Ri is selected from the group consisting of C_5-30 alkyl, C_5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R₂ and R₃ are independently selected from the group consisting of H, Ci-₁₄ alkyl, C_2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R₃, together with the atom to which they are attached, form a heterocycle or carbocycle;

R₄ is selected from the group consisting of a C_3-6 carbocycle, -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, -CQ(R)₂, and unsubstituted C_1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -0(CH₂)nN(R)₂, -C(0)OR, -OC(0)R, -CX3, -CX₂H, -CXH₂, -CN, -N(R)₂, -C(0)N(R)2, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -N(R)R₈, -0(CH₂)nOR, -N(R)C(=NR₉)N(R)2, -N(R)C(=CHR₉)N(R)₂, -OC(0)N(R)₂, -N(R)C(0)OR, -N(OR)C(0)R, -N(OR)S(0)₂R, -N(OR)C(0)OR, -N(OR)C(0)N(R)₂, -N(OR)C(S)N(R)₂, -N(0R)C(=NR₉)N(R)₂, -N(0R)C(=CHR₉)N(R)₂, -C(=NR₉)N(R)2, -C(=NR₉)R, -C(0)N(R)OR, and -C(R)N(R)₂C(0)OR, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

-N(R’)C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(OR’)0-, -S(0)₂-, -S-S-, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

R₈ is selected from the group consisting of C_3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C_1-6 alkyl, -OR, -S(0)₂R, -S(0)₂N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. In some embodiments, a subset of compounds of Formula (I) includes those in which when R₄ is -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, or -CQ(R)₂, then (i) Q is not -N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, another subset of compounds of Formula (I) includes those in which

Ri is selected from the group consisting of C_5-30 alkyl, C₅-_2o alkenyl, -R*YR”, -YR”, and -R”M’R’;

R₂ and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C_2-i4 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is selected from the group consisting of a C3-6 carbocycle, -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, -CQ(R)₂, and unsubstituted C_1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -0(CH₂)„N(R)₂, -C(0)OR, -OC(0)R, -CX3, -CX₂H, -CXH₂, -CN, -C(0)N(R)₂, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)C(0)N(R)₂, -N(R)C(S)N(R)₂, -CRN(R)₂C(0)OR, -N(R)R₈, -0(CH₂)„OR, -N(R)C(=NR₉)N(R)₂, -N(R)C(=CHR₉)N(R)₂, -OC(0)N(R)₂, -N(R)C(0)OR, -N(OR)C(0)R, -N(OR)S(0)₂R, -N(OR)C(0)OR, -N(OR)C(0)N(R)₂, -N(OR)C(S)N(R)₂, -N(OR)C(=NR₉)N(R)₂, -N(OR)C(=CHR₉)N(R)₂, -C(=NR₉)N(R)₂, -C(=NR₉)R, -C(0)N(R)OR, and a 5- to 14-membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S which is substituted with one or more substituents selected from oxo (=0), OH, amino, mono- or di-alkylamino, and Ci-3 alkyl, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H;

M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

-N(R’)C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(0R’)0-, -S(0)₂-, -S-S-, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H;

R₈ is selected from the group consisting of C3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, N0₂, C_1-6 alkyl, -OR, -S(0)₂R, -S(0)₂N(R)₂, C_2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and

H; each R’ is independently selected from the group consisting of C_1-18 alkyl, C_2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

Ri is selected from the group consisting of C_5-30 alkyl, C_5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

R2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is selected from the group consisting of a C3-6 carbocycle, -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, -CQ(R)2, and unsubstituted C_1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -0(CH₂)nN(R)₂, -C(0)OR, -OC(0)R, -CX3, -CX₂H, -CXH₂, -CN, -C(0)N(R)₂, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -CRN(R)₂C(0)OR, -N(R)R₈, -0(CH₂)_nOR, -N(R)C(=NR₉)N(R)2, -N(R)C(=CHR₉)N(R)₂, -0C(0)N(R)2, -N(R)C(0)OR, -N(OR)C(0)R, -N(OR)S(0)₂R, -N(OR)C(0)OR, -N(0R)C(0)N(R)2, -N(0R)C(S)N(R)2, -N(0R)C(=NR₉)N(R)₂, -N(0R)C(=CHR₉)N(R)₂, -C(=NR₉)R, -C(0)N(R)OR, and -C(=NR₉)N(R)₂, and each n is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is -(CH₂)_nQ in which n is 1 or 2, or (ii) R₄ is -(CH₂)_nCHQR in which n is 1, or (iii) R₄ is -CHQR, and -CQ(R)2, then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocyclo alkyl ; each R₅ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

R₈ is selected from the group consisting of C3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, NO2, C_1-6 alkyl, -OR, -S(0)2R, -S(0)₂N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C_1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of C_1-12 alkyl and C_2-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

R2 and R3 are independently selected from the group consisting of H, C_1-14 alkyl, C_2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is selected from the group consisting of a C_3-6 carbocycle, -(CH₂ )_nQ, -(CFH2)_nCHQR, -CHQR, -CQ(R)2, and unsubstituted C_1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -0(CH₂)nN(R)₂, -C(0)OR, -OC(0)R, -CX₃, -CX₂H, -CXH₂, -CN, -C(0)N(R)₂, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -CRN(R)₂C(0)OR, -N(R)R₈, -0(CH₂)_nOR, -N(R)C(=NR₉)N(R)2, -N(R)C(=CHR₉)N(R)₂, -0C(0)N(R)2, -N(R)C(0)OR, -N(OR)C(0)R, -N(OR)S(0)₂R, -N(OR)C(0)OR, -N(0R)C(0)N(R)2, -N(0R)C(S)N(R)2,

-N(0R)C(=NR₉)N(R)₂, -N(0R)C(=CHR₉)N(R)₂, -C(=NR₉)R, -C(0)N(R)0R, and -C(=NR₉)N(R)₂, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H;

M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

R₈ is selected from the group consisting of C3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, N0₂, C_1-6 alkyl, -OR, -S(0)₂R, -S(0)₂N(R)₂, C_2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C₂-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-is alkyl, C_2-is alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-i₂ alkyl and C_2-i2 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

Ri is selected from the group consisting of C_5-30 alkyl, C_5-2o alkenyl, -R*YR”, -YR”, and -R”M’R’;

R₂ and R3 are independently selected from the group consisting of H, C_2-i4 alkyl, C_2-i4 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R₄ is -(CH₂)_nQ or -(CH₂)_nCHQR, where Q is -N(R)₂, and n is selected from 3, 4, and 5; each R₅ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

R₇ is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C_1-18 alkyl, C_2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-₁₂ alkyl and Ci-₁₂ alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

R₂ and R₃ are independently selected from the group consisting of Ci-₁₄ alkyl, C_2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R₃, together with the atom to which they are attached, form a heterocycle or carbocycle;

R₄ is selected from the group consisting of -(CH₂)_nQ, -(C H₂)_nCHQR, -CHQR, and -CQ(R)₂, where Q is -N(R)₂, and n is selected from 1, 2, 3, 4, and 5; each R₅ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R₆ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-,

R₇ is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C_1-18 alkyl, C_2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C_3-14 alkyl and C_3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and Ci-12 alkenyl; each Y is independently a C_3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes those of Formula

(IA):

(IA), or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or M’; R₄ is unsubstituted C_1-3 alkyl, or -(CH2)_nQ, in which Q is OH, -NHC(S)N(R)2, -NHC(0)N(R)2, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)RS, -NHC(=NR₉)N(R)₂, -NHC(=CHR₉)N(R)₂, -0C(0)N(R)2, -N(R)C(0)0R, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-, -P(0)(0R’)0-, -S-S-, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, and C2-14 alkenyl.

(II):

(II) or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; Mi is a bond or M’; R₄ is unsubstituted C_1-3 alkyl, or -(CH2)_nQ, in which n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)₂, -NHC(0)N(R)₂, -N(R)C(0)R, -N(R)S(0)₂R, -N(R)R₈, -NHC(=NR₉)N(R)₂, -NHC(=CHR₉)N(R)₂, -0C(0)N(R)₂, -N(R)C(0)0R, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(0)0-, -OC(O)-, -C(0)N(R’)-, -P(0)(0R’)0-, -S-S-, an aryl group, and a heteroaryl group; and R₂ and R3 are independently selected from the group consisting of H, Ci-14 alkyl, and C_2-i4 alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes those of Formula (Da), (lib), (lie), or (IIe):

or a salt or isomer thereof, wherein R₄ is as described herein.

(lid):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R’, R”, and R2 through R₆ are as described herein. For example, each of R2 and R3 may be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.

