CORONAVIRUS VACCINE COMPOSITIONS AND METHODS
Provided herein are nucleic acid molecules encoding viral replication proteins and antigenic coronavirus proteins or fragments thereof. Also provided herein are compositions that include nucleic acid molecules encoding viral replication and antigenic proteins, and lipids. Nucleic acid molecules provided herein are useful for inducing immune responses.
1 . A nucleic acid molecule comprising:
(i) a first polynucleotide encoding one or more viral replication proteins, wherein the first polynucleotide is codon-optimized as compared to a wild-type polynucleotide encoding the one or more viral replication proteins, and wherein the first polynucleotide comprises a sequence having at least 80% identity to the sequence of SEQ ID NO:72; and
(ii) a second polynucleotide comprising a transgene encoding an antigenic protein or a fragment thereof, wherein the antigenic protein is a coronavirus protein.
2 .- 8 . (canceled)
9 . The nucleic acid molecule of claim 1 , further comprising
(a) a 5′ untranslated region (UTR); or
(b) a 5′ untranslated region (UTR) comprising an alphavirus 5′ UTR sequence; or
(c) 5′ untranslated region (UTR) comprising a sequence having at least 80% identity to the sequence of SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75; or
(d) a 3′ untranslated region (UTR); or
(e) a 3′ untranslated region (UTR) comprising an alphavirus 3′ UTR sequence; or
(f) a 3′ untranslated region (UTR) comprising an alphavirus 3′ UTR sequence, wherein the 3′ UTR further comprises a poly-A sequence.
10 .- 19 . (canceled)
20 . The nucleic acid molecule of claim 1 , wherein the antigenic protein is a SARS-CoV-2 protein, a SARS-CoV-2 spike glycoprotein, or a variant SARS-CoV-2 spike glycoprotein.
21 .- 28 . (canceled)
29 . The nucleic acid molecule of claim 1 , wherein the first polynucleotide is located 5′ of the second polynucleotide.
30 . The nucleic acid molecule of claim 29 , further comprising
(a) an intergenic region located between the first polynucleotide and the second polynucleotide; or
(b) an intergenic region located between the first polynucleotide and the second polynucleotide, wherein the intergenic region comprises a sequence having at least 85% identity to the sequence of SEQ ID NO:77.
31 . (canceled)
32 . The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule is
(a) a DNA molecule; or
(b) an RNA molecule, wherein T is substituted with U.
33 . The nucleic acid molecule of claim 32 , wherein the DNA molecule further comprises a promoter, wherein the promoter is a T7 promoter, a T3 promoter, or an SP6 promoter.
34 . (canceled)
35 . (canceled)
36 . The nucleic acid molecule of claim 32 , wherein the RNA molecule is a self-replicating RNA molecule.
37 . The nucleic acid molecule of claim 32 , wherein the RNA molecule further comprises a 5′ cap having a Cap 1 structure, a Cap 1 ( m6 A) structure, a Cap 2 structure, or a Cap 0 structure.
38 .- 75 . (canceled)
76 . A composition comprising the nucleic acid molecule of claim 1 and a lipid formulation.
77 .- 80 . (canceled)
81 . The composition of claim 76 , wherein the lipid formulation is a lipid nanoparticle.
82 .- 87 . (canceled)
88 . The composition of claim 76 , wherein the lipid formulation comprises an ionizable cationic lipid.
89 . The composition of claim 88 , wherein the ionizable cationic lipid has a structure of Formula I:
or a pharmaceutically acceptable salt or solvate thereof, wherein R 5 and R 6 are each independently selected from the group consisting of a linear or branched C 1 -C 31 alkyl, C 2 -C 31 alkenyl or C 2 -C 31 alkynyl and cholesteryl; L 5 and L 6 are each independently selected from the group consisting of a linear C 1 -C 20 alkyl and C 2 -C 20 alkenyl; X 5 is —C(O)O—, whereby —C(O)O—R 6 is formed or —OC(O)— whereby —OC(O)—R 6 is formed; X 6 is —C(O)O— whereby —C(O)O—R 5 is formed or —OC(O)— whereby —OC(O)—R 5 is formed; X 7 is S or O; L 7 is absent or lower alkyl; R 4 is a linear or branched C 1 -C 6 alkyl; and R 7 and R 8 are each independently selected from the group consisting of a hydrogen and a linear or branched C 1 -C 6 alkyl.
