Vaccine for eliciting immune response comprising RNA encoding an immunogen and lipid formulations comprising mole percentage of lipids
Provided are vaccines for eliciting an immune response. The vaccines for eliciting an immune response comprise RNA encoding an immunogen, which is delivered in a liposome for the purposes of immunisation. The liposome includes lipids which have a pKa in the range of 5.0 to 7.6 and, preferably, a tertiary amine. These liposomes can have essentially neutral surface charge at physiological pH and are effective for immunisation.
1. A formulation comprising:
ribonucleic acid (RNA) molecules comprising a sequence that encodes an immunogen; and
lipids comprising: (a) from 20 mole % to 70 mole % cationic lipid, (b) an anionic or a zwitterionic lipid, (c) a polyethylene glycol-conjugated (PEG-conjugated) lipid, and (d) from 5 mole % to 80 mole % cholesterol; wherein:
the lipids encapsulate at least half of the RNA molecules;
the formulation is immunogenic in vivo by eliciting an antibody response against the immunogen in vivo; and
the cationic lipid comprises a tertiary amine and has a pK a from 6.07 to 7.6; and
whereby the pKa is determined at standard temperature and pressure by the following:
(1) admixing 400 μL of 2 mM cationic lipid that is in ethanol and 800 μL of 0.3 mM fluorescent probe 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS), which is in 90 volume % ethanol and 10 volume % methanol, thereby obtaining a lipid/TNS mixture;
(2) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a first buffer consisting essentially of a sodium salt buffer comprising 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, wherein the first buffer has a pH from 4.44 to 4.52, which has been adjusted with 12N hydrochloric acid or 6N sodium hydroxide, thereby obtaining a first mixture, and dispensing 100 μL of the first mixture in a first well of a 96-well plate, which has a clear bottom;
(3) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a second buffer consisting essentially of the sodium salt buffer, wherein the second buffer has a pH of 5.27, which has been adjusted with 12N hydrochloric acid or 6N sodium hydroxide, thereby obtaining a second mixture, and dispensing 100 μL of the second mixture in a second well of the 96-well plate;
(4) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a third buffer consisting essentially of the sodium salt buffer, wherein the third buffer has a pH from 6.15 to 6.21, which has been adjusted with 12N hydrochloric acid or 6N sodium hydroxide, thereby obtaining a third mixture, and dispensing 100 μL of the third mixture in a third well of the 96-well plate;
(5) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a fourth buffer consisting essentially of the sodium salt buffer, wherein the fourth buffer has a pH of 6.57, which has been adjusted with 12N hydrochloric acid or 6N sodium hydroxide, thereby obtaining a fourth mixture, and dispensing 100 μL of the fourth mixture in a fourth well of the 96-well plate;
(6) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a fifth buffer consisting essentially of the sodium salt buffer, wherein the fifth buffer has a pH from 7.10 to 7.20, which has been adjusted with 12N hydrochloric acid or 6N sodium hydroxide, thereby obtaining a fifth mixture, and dispensing 100 μL of the fifth mixture in a fifth well of the 96-well plate;
(7) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a sixth buffer consisting essentially of the sodium salt buffer, wherein the sixth buffer has a pH from 7.72 to 7.80, which has been adjusted with 12N hydrochloric acid or 6N sodium hydroxide, thereby obtaining a sixth mixture, and dispensing 100 μL of the sixth mixture in a sixth well of the 96-well plate;
(8) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of a seventh buffer consisting essentially of the sodium salt buffer, wherein the seventh buffer has a pH from 8.27 to 8.33, which has been adjusted with 12N hydrochloric acid or 6N sodium hydroxide, thereby obtaining a seventh mixture, and dispensing 100 μL of the seventh mixture in a seventh well of the 96-well plate;
(9) admixing 7.5 μL of the lipid/TNS mixture and 242.5 μL of an eighth buffer consisting essentially of the sodium salt buffer, wherein the eighth buffer has a pH from 10.47 to 11.12, which has been adjusted with 12N hydrochloric acid or 6N sodium hydroxide, thereby obtaining an eighth mixture, and dispensing 100 μL of the eighth mixture in an eighth well of the 96-well plate;
(10) measuring the fluorescence at a wavelength of 431 nm with an excitation wavelength of 322 nm and a cut-off below a wavelength of 420 nm of each of the first through eighth wells and an empty well of the 96-well plate, thereby obtaining a measured fluorescence for each of the p empty well and the first through eighth wells;
(11) subtracting the measured fluorescence of the empty well from each of the measured fluorescences of the first through eighth wells, thereby obtaining a blank-subtracted fluorescence for each of the first through eighth mixtures;
(12) normalizing each of the blank-subtracted fluorescences of the first through eighth mixtures to the blank-subtracted fluorescence of the first mixture thereby obtaining a relative fluorescence for each of the first through eighth mixtures, the relative fluorescence being 1 for the first mixture;
(13) obtaining a line of best fit of the pHs of the first through eighth buffers versus the respective relative fluorescences of the first through eighth mixtures; and
(14) defining the pKa as the pH on the line of best fit at which a relative fluorescence of 0.5 is obtained.