In some embodiments, an ionizable cationic lipid of the disclosure comprises heptadecan- 9-yl 8 ((2 hydroxyethyl)(6 oxo 6-(undecyloxy)hexyl)amino)octanoate. Thus, in some embodiments, an ionizable cationic lipid of the disclosure comprises a compound having structure:

In some embodiments, an ionizable cationic lipid of the disclosure comprises a compound having structure:

In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-0-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,l,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1 -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

In some embodiments, a PEG modified lipid of the disclosure comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is DMG-PEG, PEG-c- DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG.

In some embodiments, a sterol of the disclosure comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof.

In some embodiments, a LNP of the disclosure comprises an ionizable cationic lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG.

In some embodiments, the lipid nanoparticle comprises 45 - 55 mole percent (mol%) ionizable cationic lipid. For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable cationic lipid.

In some embodiments, the lipid nanoparticle comprises 5 - 15 mol% DSPC. For example, the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.

In some embodiments, the lipid nanoparticle comprises 35 - 40 mol% cholesterol. For example, the lipid nanoparticle may comprise 35, 36, 37, 38, 39, or 40 mol% cholesterol.

In some embodiments, the lipid nanoparticle comprises 1 - 2 mol% DMG-PEG. For example, the lipid nanoparticle may comprise 1, 1.5, or 2 mol% DMG-PEG.

In some embodiments, the lipid nanoparticle comprises 50 mol% ionizable cationic lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

In some embodiments, a LNP of the disclosure comprises an N:P ratio of from about 2:1 to about 30:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio of about 6:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio of about 3:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of from about 10:1 to about 100:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 20:1. In some embodiments, a LNP of the disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10:1.

In some embodiments, a LNP of the disclosure has a mean diameter from about 50 nm to about 150 nm.

In some embodiments, a LNP of the disclosure has a mean diameter from about 70 nm to about 120 nm.

Multivalent Vaccines

The compositions, as provided herein, may include RNA or multiple RNAs encoding two or more antigens of the same or different species. In some embodiments, composition includes an RNA or multiple RNAs encoding two or more coronavirus antigens. In some embodiments, the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more coronavirus antigens.

In some embodiments, two or more different RNA (e.g., mRNA) encoding antigens may be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNA encoding antigens may be formulated in separate lipid nanoparticles (each RNA formulated in a single lipid nanoparticle). The lipid nanoparticles may then be combined and administered as a single vaccine composition (e.g., comprising multiple RNA encoding multiple antigens) or may be administered separately.

Combination Vaccines

The compositions, as provided herein, may include an RNA or multiple RNAs encoding two or more antigens of the same or different virus(es) or viral strain(s). In some embodiments, a composition includes RNA encoding at least one coronavirus antigen and at least one antigen of a different virus. In some embodiments, a composition includes RNA encoding at a first coronavirus antigen and a second coronavirus antigen, wherein the first and second coronavirus antigens are different from each other. Thus, compositions (e.g., RNA vaccines) of the present disclosure may target one or more antigen(s) of the same strain/species, or one or more antigen(s) of different strains/species, e.g., antigens which induce immunity to organisms which are found in the same geographic areas where the risk of coronavirus infection is high or organisms to which an individual is likely to be exposed to when exposed to a coronavirus.

Pharmaceutical Formulations

Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention or treatment of coronavirus in humans and other mammals, for example. The compositions provided herein can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and/or treat a coronavirus infection.

In some embodiments, the coronavirus vaccine containing RNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide (antigen).

An “effective amount” of a composition (e.g., comprising RNA) is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the RNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject. Typically, an effective amount of a composition provides an induced or boosted immune response as a function of antigen production in the cells of the subject. In some embodiments, an effective amount of the composition containing RNA polynucleotides having at least one chemical modifications are more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation and/or expression from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.

The term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable carrier," after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

In some embodiments, the compositions (comprising polynucleotides and their encoded polypeptides) in accordance with the present disclosure may be used for treatment or prevention of a coronavirus infection. A composition may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the amount of RNA provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

The vaccine may be administered to seropositive or seronegative subjects. For example, a subject may be naive and not have antibodies that react with a virus having an antigen, wherein the antigen is the viral antigen or fragment thereof encoded by the mRNA of the vaccine (e.g., a SARS-CoV-2 antigen disclosed herein). Such a subject is said to be seronegative with respect to that vaccine. Alternatively, the subject may have preexisting antibodies to viral antigen encoded by the mRNA of the vaccine because they have previously had an infection with virus carrying the antigen or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against the antigen (e.g., a SARS-CoV-2 antigen disclosed herein). Such a subject is said to be seropositive with respect to that vaccine. In some instances the subject may have been previously exposed to a virus but not to a specific variant or strain of the virus or a specific vaccine associated with that variant or strain. Such a subject is considered to be seronegative with respect to the specific variant or strain.

Thus, the present disclosure provides compositions (e.g., mRNA vaccines) that elicit potent neutralizing antibodies against an antigen (e.g., a SARS-CoV-2 antigen disclosed herein) in a subject. Such a composition can be administered to seropositive or seronegative subjects in some embodiments. A seronegative subject may be naive and not have antibodies that react with the specific virus (e.g., SARS-CoV-2) which the subject is being immunized against. A seropositive subject may have preexisting antibodies to the specific virus (e.g., SARS-CoV-2) because they have previously had an infection with that virus, variant or strain or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against that virus, variant, or strain.

In some embodiments, an initial dose is administered followed by a booster dose. A booster dose is a dose that is given at a certain interval after completion of the primary dose or series of doses that is/are intended to boost immunity to, and therefore prolong protection against, the disease (e.g., COVID-19) that is to be prevented. A booster dose may be given after an earlier administration of an immunizing composition. The time of administration between the initial administration of an immunizing composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month (e.g., 28 days, 29 days, 30 days, or 31 days), 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years. In exemplary embodiments, the time of administration between the initial administration of the immunizing composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year.

In some embodiments, a composition may be administered intramuscularly, intranasally or intradermally, similarly to the administration of inactivated vaccines known in the art.

A composition may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As a non-limiting example, the RNA vaccines may be utilized to treat and/or prevent a variety of infectious disease. RNA vaccines have superior properties in that they produce much larger antibody titers, better neutralizing immunity, produce more durable immune responses, and/or produce responses earlier than commercially available vaccines.

Provided herein are immunizing compositions including RNA and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.

The RNA may be formulated or administered alone or in conjunction with one or more other components. For example, an immunizing composition may comprise other components including, but not limited to, adjuvants.

In some embodiments, an immunizing composition does not include an adjuvant (they are adjuvant free).

An RNA may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, vaccine compositions comprise at least one additional active substances, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety). In some embodiments, an immunizing composition is administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the RNA vaccines or the polynucleotides contained therein, for example,

RNA polynucleotides (e.g., mRNA polynucleotides) encoding antigens.

Formulations of the vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA polynucleotide) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

In some embodiments, an RNA is formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

Dosing/Administration

Provided herein are immunizing compositions (e.g., RNA vaccines), methods, kits and reagents for prevention and/or treatment of coronavirus infection in humans and other mammals. Immunizing compositions can be used as therapeutic or prophylactic agents. In some embodiments, immunizing compositions are used to provide prophylactic protection from coronavirus infection. In some embodiments, immunizing compositions are used to treat a coronavirus infection. In some embodiments, embodiments, immunizing compositions are used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.

A subject may be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject.

In some embodiments, an immunizing composition (e.g., RNA a vaccine) is administered to a subject (e.g., a mammalian subject, such as a human subject) in an effective amount to induce an antigen-specific immune response. The RNA encoding the coronavirus antigen is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject.

Prophylactic protection from a coronavirus can be achieved following administration of an immunizing composition (e.g., an RNA vaccine) of the present disclosure. Immunizing compositions can be administered once, twice, three times, four times or more but it is likely sufficient to administer the vaccine once (optionally followed by a single booster). It is possible, although less desirable, to administer an immunizing compositions to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

A method of eliciting an immune response in a subject against a coronavirus antigen (or multiple antigens) is provided in aspects of the present disclosure. In some embodiments, a method involves administering to the subject an immunizing composition comprising a RNA (e.g., mRNA) having an open reading frame encoding a coronavirus antigen, thereby inducing in the subject an immune response specific to the coronavirus antigen, wherein anti-antigen antibody titer in the subject is increased following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the antigen. An “anti-antigen antibody” is a semm antibody the binds specifically to the antigen.

A prophylactically effective dose is an effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine. A traditional vaccine, as used herein, refers to a vaccine other than the mRNA vaccines of the present disclosure. For instance, a traditional vaccine includes, but is not limited, to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc. In exemplary embodiments, a traditional vaccine is a vaccine that has achieved regulatory approval and/or is registered by a national dmg regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA).

In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the coronavirus or an unvaccinated subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the coronavirus or an unvaccinated subject.