90 . (canceled)
91 . The composition of claim 88 , wherein the ionizable cationic lipid has a structure of:
or a pharmaceutically acceptable salt thereof.
92 . The composition of claim 76 , wherein the lipid formulation encapsulates the nucleic acid molecule or is complexed to the nucleic acid molecule.
93 . (canceled)
94 . The composition of claim 76 , wherein the lipid formulation comprises
(a) a helper lipid;
(b) a phospholipid;
(c) a polyethylene glycol (PEG)-lipid conjugate; or
(d) any combination thereof.
95 . (canceled)
96 . The composition of claim 94 , wherein the phospholipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC).
97 .- 101 . (canceled)
102 . The composition of claim 76 , wherein the lipid portion of the lipid formulation comprises about 40 mol % to about 60 mol % of t+e an ionizable cationic lipid, about 4 mol % to about 16 mol % DSPC, about 30 mol % to about 47 mol % cholesterol, and about 0.5 mol % to about 3 mol % PEG2000-DMG.
103 . (canceled)
104 . (canceled)
105 . The composition of claim 76 , wherein the composition has a total lipid:nucleic acid molecule weight ratio of about 50:1 to about 10:1.
106 .- 109 . (canceled)
110 . The composition of claim 76 , wherein the composition comprises
(a) a HEPES or TRIS buffer at a pH of about 7.0 to about 8.5;
(b) a HEPES or TRIS buffer at a concentration of about 7 mg/mL to about 15 mg/mL;
(c) about 2.0 mg/mL to about 4.0 mg/mL of NaCl;
(d) one or more cryoprotectants;
(e) one or more cryoprotectants selected from sucrose, glycerol, or a combination of sucrose and glycerol; or
(f) any combination thereof.
111 .- 115 . (canceled)
116 . The composition of claim 76 , wherein
(a) the composition is a lyophilized composition; or
(b) the composition is a lyophilized composition comprising one or more lyoprotectants: or
(c) the composition is a lyophilized composition comprising a poloxamer, potassium sorbate, sucrose, or any combination thereof.
117 .- 119 . (canceled)
120 . The composition of claim 116 , wherein the lyophilized composition comprises
(a) about 0.01% to about 1.0% w/w of the nucleic acid molecule;
(b) about 1.0% to about 5.0% w/w lipids;
(c) about 0.5% to about 2.5% w/w of TRIS buffer;
(d) about 0.75% to about 2.75% w/w of NaCl;
(e) about 85% to about 95% w/w of a sugar;
(f) about 0.01% to about 1.0% w/w of a poloxamer;
(g) about 1.0% to about 5.0% w/w of potassium sorbate; or
(h) any combination thereof.
121 .- 132 . (canceled)
133 . A method of administering the composition of claim 76 to a subject in need thereof, wherein the composition is lyophilized and is reconstituted prior to administration.
134 . A method of preventing or ameliorating COVID-19, comprising administering the composition of claim 76 to a subject in need thereof.
135 . (canceled)
136 . (canceled)
137 . A method of administering a booster dose to a vaccinated subject, comprising administering the composition of claim 76 to a subject who was previously vaccinated against coronavirus.
138 . The method of claim 134 , wherein
(a) the composition is administered at a dosage of about 0.01 μg to about 1,000 μg of nucleic acid; or
(b) wherein the composition is administered at a dosage of about 1, 2, 5, 7.5, or 10 μg of nucleic acid.
139 . (canceled)
140 . A method of inducing an immune response against a coronavirus in a subject comprising:
administering to the subject an effective amount of the nucleic acid molecule of claim 1 .
141 . (canceled)
142 . A method of inducing an immune response in a subject comprising:
administering to the subject an effective amount of the composition of claim 76 .
143 .- 145 . (canceled)