2. The formulation of claim 1 , wherein the immunogen comprises a viral immunogen, a bacterial immunogen, a fungal immunogen, or a parasitic immunogen.
3. The formulation of claim 1 , wherein the immunogen comprises two or more different immunogens.
4. The formulation of claim 1 , wherein the formulation is further immunogenic in vivo by eliciting a cell-mediated immune response against the immunogen in vivo.
5. The formulation of claim 1 , wherein the PEG-conjugated lipid comprises a PEG that has a molecular weight of 2000 Daltons.
6. The formulation of claim 1 , wherein the lipids comprise from 1 mole % to 6 mole % PEG-conjugated lipid.
7. The formulation of claim 6 , wherein the PEG-conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.
8. The formulation of claim 1 , wherein the PEG-conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.
9. The formulation of claim 1 , wherein the lipids comprise at most 50 mole % zwitterionic lipid.
10. The formulation of claim 1 , wherein the lipids comprise from 10 mole % to 20 mole % zwitterionic lipid.
11. The formulation of claim 1 , wherein the zwitterionic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or dimyristoyl phosphatidylethanolamine (DMPE).
12. The formulation of claim 11 , wherein the zwitterionic lipid is DSPC and the immunogen is a viral immunogen.
13. The formulation of claim 12 , wherein the lipids comprise from 40 mole % to 60 mole % tertiary amine cationic lipid.
14. The formulation of claim 13 , wherein the lipids comprise from 35 mole % to 50 mole % cholesterol.
15. The formulation of claim 14 , wherein the RNA molecules further comprise a 5′ cap nucleoside, a triphosphate bridge, and a 5′ first ribonucleoside, and wherein the 5′ cap nucleoside is linked 5′-to-5′ to the 5′ first ribonucleoside by the triphosphate bridge.
16. The formulation of claim 15 , wherein the 5′ first ribonucleoside comprises a 2′-methylated ribose.
17. The formulation of claim 1 , wherein the lipids comprise from 20 mole % to 70 mole % cholesterol.
18. The formulation of claim 1 , wherein the lipids comprise from 30 mole % to 50 mole % cholesterol.
19. The formulation of claim 1 , wherein the lipids comprise from 35 mole % to 50 mole % cholesterol.
20. The formulation of claim 1 , wherein the pKa is from 6.07 to 6.8.
21. The formulation of claim 1 , wherein the lipids comprise from 30 mole % to 60 mole % cationic lipid.
22. The formulation of claim 1 , wherein the lipids comprise from 40 mole % to 60 mole % cationic lipid.
23. The formulation of claim 1 , wherein the lipids comprise:
from 30 mole % to 60 mole % cationic lipid,
at most 30 mole % zwitterionic lipid,
from 30 mole % to 50 mole % cholesterol, and
from 1 mole % to 6 mole % the PEG-conjugated lipid.
24. The formulation of claim 23 , wherein the zwitterionic lipid is DSPC or DMPE.
25. The formulation of claim 23 , wherein the PEG-conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.
26. The formulation of claim 1 , wherein the RNA molecules further comprise a 3′ poly(adenosine monophosphate) tail.
27. The formulation of claim 1 , wherein the RNA molecules further comprise a 5′ cap nucleoside, a triphosphate bridge, and a 5′ first ribonucleoside, and wherein the 5′ cap nucleoside is linked 5′-to-5′ to the 5′ first ribonucleoside by the triphosphate bridge.
28. The formulation of claim 27 , wherein the 5′ first ribonucleoside comprises a 2′-methylated ribose.
29. The formulation of claim 1 , wherein the RNA molecules further comprise a sequence that encodes a replicase, and the RNA molecules are self-replicating RNA molecules.
30. The formulation of claim 1 , wherein the lipids comprise:
from 30 mole % to 60 mole % cationic lipid,
at most 30 mole % zwitterionic lipid,
from 30 mole % to 50 mole % cholesterol, and
from 1 mol % to 6 mol % PEG-conjugated lipid, and
wherein the zwitterionic lipid is DSPC, and
wherein the PEG-conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.