A method of eliciting an immune response in a subject against a coronavirus is provided in other aspects of the disclosure. The method involves administering to the subject an immunizing composition (e.g., an RNA vaccine) comprising a RNA polynucleotide comprising an open reading frame encoding a coronavirus antigen, thereby inducing in the subject an immune response specific to the coronavirus, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the coronavirus at 2 times to 100 times the dosage level relative to the immunizing composition.

In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times, 5 times, 10 times, 50 times, or 100 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure.

In other embodiments, the immune response is assessed by determining [protein] antibody titer in the subject. In other embodiments, the ability of serum or antibody from an immunized subject is tested for its ability to neutralize viral uptake or reduce coronavirus transformation of human B lymphocytes. In other embodiments, the ability to promote a robust T cell response(s) is measured using art recognized techniques.

Other aspects the disclosure provide methods of eliciting an immune response in a subject against a coronavirus by administering to the subject an immunizing composition (e.g., an RNA vaccine) comprising an RNA having an open reading frame encoding a coronavirus antigen, thereby inducing in the subject an immune response specific to the coronavirus antigen, wherein the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the coronavirus. In some embodiments, the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to an immunizing composition of the present disclosure.

In some embodiments, the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

Also provided herein are methods of eliciting an immune response in a subject against a coronavirus by administering to the subject an RNA having an open reading frame encoding a first antigen, wherein the RNA does not include a stabilization element, and wherein an adjuvant is not co-formulated or co-administered with the vaccine.

An immunizing composition (e.g., an RNA vaccine) may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous administration. The present disclosure provides methods comprising administering RNA vaccines to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The RNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the RNA may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

The effective amount of the RNA, as provided herein, may range from about 50 μg - 500 pg, administered as a single dose or as multiple (e.g., booster) doses. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 50 μg mRNA. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 100 μg mRNA. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises at least 25 pg mRNA. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises less than 100 μg mRNA. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises 100 μg or less mRNA. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 250 μg mRNA.

In some embodiments, a total amount of mRNA administered to a subject is about 50 μg, about 100 μg, about 200 μg, about 250 μg, or about 500 μg mRNA. In some embodiments, a total amount of mRNA administered to a subject is about 50 μg. In some embodiments, a total amount of mRNA administered to a subject is about 100 μg. In some embodiments, a total amount of mRNA administered to a subject is about 250 μg. In some embodiments, a total amount of mRNA administered to a subject is about 500 μg.

The RNA described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

Vaccine Efficacy

Some aspects of the present disclosure provide formulations of the immunizing compositions (e.g., RNA vaccines), wherein the RNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to a coronavirus antigen). “An effective amount” is a dose of the RNA effective to produce an antigen- specific immune response. Also provided herein are methods of inducing an antigen- specific immune response in a subject.

As used herein, an immune response to a vaccine or LNP of the present disclosure is the development in a subject of a humoral and/or a cellular immune response to a (one or more) coronavirus protein(s) present in the vaccine. For purposes of the present disclosure, a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T- lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells. One important aspect of cellular immunity involves an antigen- specific response by cytolytic T- cells (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves and antigen- specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and/or other white blood cells including those derived from CD4+ and CD8+ T-cells.

In some embodiments, the antigen- specific immune response is characterized by measuring an anti-coronavirus antigen antibody titer produced in a subject administered an immunizing composition as provided herein. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen or epitope of an antigen. Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.

In some embodiments, an antibody titer is used to assess whether a subject has had an infection or to determine whether immunizations are required. In some embodiments, an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections. In accordance with the present disclosure, an antibody titer may be used to determine the strength of an immune response induced in a subject by an immunizing composition (e.g., RNA vaccine).

In some embodiments, an anti-coronavirus antigen antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, anti-coronavirus antigen antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to a control. In some embodiments, the anti-coronavirus antigen antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control. In some embodiments, the anti-coronavirus antigen antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the anti-coronavirus antigen antibody titer produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.

In some embodiments, the anti-coronavirus antigen antibody titer produced in a subject is increased at least 2 times relative to a control. For example, the anti-coronavirus antigen n antibody titer produced in a subject may be increased at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times relative to a control. In some embodiments, the anti-coronavirus antigen antibody titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control. In some embodiments, the anti-coronavirus antigen antibody titer produced in a subject is increased 2-10 times relative to a control. For example, the anti-coronavirus antigen antibody titer produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.

In some embodiments, antibody-mediated immunogenicity in a subject is assessed at one or more time points (e.g., Day 1 - Day 196, e.g., Day 1, Day 8, Day 29, Day 57, and/or Day 196). Methods of assessing antibody-mediated immunogenicity are known and include geometric mean concentration (GMC) of antibody to antigen, geometric mean fold rise (GMFR) in serum antibody, geometric mean titer (GMT), median, minimum, maximum, 95% confidence interval (Cl), geometric mean ratio (GMR) of post-baseline / baseline titers, and seroconversion rate.

The GMC is the average antibody concentration for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data. GMT is the average antibody titer for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data.

A control, in some embodiments, is an anti-coronavirus antigen antibody titer produced in a subject who has not been administered an immunizing composition (e.g., RNA vaccine), or who has been administered a saline placebo (un unvaccinated subject). In some embodiments, a control is an anti-coronavirus antigen antibody titer produced in a subject administered a recombinant or purified protein vaccine (e.g., protein subunit vaccine). Recombinant protein vaccines typically include protein antigens that either have been produced in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of the pathogenic organism. A control may be, for example, a subject administered a live attenuated viral vaccine or an inactivated viral vaccine.

In some embodiments, the ability of an immunizing composition (e.g., RNA vaccine) to be effective is measured in a murine model. For example, an immunizing composition may be administered to a murine model and the murine model assayed for induction of neutralizing antibody titers. Viral challenge studies may also be used to assess the efficacy of a vaccine of the present disclosure. For example, an immunizing composition may be administered to a murine model, the murine model challenged with virus, and the murine model assayed for survival and/or immune response (e.g., neutralizing antibody response, T cell response (e.g., cytokine response)).

In some embodiments, an effective amount of an immunizing composition (e.g., RNA vaccine) is a dose that is reduced compared to the standard of care dose of a recombinant protein vaccine. A “standard of care,” as provided herein, refers to a medical or psychological treatment guideline and can be general or specific. “Standard of care” specifies appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician/ clinician should follow for a certain type of patient, illness or clinical circumstance. A “standard of care dose,” as provided herein, refers to the dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent coronavirus infection or a related condition, while following the standard of care guideline for treating or preventing coronavirus infection or a related condition.

In some embodiments, the anti-coronavirus antigen antibody titer produced in a subject administered an effective amount of an immunizing composition is equivalent to an anti- coronavirus antigen antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine.

Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1 ;201 ( 11 ) : 1607 -10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas:

Efficacy = (ARU - ARV)/ARU x 100; and

Efficacy = (1-RR) x 100.

Likewise, vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1 ;201 ( 11 ) : 1607 -10). Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination:

Effectiveness = (1 - OR) x 100.

In some embodiments, efficacy of the immunizing composition (e.g., RNA vaccine) is at least 60% relative to unvaccinated control subjects. For example, efficacy of the immunizing composition may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100% relative to unvaccinated control subjects.

Sterilizing Immunity. Sterilizing immunity refers to a unique immune status that prevents effective pathogen infection into the host. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 1 year. For example, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 2 years, at least 3 years, at least 4 years, or at least 5 years. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject at an at least 5-fold lower dose relative to control. For example, the effective amount may be sufficient to provide sterilizing immunity in the subject at an at least 10-fold lower, 15-fold, or 20-fold lower dose relative to a control.

Detectable Antigen. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to produce detectable levels of coronavirus antigen as measured in serum of the subject at 1-72 hours post administration.

Titer. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti-coronavirus antigen). Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.

In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to produce a 1,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the coronavirus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 1,000-5,000 neutralizing antibody titer produced by neutralizing antibody against the coronavirus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 5,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the coronavirus antigen as measured in serum of the subject at 1-72 hours post administration.

In some embodiments, the neutralizing antibody titer is at least 100 NT50. For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NT50. In some embodiments, the neutralizing antibody titer is at least 10,000 NT50.

In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL). For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU/mL. In some embodiments, the neutralizing antibody titer is at least 10,000 NU/mL.

In some embodiments, an anti-coronavirus antigen antibody titer produced in the subject is increased by at least 1 log relative to a control. For example, an anti-coronavirus antigen antibody titer produced in the subject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 log relative to a control.

In some embodiments, an anti-coronavirus antigen antibody titer produced in the subject is increased at least 2 times relative to a control. For example, an anti-coronavirus antigen antibody titer produced in the subject is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 times relative to a control.

In some embodiments, a geometric mean, which is the nth root of the product of n numbers, is generally used to describe proportional growth. Geometric mean, in some embodiments, is used to characterize antibody titer produced in a subject.

