Abstract
Antigenic polysaccharides are modified to generate a terminally-located aldehyde group by controlled oxidation of vicinal hydroxyl groups, e.g. of unlinked terminal non-reducing sialic acid residues. In some cases where there is a reducing end group, e.g. of the type N-acetylmannosamine residue, it can be made into the most susceptible site for oxidation by initially reducing it to its open chain hydroxyl form, e.g. N-acetylmannosaminitol. The vicinal hydroxyl oxidation is controlled to yield a reactive aldehyde group which is then covalently linked to a free amino group of a selected protein by reductive amination. The resulting polysaccharide-protein conjugates are soluble and have been found to have enhanced antigenicity compared to the polysaccharide alone. This terminal aldehyde:free amine group reductive amination can be applied to various polysaccharide antigens and various well-tolerated proteins, preferably protein immunogens. For example, meningococcal group A, B and...
Filing date: May 27, 1981
Issue date: Oct 26, 1982
Inventors: Harold J. Jennings, Czeslaw Lugowski
Assignee: Canadian Patents & Development Ltd.
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What is claimed is:
1. A method of preparing antigenic polysaccharide: protein conjugates, comprising:
- (a) providing an antigenic polysaccharide which has reactive vicinal hydroxyl groups in a terminal portion of the molecule and has a MW above about 2000;
- (b) subjecting said vicinal hydroxyl groups at a terminal location to controlled oxidation only sufficient to generate a terminal reactive aldehyde group therefrom on the polysaccharide;
- (c) selecting a physiologically-tolerated protein having a free amino group and reacting said amino group with said aldehyde group by reductive amination, to covalently link said polysaccharide and protein; and
- (d) recovering a substantially non-crosslinked, soluble, antigenic polysaccharide:protein conjugate.
2. The method of claim 1 wherein the antigenic polysaccharide is selected from the group derived from meningococci, Haemophilus influenza, pneumococci, .beta.-hemolytic streptococci, and E. coli.
3. The method of claim 1 wherein the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, and immunogenic proteins derived from bacteria selected from .beta.-hemolytic streptococci, Haemophilus influenza, meningococci, pneumococci, and E. coli.
4. The method of claim 1 wherein the polysaccharide initially has no terminal vicinal hydroxyl groups but has a terminal reducing sugar residue which is reduced to form reactive vicinal hydroxyl groups in (a).
5. The method of claim 4 wherein the sugar residue is N-acetylmannosamine residue.
6. The method of claim 4 wherein the reduction is effected using sodium borohydride at a pH of about 7.5-10.
7. The method of claim 6 wherein the pH is about 8-9.
8. The method of claim 1 wherein the controlled oxidation in (b) is effected using periodate reagent for a limited time.
9. The method of claim 8 wherein the polysaccharide is derived from meningococci, and the time for controlled oxidation is within 10-15 minutes.
10. The method of claim 1 wherein meningococcal group A, B or C polysaccharides having terminal vicinal hydroxyl groups are utilized and are oxidized sufficiently to convert only the terminal vicinal hydroxyl groups to a terminal aldehyde group, reductive amination carried out to link covalently the terminal aldehyde group to an amino group on the selected protein tetanus toxoid molecule, and recovering a soluble conjugate thereof of enhanced antigenicity.
11. An antigenic-polysaccharide:protein conjugate wherein the polysaccharide and protein are covalently linked through a ##STR3## linkage to a terminal portion of the polysaccharide without significant cross-linking, said antigenic polysaccharide having a MW above about 2000.
12. The conjugate of claim 11 wherein the antigenic polysaccharide is selected from the group derived from meningococci, Haemophilus influenza, pneumococci, .beta.-hemolytic streptococci, and E. coli.
13. The conjugate of claim 11 wherein the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, and immunogenic proteins derived from bacteria selected from .beta.-hemolytic streptococci, Haemophilus influenza, meningococci, pneumococci and E. coli.
14. The conjugate of claim 11 wherein the antigenic polysaccharide is selected from meningococcal Group A, B or C polysaccharides and the protein is tetanus toxoid.
15. The conjugate of claim 11 wherein the antigenic polysaccharide is derived from Haemophilus influenza and the protein is tetanus toxoid.
16. The conjugate of claim 11 wherein said linkage is through an end-group terminal reducing sugar moiety on the polysaccharide.
17. The conjugate of claim 16 wherein said reducing sugar moiety is N-acetylmannosaminitol.
18. The conjugate of claim 11 wherein said linkage is through a lysine amino group on the protein.
19. The conjugate of claim 11 wherein the polysaccharide and protein are derived from the same bacteria.
20. A method of immunizing against infection susceptible humans or animals comprising, administering a vaccine comprising the conjugate of claim 11 in an immunogenic amount by intramuscular or subcutaneous injection.
21. The method of claim 20 wherein the dosage of the conjugate is equivalent to from about 5 to about 25 micrograms for the human infant.
22. The method of claim 21 wherein human infants are immunized with a vaccine comprising at least one of (a) the conjugate of meningococcal polysaccharide, and (b) the conjugate of H. influenza polysaccharide.
23. A vaccine comprising at least one conjugate as defined in claim 11.
24. A vaccine for infants comprising the conjugate of claim 11 wherein the initial non-conjugated polysaccharide is of the type for which the immune response is non-thymus-controlled.
25. A human infant vaccine comprising the conjugate of claim 11 wherein the polysaccharide comprises at least one of meningococcal polysaccharide and Haemophilus influenza polysaccharide.
26. A vaccine as in claim 24 in a dosage unit form wherein the conjugate is present in the equivalent of from about 5 to about 25 micrograms based on the human infant.