Vaccine for eliciting immune response comprising lipid formulations and RNA encoding multiple immunogens
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:
a first species of ribonucleic acid (RNA) molecules comprising a sequence that encodes a first immunogen;
a second species of RNA molecules comprising a sequence that encodes a second immunogen; and
lipids comprising: (a) a cationic lipid, (b) an anionic lipid or a zwitterionic lipid, (c) a polyethylene glycol-conjugated (PEG-conjugated) lipid, and (d) a cholesterol, wherein:
the lipids encapsulate at least half of the first species of RNA molecules and at least half of the second species of RNA molecules;
the cationic lipid comprises a tertiary amine and has a pK a from 6.07 to 7.6; and
the formulation is immunogenic in vivo by eliciting an antibody response against the first immunogen and the second immunogen in vivo; and
whereby the pK a is determined at standard temperature and pressure by the following:
(1) admixing 400 μL of 2 mM of the cationic lipid that is in ethanol and 800 μL of 0.3 mM of 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 consisting of 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 empty well and the first through eighth wells;
(11) subtracting the 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) determining the pK a 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 first immunogen comprises a viral immunogen, a bacterial immunogen, a fungal immunogen, or a parasitic immunogen.
3. The formulation of claim 2 , wherein the second immunogen comprises a viral immunogen, a bacterial immunogen, a fungal immunogen, or a parasitic immunogen.
4. The formulation of claim 3 , wherein the first immunogen and second immunogen comprise viral immunogens.
5. The formulation of claim 4 , wherein the lipids comprise from 40 mole % to 60 mole % tertiary amine cationic lipid.
6. The formulation of claim 5 , wherein the lipids comprise from 35 mole % to 50 mole % cholesterol.
7. The formulation of claim 1 , wherein the formulation is further immunogenic in vivo by eliciting a cell-mediated immune response against the first immunogen or the second immunogen in vivo.
8. The formulation of claim 1 further comprising a third species of RNA molecules comprising a sequence that encodes a third immunogen and wherein the lipids encapsulate at least half of the third species of RNA molecules.
9. The formulation of claim 8 , wherein the third immunogen comprises a viral immunogen, a bacterial immunogen, a fungal immunogen, or a parasitic immunogen.
10. The formulation of claim 9 , wherein the first species of RNA molecules further comprise a sequence that encodes a replicase, and wherein the first species of RNA molecules are self-replicating.
11. The formulation of claim 10 , wherein the second species of RNA molecules further comprise a sequence that encodes the replicase, and wherein the second species of RNA molecules are self-replicating.
12. The formulation of claim 11 , wherein the third species of RNA molecules further comprise a sequence that encodes the replicase, and wherein the third species of RNA molecules are self-replicating.
13. The formulation of claim 1 , wherein the zwitterionic lipid is 1,2-di stearoyl-sn-glycero-3-phosphocholine (DSPC).
14. The formulation of claim 13 , wherein the lipids comprise from 40 mole % to 60 mole % tertiary amine cationic lipid; wherein the first immunogen and second immunogen comprise viral immunogens.
15. The formulation of claim 1 , wherein the PEG-conjugated lipid comprises a PEG that has a molecular weight of 2000 Daltons.
16. The formulation of claim 1 , wherein the PEG-conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.
17. The formulation of claim 1 , wherein the first immunogen comprises a cytomegalovirus (CMV) immunogen, a herpes simplex virus (HSV) immunogen, a varicella-zoster virus (VZV) immunogen, an Epstein-Barr virus (EBV) immunogen, a papillomavirus immunogen, a polyomavirus immunogen, a coronavirus immunogen, an oncovirus immunogen, a lentivirus immunogen, a flavivirus immunogen, an orthomyxovirus immunogen, a paramyxovirus immunogen, or a picornavirus immunogen.
18. The formulation of claim 17 , wherein the second immunogen comprises the CMV immunogen, the HSV immunogen, the VZV immunogen, the EBV immunogen, the papillomavirus immunogen, the polyomavirus immunogen, the coronavirus immunogen, the oncovirus immunogen, the lentivirus immunogen, the flavivirus immunogen, the orthomyxovirus immunogen, the paramyxovirus immunogen, or the picornavirus immunogen.
19. The formulation of claim 18 , wherein the first immunogen comprises the coronavirus immunogen and the second immunogen comprises the influenza virus immunogen.
20. The formulation of claim 17 , wherein the first immunogen and the second immunogen comprise CMV immunogens.
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 pK a is from 6.07 to 6.8.
23. The formulation of claim 1 , wherein the lipids comprise at most 30 mole % zwitterionic lipid, and wherein the zwitterionic lipid is DSPC or dimyristoyl phosphatidylethanolamine (DMPE).
24. The formulation of claim 1 , wherein the lipids comprise from 30 mole % to 50 mole % cholesterol.
25. The formulation of claim 1 , wherein the lipids comprise from 1 mole % to 6 mole % PEG-conjugated lipid.
26. The formulation of claim 25 , and wherein the PEG-conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.
27. The formulation of claim 1 , wherein the lipids comprise:
at most 30 mole % zwitterionic lipid,
from 30 mole % to 50 mole % cholesterol, and
from 1 mole % to 6 mole % PEG-conjugated lipid,
wherein the zwitterionic lipid is DSPC, and
wherein the PEG-conjugated lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.
28. The formulation of claim 1 , wherein the first species and the second species of RNA molecules further comprise a 5′ cap nucleoside, a triphosphate bridge, and a 5′ first ribonucleoside; wherein the 5′ cap nucleoside is linked 5′-to-5′ to the 5′ first ribonucleoside by the triphosphate bridge; and wherein the first ribonucleoside comprises a 2′-methylated ribose.
29. The formulation of claim 1 , wherein the lipids comprise from 40 mole % to 60 mole % tertiary amine cationic lipid.
30. The formulation of claim 29 , wherein the lipids comprise from 35 mole % to 50 mole % cholesterol.