EXAMPLES

Example 1: Phase 2 Clinical Trial

Study Design and Methodology

The study was randomized, observer-blind, and placebo-controlled, with adult participants at least 18 years of age.

Two dose levels, 50 μg and 100 μg, were evaluated in this study, based in part on initial safety data from the Phase 1 Division of Microbiology and Infectious Diseases (DMID) study of a SARS-CoV-2 mRNA vaccine (SEQ ID NO: 6, ORF SEQ ID NO: 7, encoding S protein SEQ ID NO: 8). In the present study, the SARS-CoV-2 mRNA vaccine (SEQ ID NO: 6, ORF SEQ ID NO: 7, encoding the prefusion stabilized S protein of SARS-CoV-2, SEQ ID NO: 8) was encapsulated in a lipid nanoparticle as described herein. The study included 2 age cohorts: Cohort 1 with 300 participants (> 18 to < 55 years old) and Cohort 2 with 300 participants (> 55 years old). Approximately 600 participants received either the SARS CoV-2 mRNA vaccine or saline placebo control according to a 1:1:1 randomization ratio; i.e., within each age cohort,

100 participants received 50 μg SARS CoV-2 mRNA, 100 participants received 100 μg SARS CoV-2 mRNA, and 100 participants received saline placebo.

The study was initiated with a parallel enrollment of all 300 participants in Cohort 1 (> 18 to < 55 years old) and a sentinel group of 50 participants in Cohort 2 (> 55 years old) receiving study treatment. Before initiating study treatment of the remaining participants in Cohort 2, safety data through Day 7 from the sentinel group of Cohort 2 and all available data from Cohort 1 was reviewed by the Safety Monitoring Committee (SMC).

In addition to the SMC’s review, prior to expansion in Cohort 2, there was a pause for the review of the following:

• Safety data through Day 7 from the sentinel group of Cohort 2

• All available safety data from Cohort 1

As no safety concerns were found, expansion enrollment (N=250) of Cohort 2 proceeded.

The full study comprised 10 scheduled study site visits: Screening, Day 1, Day 8, Day 15, Day 29 (Month 1), Day 36, Day 43, Day 57 (Month 2), Day 197 (Month 7), and Day 365 (Month 13). There were also scheduled biweekly safety phone calls to collect medically attended adverse events (MAAEs), adverse events (AEs) leading to withdrawal, serious AEs (SAEs), concomitant medications associated with these events, and receipt of non-study vaccinations. These phone calls were scheduled biweekly from Day 71 through Day 183 and from Day 211 through Day 351. The study duration was approximately 14 months for each participant: a screening period of up to 1 month and a study period of 13 months that included the first dose of vaccine on Day 1 and the second dose on Day 29. The participant's final visit was on Day 365 (Month 13), 12 months after the second dose of vaccine on Day 29 (Month 1).

To test for the presence of SARS-CoV-2, nasal swab samples were collected at the Screening Visit (Day 0) and also at Day 1, Day 29, and Day 57. During the course of the study, participants meeting pre-specified disease criteria that suggested possible SARS-CoV-2 infection were asked to contact the study site to arrange for a prompt, thorough, and careful assessment.

Each participant received 2 injections of SARS CoV-2 mRNA vaccine or placebo by 0.5 ml intramuscular (IM) injection on Day 1 and Day 29 to the same arm (e.g., the nondominant arm). Normal saline was used to dilute the doses prior to administration. Participants were monitored for at least 60 minutes post-injection. Assessments included vital sign measurements and monitoring of local and/or systemic reactions. Vaccine accountability, dose preparation, and vaccine administration was performed by unblinded pharmacy personnel who did not participate in any other aspects of the study. The remainder of the study staff, all participants, and Sponsor personnel (or its designees) remained blinded to dosing assignment.

All participants were followed for safety and reactogenicity and provide pre- and post-injection blood specimens for immunogenicity through 12 months after the last dose of investigational product. There were 2 planned interim analyses.

The end of study (EOS) was defined as completion of the last visit of the last participant in the study or the last scheduled procedure for the last participant in this study. Participants were considered to have completed the study if they complete the final visit on Day 365 (Month 13),

12 months after the second injection on Day 29 (Month 1).

At each dosing visit, participants were instructed (Day 1) or reminded (Day 29) how to document and report solicited adverse reactions (ARs) within a provided electronic diary (eDiary). Solicited ARs were assessed for 7 days (the day of injection and the following 6 days) after each injection and unsolicited AEs were assessed for 28 days after each injection; SAEs and MAAEs were assessed throughout the study. Further, the results of safety laboratory tests, vital sign measurements, physical examination findings, and assessments for SARS-CoV-2 infection from day 1 through study completion were analyzed. Solicited local ARs included pain, erythema, and swelling/induration at the injection site, and localized axillary swelling or tenderness ipsilateral to the injection arm. Solicited systemic ARs were headache, fatigue, myalgia, arthralgia, nausea/vomiting, rash, fever, and chills. The Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials (DHHS 2007) was used in this study with modification for rash, solicited ARs, and vital signs.

Participants have blood sampled at 9 scheduled study site visits during the study, for safety and immunogenicity assessments or other medical concerns according to the investigator’s judgment. In addition, participants may have blood sampled at unscheduled visits for acute respiratory symptoms.

Objectives

The primary objectives were to (1) evaluate the safety and reactogenicity of 2 dose levels of the SARS-CoV-2 mRNA vaccine (SEQ ID NO: 6, ORF SEQ ID NO: 7, encoding S protein SEQ ID NO: 8), each administered in 2 doses 28 days apart, and (2) evaluate the immunogenicity of 2 dose levels of the SARS-CoV-2 vaccine, each administered in 2 doses 28 days apart, as assessed by the titer of specific binding antibody (bAb). The secondary objective was to evaluate the immunogenicity of 2 dose levels of the SARS-CoV-2 vaccine, each administered in 2 doses 28 days apart, as assessed by the titer of neutralizing antibody (nAb).

The exploratory objectives were to (1) profile the relative proportion of S protein- specific serum immunoglobulin G (IgG), (2) describe the ratio or profile of specific bAb relative to nAb in serum, (3) describe initial immunogenicity responses following the first dose (Day 1) and prior to the second dose (Day 29), and (4) characterize the clinical profile and immune response of participants infected by SARS-CoV-2.

Study Population

Participants (males and females 18 years of age or older at time of consent) were included in the study if they are in good health according to the assessment of the investigator and can comply with study procedures. Negative pregnancy tests were required at Screening and before vaccine administration for female participants of childbearing potential.

Safety Assessments

Safety assessments included monitoring and recording of the following for each participant:

• Solicited local and systemic ARs that occur during the 7 days following each injection (ie, the day of injection and 6 subsequent days). Solicited ARs were recorded daily using eDiaries.

• Unsolicited AEs observed or reported during the 28 days following each injection (i.e., the day of injection and 27 subsequent days). Unsolicited AEs are AEs that were not included in the protocol-defined solicited ARs.

• AEs leading to discontinuation from dosing and/or study participation from Day 1 through Day 365 or withdrawal from the study.

MAAEs from Day 1 through Day 365 or withdrawal from the study.

SAEs from Day 1 through Day 365 or withdrawal from the study.

Results of safety laboratory tests.

Vital sign measurements.

Physical examination findings.

Assessments for SARS CoV 2 infection from Day 1 through study completion.

Safety and reactogenicity were assessed by clinical review of all relevant parameters including solicited ARs (local and systemic events), unsolicited AEs, SAEs, MAAEs, AEs leading to discontinuation, safety laboratory test results, vital signs, and physical examination findings. Solicited ARs and unsolicited AEs were coded by system organ class (SOC) and preferred term according to the Medical Dictionary for Regulatory Activities (MedDRA) for Adverse Reaction Terminology. The Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventative Vaccine Clinical Trials was used in this study with modification for rash, solicited ARs, unsolicited AE, and vital signs.

Rash are graded in the following manner:

• Grade 0 = no rash

• Grade 1 = localized without associated symptoms

• Grade 2 = maculopapular rash covering <50% body surface area

• Grade 3 = urticarial rash covering > 50% body surface area

• Grade 4 = generalized exfoliative, ulcerative or bullous dermatitis.

All safety analyses were based on the Safety Set, except summaries of solicited ARs which are based on the Solicited Safety Set. All safety analyses are provided by age cohort unless otherwise specified.

The number and percentage of participants with any solicited local AR, with any solicited systemic AR and with any solicited AR during the 7 day follow-up period after each injection were provided with a 2 sided 95% exact confidence interval (Cl) using the Clopper- Pearson method.

Number and percentage of participants with unsolicited AEs, SAEs, MAAEs, Grade 3 or higher ARs and AEs, and AEs leading to discontinuation from study vaccine or participation in the study were summarized.

