically acceptable excipient.