METHODS OF ELICITING AN IMMUNE RESPONSE
Provided are methods for eliciting an immune response, the methods comprising administering a vaccine to a subject. 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 method of eliciting an immune response against an immunogen in a mammal, the method comprising administering to the mammal an effective amount of a formulation to elicit the immune response, the formulation comprising:
ribonucleic acid (RNA) molecules comprising a sequence that encodes the immunogen; and
lipid nanoparticles (LNPs) comprising lipids comprising first lipids, second lipids, polyethylene glycol-conjugated (PEG-conjugated) lipids, and cholesterol; wherein:
at least half of the RNA molecules are comprised within the LNPs;
the second lipids comprise anionic lipids or neutral zwitterionic lipids;
the first lipids comprise a tertiary amine and have a pKa from 5.0 to 7.6;
at least half of the first lipids are neutrally charged when the first lipids are at a pH that is above the pKa; and
at least half of the first lipids are positively charged when the first lipids are at a pH that is below the pKa; and
whereby the pKa is defined by the following:
(1) admixing the first lipids with ethanol and fluorescent probe 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS), thereby obtaining a lipid/TNS mixture;
(2) separately admixing each of a plurality of a sodium salt buffer with a portion of the lipid/TNS mixture, wherein the sodium salt buffer comprises 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, wherein each of the plurality of the sodium salt buffer has a different pH and the plurality of the sodium salt buffer has a range of pH from 4.4 to 11.12, thereby obtaining a plurality of pH-varied lipid/TNS mixtures;
(3) measuring the absolute 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 plurality of the pH-varied lipid/TNS mixtures, thereby obtaining an absolute fluorescence for each of the plurality of the pH-varied lipid/TNS mixtures;
(4) measuring the absolute 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 an empty vessel used in the measuring of (3), thereby obtaining a blank fluorescence;
(5) subtracting the blank fluorescence from each of the absolute fluorescences of the plurality of the pH-varied lipid/TNS mixtures, thereby obtaining a blank-subtracted fluorescence for each of the plurality of the pH-varied lipid/TNS mixtures;
(6) normalizing each of the blank-subtracted fluorescences of the plurality of the pH-varied lipid/TNS mixtures to the blank-subtracted fluorescence of the pH-varied lipid/TNS mixture that was obtained from the admixing in (2) with the sodium salt buffer that had the lowest pH of the first sodium salt buffers, thereby obtaining a relative fluorescence for each of the plurality of the pH-varied lipid/TNS mixtures, the relative fluorescence being 1 for the pH-varied lipid/TNS mixture that was obtained from the admixing in (2) with the sodium salt buffer that had the lowest pH of the first sodium salt buffers;
(7) obtaining a line of best fit of the pHs of the sodium salt buffers versus the respective relative fluorescences of the plurality of pH-varied lipid/TNS mixtures; and
(8) defining the pKa as the pH on the line of best fit at which a relative fluorescence of 0.5 is obtained.
2 . The method of claim 1 , wherein the immunogen comprises two or more different immunogens.
3 . The method of claim 1 , wherein the immunogen comprises a viral immunogen, a bacterial immunogen, a fungal immunogen, or a parasitic immunogen.
4 . The method of claim 3 , wherein the viral immunogen comprises a hepadnavirus immunogen and the immune response is at least against hepadnavirus.
5 . The method of claim 3 , wherein the viral immunogen comprises a herpesvirus immunogen and immune response is at least against herpesvirus.
6 . The method of claim 3 , wherein the viral immunogen comprises a papillomavirus immunogen and the immune response is at least against papillomavirus.
7 . The method of claim 3 , wherein the viral immunogen comprises a coronavirus immunogen and the immune response is at least against coronavirus.
8 . The method of claim 3 , wherein the viral immunogen comprises a cytomegalovirus immunogen and the immune response is at least against cytomegalovirus.
9 . The method of claim 3 , wherein the viral immunogen comprises an Epstein-Barr virus immunogen and the immune response is at least against Epstein-Barr virus.
10 . The method of claim 3 , wherein the bacterial immunogen comprises a Helicobacter pylori immunogen and the immune response is at least against Helicobacter pylori.
11 . The method of claim 3 , wherein the fungal immunogen comprises a Malassezia spp. immunogen and the immune response is at least against Malassezia spp.
12 . The method of claim 1 , wherein the immunogen comprises a tumor antigen and the immune response is at least against a tumor expressing the tumor antigen.
13 . The method of claim 1 , wherein the immunogen comprises a viral immunogen, and wherein the viral immunogen comprises a polyomavirus immunogen, an oncovirus immunogen, a lentivirus immunogen, a flavivirus immunogen, an orthomyxovirus immunogen, a paramyxovirus immunogen, or a picornavirus immunogen.
14 . The method of claim 1 , wherein from 0.5 ml to 1.0 ml of the formulation is administered.
15 . The method of claim 1 , wherein the method comprises administering multiple unit doses of the formulation four weeks apart to the mammal.
16 . The method of claim 1 , further comprising administering a booster dose of the effective amount of the formulation to the mammal.
17 . The method of claim 16 , wherein administering the booster dose to the mammal is at least 6 months after administering the effective amount of the formulation.
18 . The method of claim 1 , wherein the immune response comprises an antibody response.
19 . The method of claim 18 , wherein the antibody response comprises a neutralizing antibody response.
20 . The method of claim 1 , wherein the LNPs comprise from 35 mol % to 50 mol % of the cholesterol.
21 . The method of claim 1 , wherein the LNPs comprise from 1 mol % to 6 mol % of the PEG-conjugated lipids.
22 . The method of claim 1 , wherein the immune response comprises a CD8+ T cell response.
23 . The method of claim 22 , wherein the CD8+ T cell response results in production of cytokines in the mammal.
24 . The method of claim 23 , wherein the cytokines comprise IFNγ, TNF-α, or IL-2.
25 . The method of claim 1 , wherein the PEG-conjugated lipids comprise a PEG that has a molecular weight of 2000 Daltons.
26 . The method of claim 1 , wherein the PEG-conjugated lipids comprise 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.
27 . The method of claim 1 , wherein the neutral zwitterionic lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine or dimyristoyl phosphatidylethanolamine.
28 . The method of claim 1 , wherein the RNA molecules further comprise a 7′-methylguanosine, a tri-phosphate bridge, and a 5′ first ribonucleoside, and wherein the 7′-methylguanosine is linked 5′-to-5′ to the 5′ first ribonucleoside by the triphosphate bridge.
29 . The method of claim 1 , wherein the pKa is from 5.6 to 6.8.
30 . The method of claim 25 , wherein the PEG-conjugated lipids comprise 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.