Number of events of solicited ARs, unsolicited AEs/SAEs, and MAAEs were reported in summarization tables accordingly.

For all other safety parameters, descriptive summary statistics were provided.

For treatment-emergent safety laboratory tests results, the raw values and change from baseline values were summarized by age cohort, injection group and visit at each timepoint.

The number and percentage of participants who have chemistry, hematology, coagulation, and vital signs results below or above the laboratory normal ranges are tabulated by timepoint.

Demographic variables (e.g., age, height, weight, and body mass index (BMI)) and baseline characteristics were summarized by injection group for each age cohort (when appropriate) by descriptive statistics (mean, standard deviation for continuous variable, and number and percentage for categorical variables). Immunogenicity Assessments

Immunogenicity assessments included the following:

• Serum bAb titer against SARS-CoV-2 as measured by enzyme-linked immunosorbent assay (ELISA) specific to the SARS CoV 2 spike protein

• Serum nAb titer against SARS-CoV-2 as measured by pseudovirus and/or live virus neutralization assays

The analyses of immunogenicity were based on the Per-Protocol (PP) Set. For each age cohort, if the number of participants in the Full Analysis Set (FAS) and PP Set differ (defined as the difference divided by the total number of participants in the PP Set) by more than 10%, supportive analyses of immunogenicity may be conducted using the FAS.

For the primary immunogenicity endpoint, geometric mean titer (GMT) of specific bAb with corresponding 95% Cl at each timepoint and geometric mean fold rise (GMFR) of specific bAb with corresponding 95% Cl at each post-baseline timepoint over pre-injection baseline at Day 1 were provided by injection group and age cohort. Descriptive summary statistics including median, minimum, and maximum were also provided.

For the secondary immunogenicity endpoint, GMT of specific nAb with corresponding 95% Cl at each timepoint and GMFR of specific nAb with corresponding 95% Cl at each postbaseline timepoint over pre-injection baseline at Day 1 were provided by injection group and age cohort. Descriptive summary statistics including median, minimum, and maximum were also provided. For summarizations of GMT values, antibody values reported as below the limit of detection (LOD) or lower limit of quantification (LLOQ) arweree replaced by 0.5 x LOD or 0.5 x LLOQ. Values that are greater than the upper limit of quantification (ULOQ) were converted to the ULOQ.

The number and percentage of participants with GMFR > 2, GMFR > 3, and GMFR > 4 of serum SARS-CoV-2-specific nAb titers and participants with seroconversion from baseline were provided with 2-sided 95% Cl using the Clopper-Pearson method at each post baseline timepoint. Seroconversion at a participant level was defined as a change of nAb titer from below the LOD or LLOQ to equal to or above LOD or LLOQ (respectively), or a 4-times or higher log- transformed titer ratio in participants with pre-existing nAb titers.

Exploratory analyses of each dose level of t versus placebo on bAb and nAb titers may be performed.

Vaccine, Dosage, and Route of Administration

The SARS-CoV-2 mRNA vaccine (SEQ ID NO: 6, ORF SEQ ID NO: 7, encoding S protein SEQ ID NO: 8) is a lipid nanoparticle (LNP) dispersion of an mRNA encoding the prefusion stabilized spike protein SARS-CoV-2 formulated in LNPs composed of four (4) lipids (50 mol% ionizable lipid heptadecan-9-yl 8 ((2 hydroxy ethyl) (6 oxo 6- (undecyloxy)hexyl)amino)octanoate (Compound 1); 10 mol% 1,2 distearoyl sn glycero-3 phosphocholine (DSPC); 38.5 mol% cholesterol; and 1.5 mol% 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG)). The SARS-CoV-2 mRNA vaccine was provided as a sterile liquid for injection, white to off white dispersion in appearance, at a concentration of 0.5 mg/mL in 20 mM Tris buffer containing 87 mg/mL sucrose and 10.7 mM sodium acetate at pH 7.5.

The placebo is 0.9% sodium chloride (normal saline) injection, United States Pharmacopeia (USP).

The SARS-CoV-2 mRNA vaccine was administered as an intramuscular injection into the deltoid muscle on a 2-dose vaccination schedule on Day 1 and Day 29, with at least a 28-day interval between doses. Each vaccination contains 0.5 mL of 50 μg or 100 μg of the SARS- CoV-2 mRNA vaccine or saline placebo. Preferably, the vaccine was administered into the nondominant arm and the second dose of vaccine was administered in the same arm as used for the first dose.

Unblinded pharmacy personnel, who do not participate in any other aspect of the study, performed vaccine accountability, dose preparation, and vaccine administration.

Sample Size

There was no hypothesis testing in this study. The number of proposed participants is considered sufficient to provide a descriptive summary of the safety and immunogenicity of different dose levels of the vaccine.

Approximately 600 participants were randomly assigned in a 1 : 1 : 1 ratio to 50 μg SARS- CoV-2 mRNA vaccine, 100 μg SARS-CoV-2 mRNA vaccine, or placebo. A total of 400 participants receive SARS-CoV-2 mRNA vaccine, 200 participants in each dose level, or 100 participants in each age cohort and dose level. A sample size of 400 has at least a 95% probability to observe at least 1 participant with an AE at a true 0.75% AE rate.

Primary Endpoints Primary Safety Endpoints

The primary safety objective were evaluated by the following safety endpoints:

• Solicited local and systemic ARs through 7 days after each injection.

• Unsolicited AEs through 28 days after each injection.

• MAAEs through the entire study period. • SAEs throughout the entire study period.

• Safety laboratory abnormalities at Day 29 and Day 57 (Cohort 2 only).

• Vital sign measurements and physical examination findings.

Primary Immunogenicity Endpoints

• Titer of SARS-CoV-2-specific binding antibody (bAb) measured by ELISA on Day 1, Day 29 (Ml), Day 43, Day 57 (M2), Day 197 (M7), and Day 365 (M13).

Secondary Endpoints

The secondary objectives were evaluated by the following endpoints:

• Titer of SARS-CoV-2-specific neutralizing antibody (nAb) on Day 1, Day 29 (Ml), Day 43, Day 57 (M2), Day 197 (M7), and Day 365 (M13).

• Seroconversion on Day 29 (Ml), Day 43, Day 57 (M2), Day 197 (M7), and Day 365 (M13) as measured by an increase of SARS-CoV-2-specific nAb titer either from below the limit of detection (LOD) or lower limit of quantification (LLOQ) to equal to or above LOD or LLOQ, or a 4-times higher titer in participants with pre-existing nAb titers.

Exploratory Endpoint

The exploratory endpoints were the following:

• Titers of S protein- specific bAb (IgM and IgG) and nAb in serum collected on Day 15.

• Relative amounts or profiles of S protein- specific bAb and specific nAb levels in serum

• Clinical severity and immune response of participants infected by SARS CoV-2 Detection of IgG Specific to SARS-CoV-2 Spike Protein in Human Serum

The detection and measurement of human IgG specific to SARS-CoV-2 Spike protein was done through a quantitative Enzyme-Linked Immunosorbent Assay (ELISA) developed in collaboration with PPD. Briefly, microtiter plates were coated overnight with commercially available SARS-CoV-2 S-2P Spike Protein (GenScript, Catalog #U578BFC290). After blocking, serum samples were added in duplicate and incubated for 2 hours. Bound antigen-antibody complex was detected using purified goat anti-human IgG horseradish peroxidase (HRP) conjugate. Color was developed with the addition of TMB [3,3’,5,5’-tetramethylbenzidine] substrate and color intensity was measured spectrophotometrically (OD 450nm). The color intensity was directly proportional to the IgG antibody concentration present in the serum sample. Four quality controls and a negative control (blank) were analyzed in duplicate on each assay plate. Data were processed using the PPD Preclarus® LIMS processor, in accordance with PPD standard operating procedures (SOPs). For the qualified assay, the standard curve was generated using the Anti-SARS-CoV Spike Monoclonal Antibody [clone CR3022] (Rockland, Inc., #CUST17). Quantitation of the human IgG antibody to SARS-CoV-2 spike, or antibody concentration (μg/ml), was determined by interpolation from an 11 -point dilution of CR3022 analyzed on each assay plate. Human sera from SARS-CoV-2 convalescent symptomatic patients (N=119), collected at least 15 days after infection confirmed by RT-PCR, served as reference control titers on each assay plate (BioIVT, Westbury, NY, USA and Aalto Bio Reagents Ltd, Dublin, Ireland).

