Broad spectrum influenza virus vaccine

Abstract

The disclosure relates to broad spectrum influenza virus ribonucleic acid (RNA) vaccines, as well as methods of using the vaccines and compositions comprising the vaccines.

1 - 20 . (canceled) 
     
     
         21 . An influenza virus vaccine, comprising:
 at least one ribonucleic acid polynucleotide having an open reading frame encoding at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, formulated in a lipid nanoparticle, wherein   (i) the at least one RNA polypeptide is encoded by a nucleic acid sequence identified by any one of SEQ ID NO: 505-523 or 570-573, and/or   (ii) the at least one RNA polypeptide comprises a nucleic acid sequence identified by any one of SEQ ID NO: 524-542 or 566-569, and/or   (iii) the at least one antigenic polypeptide comprises an amino acid sequence identified by any one of SEQ ID NO: 543-565.   
     
     
         22 . The vaccine of  claim 21 , wherein the vaccine is multivalent. 
     
     
         23 . (canceled) 
     
     
         24 . A multiple consensus subtype vaccine comprising at least one messenger ribonucleic acid (mRNA) polynucleotide having an open reading frame encoding at least one influenza virus antigenic polypeptide, the influenza virus antigenic polypeptide selected from a nucleoprotein (NP), a neuraminidase (NA) protein, a matrix protein 1 (M1), a matrix protein 2 (M2), a nonstructural 1 (NS1) protein, and nonstructural 2 (NS2) protein, wherein the vaccine provides cross-reactivity against a variety of influenza strains, formulated in a lipid nanoparticle-comprising compounds of Formula (I): 
       
         
           
           
               
               
           
         
         or a salt or isomer thereof, wherein: 
         R 1  is selected from the group consisting of C 5-30  alkyl, C 5-20  alkenyl, —R*YR″, —YR″, and —R″M′R′; 
         R 2  and R 3  are independently selected from the group consisting of H, C 1-14  alkyl, C 2-14  alkenyl, —R*YR″, —YR″, and —R*OR″, or R 2  and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle; 
         R 4  is selected from the group consisting of a C 3-6  carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR,-CQ(R) 2 , and unsubstituted C 1-6  alkyl, where Q is selected from a carbocycle, heterocycle, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —N(R) 2 , —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —N(R)R 8 , —O(CH 2 ) n OR, —N(R)C(═NR 9 )N(R) 2 , —N(R)C(═CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O) 2 R, —N(OR)C(O)OR, —N(OR)C(O)N(R) 2 , —N(OR)C(S)N(R) 2 , —N(OR)C(═NR 9 )N(R) 2 , —N(OR)C(═CHR 9 )N(R) 2 , —C(═NR 9 )N(R) 2 , —C(═NR 9 )R, —C(O)N(R)OR, and —C(R)N(R) 2 C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; 
         each R 5  is independently selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H; 
         each R 6  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(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group; 
         R 7  is selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H; 
         R 8  is selected from the group consisting of C 3-6  carbocycle and heterocycle; 
         R 9  is selected from the group consisting of H, CN, NO 2 , C 1-6  alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6  alkenyl, C 3-6  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, 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 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. 
       
     
     
         25 . The vaccine of  claim 24 , wherein a subset of compounds of Formula (I) includes those in which when R 4  is —(CH 2 ) n Q, —(CH 2 ) n CHQR, —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. 
     
