VACCINE FOR ELICITING IMMUNE RESPONSE COMPRISING LIPID NANOPARTICLES AND RNA COMPRISING SEGMENT ENCODING AN IMMUNOGEN
Vaccines for eliciting an immune response are provided. The vaccines include lipid nanoparticles comprising lipids which have a pKa in the range of 5.0 to 7.6 and, preferably, a tertiary amine. These lipid nanoparticles can have essentially neutral surface charge at physiological pH and are effective for immunization.
1 . A formulation comprising:
first ribonucleic acid (RNA) molecules comprising a first sequence, which encodes a first immunogen; and
lipid nanoparticles (LNPs) comprising lipids comprising first lipids, polyethylene glycol-conjugated (PEG-conjugated) lipids, and cholesterol; wherein:
at least half of the first RNA molecules are comprised within the LNPs;
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; an
(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 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 first immunogen comprises a viral immunogen.
4 . The formulation of claim 2 , wherein the first immunogen comprises a bacterial immunogen.
5 . The formulation of claim 1 , wherein the first sequence encodes a second immunogen, which differs from the first immunogen.
6 . The formulation of claim 1 , wherein the PEG-conjugated lipids comprise a PEG that has a molecular weight of 2000 Daltons.
7 . The formulation of claim 6 , wherein the PEG-conjugated lipids comprise 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.
8 . The formulation of claim 1 , wherein the first immunogen comprises a tumor polypeptide.
9 . The formulation of claim 8 , wherein the formulation is immunogenic against the tumor polypeptide.
10 . The formulation of claim 1 , wherein the first RNA molecules further comprise a 3′ poly(adenosine monophosphate) (poly(A)) tail.
11 . The formulation of claim 1 , wherein the first 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.
12 . The formulation of claim 11 , wherein the 5′ first ribonucleoside comprises a 2′-methylated ribose.
13 . The formulation of claim 1 , wherein the cholesterol is from 35 mol % to 50 mol % of the lipids.
14 . The formulation of claim 1 , wherein the first lipids have a pKa from 5.6 to 6.8.
15 . The formulation of claim 1 , wherein the first lipids are from 40 mol % to 60 mol % of the lipids.
16 . The formulation of claim 1 , wherein the formulation further comprises second RNA molecules comprising a second sequence, which encodes a third immunogen, wherein at least half of the second RNA molecules are comprised within the LNPs and the third immunogen differs from the first immunogen.
17 . The formulation of claim 16 , wherein the first immunogen and the third immunogen comprise viral immunogens.
18 . The formulation of claim 1 , wherein the lipids further comprise second lipids comprising anionic lipids or neutral zwitterionic lipids.
19 . The formulation of claim 18 , wherein the neutral zwitterionic lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine.
20 . The formulation of claim 19 , wherein the PEG-conjugated lipids comprise a PEG that has a molecular weight of 2000 Daltons.
21 . The formulation of claim 20 , wherein the PEG-conjugated lipids comprise 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.
22 . The formulation of claim 19 , wherein the cholesterol is from 35 mol % to 50 mol % of the lipids.
23 . The formulation of claim 19 , wherein the first lipids are from 40 mol % to 60 mol % of the lipids.
24 . The formulation of claim 19 , wherein the first RNA molecules further comprise a 3′ poly(A) tail.
25 . The formulation of claim 19 , wherein the first 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.
26 . The formulation of claim 25 , wherein the 5′ first ribonucleoside comprises a 2′-methylated ribose.
27 . The formulation of claim 19 , wherein the first RNA molecules further comprise a replicase sequence, which encodes a replicase, and wherein the first RNA molecules are self-replicating RNA molecules.
28 . The formulation of claim 19 , wherein the formulation is immunogenic in vivo against the immunogen.
29 . The formulation of claim 28 , wherein the formulation elicits at least an antibody response.
30 . The formulation of claim 28 , wherein the formulation elicits at least a cell-mediated immune response.