Detection of IgG Specific to SARS-CoV-2 Nucleocapsid Protein in Human Serum

The measurement of human IgG specific to SARS-CoV-2 nucleocapsid protein was also carried out by ELISA developed in collaboration with PPD. Briefly, microtiter plates were coated overnight with commercially available SARS-CoV-2 (2019-nCoV) nucleocapsid protein (NP; full length recombinant protein, MyBiosource, Catalog # MBS596190). After blocking, serum samples were added in duplicate and incubated for 2 hours. Bound antigen-antibody complex was detected using purified goat anti-human IgG horseradish peroxidase (HRP) conjugate. Color was developed by the addition of TMB [3,3’,5,5’-tctramcthylbcnzidinc] substrate and color intensity was measured spectrophotometrically (OD 450nm). The intensity of the color was directly proportional to the IgG antibody concentration present in the serum sample. Quantitation of the human IgG antibody to SARS-CoV-2 NP, or antibody concentration (arbitrary units per ml or AU/ml), was determined by interpolation from an 11 -point standard curve derived from pooled human sera from SARS-CoV-2 convalescent symptomatic patients (N=32) collected at least 15 days post-infection confirmed by RT-PCR, which served as reference control titers on each assay plate, (BioIVT, Westbury, NY, USA and Aalto Bio Reagents Ltd, Dublin, Ireland). In addition, 4 quality controls and a negative control (blank) were analyzed in duplicate on each assay plate.

SARS CoV-2 Microneutralization (MN) Assay

A SARS-CoV-2 MN assay was designed to quantify serum neutralizing antibodies against SARS-CoV-2 using an in situ ELISA readout. A total of 92, 90 and 95 participants sera from cohort 1; and 89, 89, 91 for cohort 2 have contributed both baseline and post-baseline neutralizing antibody data for this analysis. The SARS-CoV-2 MN assay was qualified for the evaluation of human serum and conducted in accordance with the provider laboratory standard operating procedures (SOPs) (Battelle) and with the use of qualified critical reagents. Briefly, dilutions of heat- inactivated serum samples and controls were incubated with an infective SARS- CoV-2 viral stock (isolate WA1/2020) prior to inoculation onto a cell culture plate containing a confluent VERO E6 cell monolayer. Following a 40 to 46-hour incubation, the inoculum was removed and an in situ ELISA performed to detect SARS-CoV-2 antigen. The optical density (OD) value of each sample well was measured using a microplate reader using a wavelength of 405 nm and a 490 nm reference. Finally, the OD values were analyzed using BioAssay in accordance with Battelle’s SOPs. The final reportable value for each sample is the MN50 and an endpoint titer.

Assessment of SARS CoV-2 Infection by Detection of SARS-CoV-2 genomic RNA by RT- PCR in Nasopharyngeal samples

A real-time reverse transcription polymerase chain reaction (rRT-PCR) test was used for the detection of SARS-CoV-2 genomic RNA in patient samples. This qualitative diagnostic assay was granted Emergency Use Authorization (fda.gov/media/136740/download) to the performing Laboratory Viracor Eurofins Clinical Diagnostics. Briefly, nucleic acid extractions were performed using a bioMerieux NucliSENS easyMAG or eMAG instrument with bioMerieux NucliSENS nucleic acid extraction reagents. RNA was reverse transcribed using oligonucleotide primers specific nucleotide sequences of the SARS-CoV-2 N gene, then amplified in the presence of thermostable DNA polymerase (Taq) enzyme and deoxy-nucleotide triphosphates (dNTPs). A dual-labeled oligonucleotide probe to an internal sequence of the amplification product was also present in the RT-PCR reaction mixture with dye label FAM at the 5' end, and a fluorescence-quenching molecule (e.g. Black Hole Quencher 1) at the 3' end of the probe. Additionally, oligonucleotide primers and a TaqMan probe for PCR detection of an internal extraction and amplification control were also present in the SARS-CoV-2 RT-PCR reaction mix. This allowed for the simultaneous detection of internal extraction/amplification control DNA in a multiplex reaction for each sample. Fluorescence intensity for both SARS- CoV-2 amplification and internal control amplification was measured in individual wells during each of the 40 amplification cycles. A sample was considered positive when the signal intensity exceeded a predetermined baseline threshold value based on cycle number, referred to as the cycle threshold CT. Detection of SARS-CoV-2 RNA in a sample was determined by a CT value of 38.

Additional Information on Convalescent Sera

Convalescent sera used in the ELISA spike protein assay was collected from a total of 119 SARS-CoV-2 symptomatic individuals, at least 15 days after a confirmed diagnosis by RT- PCR. Thirty-two samples were collected from symptomatic SARS-CoV-2 patients, also at least 15 days after a confirmed diagnosis by RT-PCR for the convalescent sera used in the microneutralization assay. These samples were included in convalescent sera panels and tested along with the vaccine trial participant samples as comparators for the ELISA-spike and microneutralization vaccine-induced responses. These sera were obtained from BioIVT (Westbury, NY, USA) and Aalto Bio Reagents Ltd (Dublin, Ireland). Results

Over approximately six weeks, 1090 participants were screened and 600 eligible participants were randomized. Of these, 300 participants were included in age cohorts 1 (>18- <55 years) and 2 (>55 years), and randomly assigned to receive 50 or 100 μg of the SARS-CoV- 2 mRNA vaccine or placebo administered as two vaccinations. There were a total of 13 participants who did not receive a second vaccination, including one who experienced an adverse event related to SARS-CoV-2 infection, 5 lost to follow-up, 1 who withdrew consent, 2 adverse events, one of which was considered serious, and 4 due to other reasons.

The baseline characteristics were generally balanced across study vaccine groups in each cohort. Mean ages of the participants were 37.4 (range 18-54) and 64.3 (range 55-87) years in cohorts 1 and 2 respectively, and 59% (cohort 1) and 71% (cohort 2) were females, and the majority of participants were white (92% and 97%).

Solicited local and systemic ARs through day 7 were mainly mild or moderate in severity in both age cohorts after the first and second vaccinations. The incidences of solicited ARs within 7 days post-vaccination were generally dose-dependent and occurred at higher frequencies in participants who received the SARS-CoV-2 mRNA vaccine than placebo. The most commonly reported local AR after vaccination 1 was pain at the injection site by 73% in the 50 μg and 86% in the 100 μg SARS-CoV-2 mRNA vaccine groups, and 14% for placebo in cohort 1, and by 58% at 50 μg SARS-CoV-2 mRNA vaccine, 81% at 100 μg SARS-CoV-2 mRNA vaccine, and 7% for placebo in cohort 2 after vaccination 1. Similarly, after vaccination 2, injection site pain was the most frequent local AR reported by 80% of the 50 μg SARS-CoV-2 mRNA vaccine, 90% of the 100 μg SARS-CoV-2 mRNA vaccine and 9% of the placebo groups in cohort 1, and in 79% on 50 ug SARS-CoV-2 mRNA vaccine and 81% on 100 ug SARS-CoV- 2 mRNA vaccine and 6% on placebo in cohort 2. The most frequent solicited systemic ARs after the first vaccination were headache and fatigue which occurred more often in the SARS-CoV-2 mRNA vaccine group than the placebo groups in both cohorts 1 and 2. The most frequent systemic ARs observed after the second vaccination in both age cohorts, were headache and fatigue. Additionally, incidences of myalgia, arthralgia, nausea/vomiting, and chills increased after the second vaccination in the SARS-CoV-2 mRNA vaccine groups of both cohorts.

Grade 3 local ARs reported after the first vaccination were infrequent and included injection site pain in 1% of participants each at the 50 and 100 μg SARS-CoV-2 mRNA vaccine doses in cohorts 1 and 2, and after the second vaccination, in 2% at the 50 μg and 1% at the 100 pg SARS-CoV-2 mRNA vaccine doses. No grade 3 local ARs were seen for placebo following the first or second vaccinations in either cohort. Additional local grade 3 ARs reported after the second vaccination included erythema (1%) at the 100 mg dose in cohort 1, and in cohort 2, erythema (4%) and swelling (1%) at the 100 mg dose. Systemic grade 3 ARs after the first vaccination included headaches in 1% of participants each in the placebo and 50 μg SARS- CoV-2 mRNA vaccine groups in cohort 1, and 3% for placebo and 2% for 50 μg SARS-CoV-2 mRNA vaccine groups in cohort 2. Additional systemic grade 3 ARs included fatigue (1% each) at 50 and 100 μg SARS-CoV-2 mRNA vaccine doses, and chills (1%) at the 100 μg SARS-CoV- 2 mRNA vaccine dose in cohort 2. The incidence of grade 3 systemic ARs were higher after the second vaccination than the first and included fever (2% and 3%), headache (3% and 4%), fatigue (5% and (11%), myalgia (7% and 11%), arthralgia (5% and 6%), and chills (2% and 1%) in the 50 and 100 μg SARS-CoV-2 mRNA vaccine groups, respectively, and fatigue (2%) for placebo in cohort 1. In cohort 2, grade 3 ARs were fever (1%) at the 100 μg dose, headache (6% and 5%), fatigue (6% and 7%), and myalgia (3% and 4%) at the 50 and 100 μg SARS-CoV- 2 mRNA vaccine doses, respectively, arthralgia (2% each) at the 50 and 100 μg doses, and chills at the 50 μg dose (1%). Mean durations for solicited ARs were similar across the placebo and SARS-CoV-2 mRNA vaccine groups and ranged from 2.4-3.1 days in cohort 1 and 2.1 to-3.7 days in cohort 2 after vaccination 1. Mean duration of ARs were also generally comparable across the SARS-CoV-2 mRNA vaccine and placebo groups after vaccination 2, ranging from 3- 4 days in younger adults (cohort 1) and 1.9-3.4 days in older adults (cohort 2).

