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 method for preparing compositions comprising one or more immunogenic polypeptides of interest, comprising:
(a) combining
(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 to provide a lipid mixture;
(b) preparing liposomes from said lipid mixture; and
(c) covalently coupling one or more immunogenic polypeptides to said at least one sterol derivative, wherein said one or more immunogenic polypeptide(s) or carbohydrate(s) are covalently linked to between 1% and 100% of said at least one sterol derivative.
2. A method according to claim 1 , wherein preparing liposomes from said lipid mixture comprises:
drying said lipid mixture;
hydrating said dried lipid mixture in an aqueous vehicle; and
sonicating, extruding, or homogenizing said hydrated lipid mixture to form liposomes.
3. A method according to claim 1 , wherein said relative percentages are 70%-85% (i): 5%-15% (ii): 10%-15% (iii).
4. A method according to claim 1 , wherein said relative percentages are about 75% (i), about 10% (ii), and about 15% (iii).
5. A method 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 .
6. A method according to claim 1 , wherein said liposomes are substantially between 50 and 500 nm in diameter.
7. A method according to claim 6 , wherein said liposomes are substantially between 50 and 200 nm in diameter.
8. A method according to claim 6 , wherein said liposomes are substantially between 50 and 150 nm in diameter.
9. A method 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.
10. A method 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.
11. A method according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a lysine residue on said immunogenic polypeptide(s).
12. A method according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a cysteine residue on said immunogenic polypeptide(s).
13. A method according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a aspartate or glutamate residue on said immunogenic polypeptide(s).
14. A method according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a serine or threonine residue on said immunogenic polypeptide(s).
15. A method according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through an N-terminal amine on said immunogenic polypeptide(s).
16. A method according to claim 1 , wherein at least one of said immunogenic polypeptide(s) are covalently linked to said one or more sterol derivative through a C-terminal carboxyl on said immunogenic polypeptide(s).
17. A method 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.
18. A method 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.
19. A method according to claim 5 , wherein R1 is —CH 2 —CH 2 —C(O)—X, wherein X is said immunogenic polypeptide, and R2 is H.
20. A method according to claim 5 , 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.
21. A method according to claim 1 , wherein the lipid in (ii) is DMPG.