Methods and compositions for liposomal formulation of antigens and uses thereof
The present invention relates to liposomal vaccine compositions, methods for the manufacture thereof, and methods for the use thereof to stimulate an immune response in an animal. These compositions comprise dimyristoylphosphatidylcholine (“DMPC”); either dimyristoylphosphatidylglycerol (“DMPG”) or dimyristoyltrimethylammonium propane (“DMTAP”) or both DMPC and DMTAP; and at least one sterol derivative providing a covalent anchor for one or more immunogenic polypeptide(s) or carbohydrate(s).
1. A composition comprising one or more immunogenic polypeptides of interest, the composition comprising:
a) an aqueous vehicle;
b) liposomes comprising
(i) dimyristoylphosphatidylcholine (“DMPC”),
(ii) one or more lipids selected from the group consisting of dimyristoylphosphatidylglycerol (“DMPG”), and dimyristoyltrimethylammonium propane (“DMTAP”), and
(iii) at least one sterol derivative,
at relative percentages of 50%-98% (i): 1%-25% (ii): 1%-25% (iii); and
c) said one or more immunogenic polypeptide(s) covalently linked to between 1% and 100% of said at least one sterol derivative.
2. A composition according to claim 1 , wherein said relative percentages are 70%-85% (i): 5%-15% (ii): 10%-15% (iii).
3. A composition according to claim 1 , wherein said relative percentages are about 75% (i), about 10% (ii), and about 15% (iii).
4. A composition according to claim 1 , wherein said sterol derivative has the following structure:
wherein:
one of R1 or R2 is a covalent linkage to said immunogenic polypeptide, wherein if R1 is said covalent linkage to said polypeptide, R2 is H, and if R2 is said covalent linkage to said immunogenic polypeptide, R1 is —CH 2 —CH 2 —CH 2 —C(H)(CH 3 ) 2 .
5. A composition according to claim 4 , wherein R1 is —CH 2 —CH 2 —C(O)—X, wherein X is said immunogenic polypeptide, and R2 is H.
6. A composition according to claim 4 , wherein R1 is —CH 2 —CH 2 —CH 2 —C(H)(CH 3 ) 2 , and R2 is —C(O)—CH 2 —CH 2 —C(O)—X, wherein X is said immunogenic polypeptide.
7. A composition according to claim 1 , wherein said liposomes are substantially between 50 and 500 nm in diameter.
8. A composition according to claim 7 , wherein said liposomes are substantially between 50 and 200 nm in diameter.
9. A composition according to claim 7 , wherein said liposomes are substantially between 50 and 150 nm in diameter.
10. A composition according to claim 1 , wherein said one or more immunogenic polypeptide(s) are covalently linked to between about 5% and about 10% of said at least one sterol derivative.
11. A composition according to claim 1 , wherein said liposomes further comprise one or more components selected from the group consisting of monophosphoryl lipid A, resiquimod, flagellin, CpG, and α-galactosylceramide.
12. A composition according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivatives through a lysine residue on said immunogenic polypeptide(s).
13. A composition according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivatives through a cysteine residue on said immunogenic polypeptide(s).
14. A composition according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivatives through a aspartate or glutamate residue on said immunogenic polypeptide(s).
15. A composition according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivatives through a serine or threonine residue on said immunogenic polypeptide(s).
16. A composition according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivatives through an N-terminal amine on said immunogenic polypeptide(s).
17. A composition according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivatives through a C-terminal carboxyl on said immunogenic polypeptide(s).
18. A composition according to claim 1 , wherein said covalent linkage to said immunogenic polypeptide comprises an (alkylene oxide) n moiety having an average length n of between 40 and 1000.
19. A composition according to claim 1 , wherein said covalent linkage to said immunogenic polypeptide has the structure —R3-X, wherein:
R3 is C 0-12 straight or branched chain alkyl, or C 0-6 straight or branched chain alkyl-(alkylene oxide) n —C 0-6 straight or branched chain alkyl, wherein
n is on average between 40 and 1000;
each said straight or branched chain alkyl optionally comprises from 1-3 chain heteroatoms and one or more substituents independently selected from the group consisting of halogen, trihalomethyl, —C 1-6 alkoxy, —NO 2 , —NH 2 , —OH, —CH2OH, —CONH2, and —C(O)(OR4) where R4 is H or C 1-3 alkyl; and
X is said immunogenic polypeptide.
20. A composition according to claim 1 , wherein the lipid in (ii) is DMPG.
21. A composition according to claim 1 , wherein said sterol derivative(s) is/are derivative(s) of cholesterol, cholesteryl chloroformate, stigmasterol, sitosterol, ergosterol, lanosterol, desmosterol, or campesterol.