There were no observed differences incidences in the reported rates of unsolicited AEs reported through 28 days after each vaccination, regardless of study vaccine, were no different across the SARS-CoV-2 mRNA vaccine and placebo groups in younger adults (77 [26%]) and in older adults (87 [29%]). The majority of AEs were generally mild and moderate in severity, and no deaths or serious AEs were reported. There were 11 (4%) severe events in cohort 1 (younger adults) and 5 (2%) in cohort 2 (older adults). The incidences of MAAEs were similar in cohorts 1 (29 [10%]) and 2 (27 [9%]) and were similar across placebo and SARS-CoV-2 mRNA vaccine doses. Unsolicited AEs related to study vaccine were reported in 31 (10%) participants in cohort 1 and 25 (8%) in cohort 2 and were higher in the SARS-CoV-2 mRNA vaccine than placebo groups. There were no SAEs or deaths, or unsolicited AEs that led to discontinuations from study vaccine or the study. No study pause rules were met. The incidence of study vaccine- related severe AEs was low in both cohorts 1 (7 [2%]) and 2 (<1%) and similar across the placebo and SARS-CoV-2 mRNA vaccine groups. Severe events occurring in >1% participants included fatigue (2%) for placebo, fatigue (1%), arthralgia (2%), and axillary pain (1%) for 50 pg mRNA, and headache (1%) for 100 μg mRNA in cohort 1, and fatigue (1%) for 50 μg mRNA in cohort 2. Study vaccine -related MAAEs also occurred infrequently in cohorts 1 (10 [3%]) and 2 (2 [1%]).

Three participants were symptomatic and had positive nasal swabs for SARS-CoV-2, two of whom had received placebo and one who received 50 μg of SARS-CoV-2 mRNA vaccine. Four participants were found to have positive nasal swabs for SARS-CoV-2 yet remained asymptomatic, three of whom had received placebo and one who received 50 μg of SARS-CoV- 2 mRNA vaccine. No participants in the 100 μg group were diagnosed with SARS-CoV-2. All were referred to the health department for isolation/contact tracing, and notification of the participants primary providers. No clinically significant laboratory abnormalities were reported. None of the placebo and SARS-CoV-2 mRNA vaccine participants had detectable anti-

SARS-CoV-2-spike bAb at baseline. Both doses of mRNA elicited increases in the levels of anti-SARS-CoV-2-spike bAb from baseline by day 29, 28 days after the first vaccination. Anti- SARS-CoV-2-spike bAb reached geometric GM mean (95% Cl) peak levels of 189 (173-207) and 239 (221-258) pg/ml in cohort 1 and 153 (135-175) and 162 (142-185) pg/ml in cohort 2, at doses of 50 and 100 μg, respectively by day 43, 14 days after the second vaccination. The geometric mean (95% Cl) levels of bAb at these doses on day 43 were higher than those of a convalescent sera control (48 [60-138] pg/ml) and remained elevated in cohorts 1 (146 [132-161] and 181 [164-200)] pg/ml) and 2 (107 [93-123)] and 121 [105-139) ] pg/ml) at the 50 and 100 pg doses through 57 days, 28 days after vaccination. The data is shown in Table 2 below.

Table 2. SARS-CoV-2 Spike Binding and Neutralizing Antibody Responses

Neutralizing antibody titers were undetectable at baseline in study participants. After the first vaccination at both the 50 and 100 μg SARS-CoV-2 mRNA vaccine doses, nAb GMTs increased from baseline by day 29, at 28 days post-vaccination 1. Maximum nAb GMTs (95% Cl) were attained in participants who received the 50 and 100 μg SARS-CoV-2 mRNA vaccine doses, respectively in cohorts 1 (1733 [1611-1865] and 1909 [1849-1971]) and 2 (1827 [1722- 1938]) and 1686 [1521-1869]), 14 days after the second vaccination on day 43, that exceeded those of the convalescent control sera (321 [235-438]). Little change in nAb GMTs was observed up to 28 days post-vaccination 2 on day 57, with titers remaining high at the 50 and 100 μg SARS-CoV-2 mRNA vaccine doses in cohorts 1 (1613 [1488-1747]) and 1692 [1586- 1805]) and 2 (1671 [1545-1807]) and 1613 [1460-1782]).

Seroconversion of the SARS-CoV-2-specific nAb responses occurred in 70% (62) and 83% (78) of recipients at the 50 and 100 μg SARS-CoV-2 mRNA vaccine doses respectively in cohort 1, and 61% (48) and 70% (60) in cohort 2 by day 29, 28 days after the first vaccination.

By day 43, 14 days after the second vaccination, seroconversion rates of 100% were observed for all participants tested in both cohorts. Seroconversion rates of 100% were also observed at day 57 in 80 and 82 participants tested on the 50 and 100 μg doses in cohort 1, respectively and 70 on each of those doses in cohort 2. The results are shown in Table 3.

Table 3. Neutralizing antibody geometric mean titers and seroconversion rates

Cl = confidence intervals, GMT = geometric mean titer, nAb=neutralizing antibody, NE=not estimable. Antibody values below the lower limit of quantification (LLOQ) were replaced by 0.5 x LLOQ. Values greater than the upper limit of quantification (ULOQ) were converted to the ULOQ. LLOQ=91.1 and ULOQ=2032. For visit day 29, visit window (-3/+7 days) was used to define per-protocol. If the visit (day 29) was disrupted and could not be completed at day 29 (-3/+7 days) as a result of the COVID-19 pandemic, the window was extended to day 29 + 21 days. Seroconversion at participant level defined as a change of nAb titer from below the lower limit of quantification (LLOQ) to equal to or above LLOQ (respectively), or a 4-times or higher ratio in participants with pre-existing nAb titers.^*Non-missing baseline data. †95% Cl based on the t-distribution of the log- transformed values for GMT back transformed to the original scale for presentation. §n, based on per-protocol set for SARS- CoV-2-specific nAb. ¶Calculated using the Clopper-Pearson method. 14.2.2.1.1.1 , Nov 5, 2020.

A post-hoc exploratory analysis of immunogenicity in subgroups of participants aged >55-<65, >65-74 and >75 years was performed (Tables 4 and 5). Increases in levels of anti- SARS-CoV-2-spike bAb and nAb at days 29 and post-second vaccination at both the 50 and 100 μg doses were generally comparable across the age subgroups and with those observed in the younger (18-55 years) study participants. Seroconversion rates were also comparable across the age groups and with those in the younger participants.

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Discussion

In this randomized-controlled phase 2 trial a SARS-CoV-2 vaccine candidate, administered as a two-dose vaccination regimen at 50 and 100 μg, exhibited robust immune responses and an acceptable safety profile in healthy adults aged 18 years and older. Local and systemic adverse reactions were mostly mild-to-moderate in severity, of <4 days of median duration and were generally reported with lower frequency in the older age groups. Anti-SARS- CoV-2 spike binding and neutralizing antibodies were induced by both doses of the SARS-CoV- 2 vaccine within 28 days after the first vaccination, and rose to peak titers by 14 days after the second vaccination that on average exceeded levels of a convalescent sera from COVID-19 patients, and remained elevated through the last timepoint assessed at 57 days. Seroconversion of neutralizing responses occurred within 28 days after the first vaccination in the majority of participants, with rates of 100% observed by 14 days after the second vaccination. Numeric values of the bAb concentrations and nAb titers were observed to be higher with the 100 μg dose as compared to the 50 μg dose. Overall, the results of this placebo-controlled, dose-confirming trial, extend and confirm previous immunogenicity and safety findings in the phase 1 study for the 50 and 100 μg doses of the SARS-CoV-2 vaccine, in an expanded cohort including participants older than 55 years of age.

The safety and reactogenicity profile of the SARS-CoV-2 vaccine through 28 days after the last dose was consistent with data previously published for other mRNA vaccines, including those of recent COVID-19 studies. Solicited local and generational symptoms were reported more frequently after the SARS-CoV-2 vaccine than the placebo, and were reported at higher frequencies after the second dose. The reported rates in participants >55 years of age tended to be lower than in subject 18-55 years of age, although the sample size was not sufficient to detect significant differences in rates between the cohorts. Unsolicited AEs occurred at similar frequencies across the mRNA groups and placebo groups in both age cohorts. The comparable safety seen in the two age cohorts in this study was also consistent with the results observed in the phase 1 trial of the SARS-CoV-2 mRNA vaccine.

Neutralizing antibody levels have been shown to correlate with protection against viral diseases; however, correlates of response to SARS-CoV-2 in humans is less well-understood. Antibody-mediated immune responses are likely to be effective against SARS-COV-2, based on evidence from studies in non-human primate challenge models and the reported results for both COVID-19 convalescent sera and monoclonal antibody therapies in early clinical trials. In that regard, it is encouraging that vaccination with the SARS-CoV-2 mRNA vaccine elicited similar neutralizing activity in younger and older adults, a finding consistent with those demonstrated in the phase 1 studies for the SARS-CoV-2 vaccine. The robust binding and neutralizing antibody titers observed at both the 50 and 100 ug doses were higher than those in human convalescent serum from COVID-19 patients. This may be attributed to the anti-spike focused response induced by the SARS-CoV-2 mRNA vaccine, whereas convalescent sera represents a broad combination of antibody-recognizing epitopes to SARS-CoV-2.

In conclusion, the results of this phase 2 trial provide additional evidence for the immunogenicity and safety of a 2-dose regimen of the SARS-CoV-2 vaccine candidate, further supporting its development as a prophylactic COVID-19 vaccine. Table 1. Sequences

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,”

“composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

Where a range of values is provided, each value between the upper and lower ends of the range are specifically contemplated and described herein.

The entire contents of International Application Nos. PCT/US2015/02740, PCT/US2016/043348, PCT/US2016/043332, PCT/US2016/058327, PCT/US2016/058324,

PCT/US2016/058314, PCT/US2016/058310, PCT/US2016/058321, PCT/US2016/058297, PCT/US2016/058319, and PCT/US2016/058314 are incorporated herein by reference.

Claims

CLAIMS What is claimed is:

1. A composition comprising:

50 mg - 250 mg of a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) that encodes a SARS-CoV-2 prefusion stabilized spike (S) protein; and a lipid nanoparticle comprising a mixture of lipids that comprises an ionizable cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid.

2. The composition of claim 1 comprising 50 mg of the mRNA.

3. The composition of claim 1 or 2, wherein the SARS-CoV-2 prefusion stabilized S protein comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 8.

4. The composition of claim 3, wherein the SARS-CoV-2 prefusion stabilized S protein comprises the sequence of SEQ ID NO: 8.

5. The composition of any one of the preceding claims, wherein the ORF comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 7.

6. The composition of any one of the preceding claims, wherein the ORF comprises the sequence of SEQ ID NO: 7.

7. The composition of any one of the preceding claims, wherein the mRNA comprises a sequence having at least 90%, at least 95%, or at least 98% identity to the sequence of SEQ ID NO: 6.

8. The composition of claim 7, wherein the mRNA comprises the sequence of SEQ ID NO:

6.

9. The composition of any one of the preceding claims, wherein the mixture of lipids comprises: ionizable lipid heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6- (undecyloxy)hexyl)amino)octanoate (Compound 1); 1,2 distearoyl sn glycero-3 phosphocholine (DSPC); cholesterol; and l-monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG).

10. The composition of any one of the preceding claims, wherein the mixture of lipids comprises: 20-60 mol% ionizable cationic lipid; 5-25 mol% non-cationic lipid; 25-55 mol% sterol; and 0.5-15 mol% PEG-modified lipid.

11. The composition of any one of the preceding claims, wherein the lipid nanoparticle comprises:

47 mol% ionizable cationic lipid; 11.5 mol% neutral lipid; 38.5 mol% sterol; and 3.0 mol% PEG-modified lipid;

48 mol% ionizable cationic lipid; 11 mol% neutral lipid; 38.5 mol% sterol; and 2.5 mol% PEG-modified lipid;

49 mol% ionizable cationic lipid; 10.5 mol% neutral lipid; 38.5 mol% sterol; and 2.0 mol% PEG-modified lipid;

50 mol% ionizable cationic lipid; 10 mol% neutral lipid; 38.5 mol% sterol; and 1.5 mol% PEG-modified lipid; or

51 mol% ionizable cationic lipid; 9.5 mol% neutral lipid; 38.5 mol% sterol; and 1.0 mol% PEG-modified lipid.

12. A composition comprising:

50 mg of a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) that encodes a SARS-CoV-2 prefusion stabilized spike (S) protein, wherein the S protein comprises an amino acid sequence having at least 95% identity to the sequence of SEQ ID NO:

8; and a lipid nanoparticle comprising a mixture of lipids that comprises 20-60 mol% ionizable lipid heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6-(undecyloxy)hexyl)amino)octanoate (Compound 1); 5-25 mol% 1,2 distearoyl sn glycero-3 phosphocholine (DSPC); 25-55 mol% cholesterol; and 0.5-15 mol% l-monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG).

13. The composition of claim 12, wherein the S protein comprise the amino acid sequence of SEQ ID NO: 8.

14. The composition of claim 12 or 13, wherein the ORF comprises a nucleotide sequence having at least 90% or at least 95% identity to the sequence of SEQ ID NO: 7.

15. The composition of claim 14, wherein the ORF comprises the nucleotide sequence of SEQ ID NO: 7.

16. The composition of any one of claims 12-15, wherein the mRNA comprises a nucleotide sequence having at least 90% or at least 95% identity to the sequence of SEQ ID NO: 6.

17. The composition of claim 16, wherein the mRNA comprises the sequence of SEQ ID NO: 6.

18. The composition of any one of claims 12-17, wherein the lipid nanoparticle comprises:

19. The composition of any one of the preceding claims, wherein the mRNA further comprises a 5’ cap analog, optionally a 7mG(5’)ppp(5’)NlmpNp cap.

20. The composition of any one of the preceding claims, wherein the mRNA further comprises a poly(A) tail, optionally having a length of 50 to 150 nucleotides.

21. The composition of any one of the preceding claims, wherein the mRNA comprises a chemical modification, optionally 1-methylpseudouridine.

22. The composition of any one of the preceding claims, wherein the composition further comprises Tris buffer, sucrose, and sodium acetate, or any combination thereof.

23. The composition of claim 22, wherein the composition further comprises 30-40 mM Tris buffer, 80-95 mg/mL sucrose, and 5-15 mM sodium acetate.

24. The composition of any one of the preceding claims, wherein the composition has a pH value of 6-8, optionally 7.5.

25. A method comprising administering to a human subject the composition of any one of the preceding claims to induce in the subject a neutralizing antibody response against SARS-CoV-2.

26. A method comprising administering to a human subject the composition of any one of the preceding claims to increase in the subject a neutralizing antibody response against SARS-CoV-

2.

27. The method of claim 25 or 26, wherein the human subject is seronegative for SARS- CoV-2.

28. The method of claim 25 or 26, wherein the human subject is seropositive for SARS-CoV-

2.

29. A method comprising administering to a human subject a composition comprising

50 mg of a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) that encodes a SARS-CoV-2 prefusion stabilized spike (S) protein; and a lipid nanoparticle comprising a mixture of lipids that comprises an ionizable cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid.

30. The method of claim 29, wherein the human subject is seronegative for SARS-CoV-2.

31. The method of claim 29, wherein the human subject is seropositive for SARS-CoV-2.

32. The method of any one of the preceding claims, wherein the composition is administered to the subject as an initial dose and as a booster dose.

33. The method of claim 32, wherein the booster dose is administered to the subject at least 28 days following the initial dose.

34. The method of any one of the preceding claims, wherein the age of the subject is 18 to 54 years or 55 years or older.

35. The method of any one of the preceding claims, wherein the subject is immunocompromised.

36. The method of any one of the preceding claims, wherein the subject has a chronic pulmonary disease, such as chronic obstructive pulmonary disease (COPD) or asthma.

37. The method of any one of the preceding claims, wherein the subject has an underlying comorbid condition, optionally selected from heart disease, diabetes, and lung disease.

38. The method of any one of the preceding claims, wherein the composition is administered to the subject via intramuscular injection, optionally into a deltoid muscle of the subject.

39. The method of claim 38, wherein the composition induces neutralizing antibody titers.

40. The method of any one of claims 32-39, wherein the composition induces anti-SARS- CoV-2 spike binding and neutralizing antibodies within 28 days after the initial dose.

41. The method of any one of claims 32-40, wherein the composition induces anti-SARS- CoV-2 spike binding and neutralizing antibodies that peak by 14 days after the booster dose.

42. The method of any one of claims 32-41, wherein seroconversion of neutralizing responses is achieved in 100% of subjects by 14 days after administration of the booster dose.