     
         26 . The vaccine of  claim 24 , wherein a subset of compounds of Formula (I) includes those in which
 R 1  is selected from the group consisting of C 5-30  alkyl, C 5-20  alkenyl, —R*YR″, —YR″, and —R″M′R′;   R 2  and R 3  are independently selected from the group consisting of H, C 1-14  alkyl, C 2-14  alkenyl, —R*YR″, —YR″, and —R*OR″, or R 2  and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;   R 4  is selected from the group consisting of a C 3-6  carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR,-CQ(R) 2 , and unsubstituted C 1-6  alkyl, where Q is selected from a C 3-6  carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —CRN(R) 2 C(O)OR, —N(R)R 8 , —O(CH 2 ) n OR, —N(R)C(═NR 9 )N(R) 2 , —N(R)C(═CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O) 2 R, —N(OR)C(O)OR, —N(OR)C(O)N(R) 2 , —N(OR)C(S)N(R) 2 , —N(OR)C(═NR 9 )N(R) 2 , —N(OR)C(═CHR 9 )N(R) 2 , —C(═NR 9 )N(R) 2 , —C(═NR 9 )R, —C(O)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 (═O), OH, amino, mono- or di-alkylamino, and C 1-3  alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;   each R 5  is independently selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H;   each R 6  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(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;   R 7  is selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H;   R 8  is selected from the group consisting of C 3-6  carbocycle and heterocycle;   R 9  is selected from the group consisting of H, CN, NO 2 , C 1-6  alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6  alkenyl, C 3-6  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, 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 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.   
     
     
         27 . The vaccine of  claim 24 , wherein a subset of compounds of Formula (I) includes those in which
 R 1  is selected from the group consisting of C 5-30  alkyl, C 5-20  alkenyl, —R*YR″, —YR″, and —R″M′R′;   R 2  and R 3  are independently selected from the group consisting of H, C 1-14  alkyl, C 2-14  alkenyl, —R*YR″, —YR″, and —R*OR″, or R 2  and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;   R 4  is selected from the group consisting of a C 3-6  carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR,-CQ(R) 2 , and unsubstituted C 1-6  alkyl, where Q is selected from a C 3-6  carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —CRN(R) 2 C(O)OR, —N(R)R 8 , —O(C H 2 ) n OR, —N(R)C(═NR 9 )N(R) 2 , —N(R)C(═CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O) 2 R, —N(OR)C(O)OR, —N(OR)C(O)N(R) 2 , —N(OR)C(S)N(R) 2 , —N(OR)C(═NR 9 )N(R) 2 , —N(OR)C(═CHR 9 )N(R) 2 , —C(═NR 9 )R, —C(O)N(R)OR, and —C(═NR 9 )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) R 4  is —(CH 2 ) n Q in which n is 1 or 2, or (ii) R 4  is —(CH 2 ) n CHQR in which n is 1, or (iii) R 4  is-CHQR, and -CQ(R) 2 , then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocycloalkyl;   each R 5  is independently selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H;   each R 6  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(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;   R 7  is selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H;   R 8  is selected from the group consisting of C 3-6  carbocycle and heterocycle;   R 9  is selected from the group consisting of H, CN, NO 2 , C 1-6  alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6  alkenyl, C 3-6  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, 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 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.   
     
     
         28 . The vaccine of  claim 24 , wherein a subset of compounds of Formula (I) includes those in which
 R 1  is selected from the group consisting of C 5-30  alkyl, C 5-20  alkenyl, —R*YR″, —YR″, and —R″M′R′;   R 2  and R 3  are independently selected from the group consisting of H, C 1-14  alkyl, C 2-14  alkenyl, —R*YR″, —YR″, and —R*OR″, or R 2  and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;   R 4  is selected from the group consisting of a C 3-6  carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR,-CQ(R) 2 , and unsubstituted C 1-6  alkyl, where Q is selected from a C 3-6  carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —CRN(R) 2 C(O)OR, —N(R)R 8 , —O(C H 2 ) n OR, —N(R)C(═NR 9 )N(R) 2 , —N(R)C(═CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O) 2 R, —N(OR)C(O)OR, —N(OR)C(O)N(R) 2 , —N(OR)C(S)N(R) 2 , —N(OR)C(═NR 9 )N(R) 2 , —N(OR)C(═CHR 9 )N(R) 2 , —C(═NR 9 )R, —C(O)N(R)OR, and —C(═NR 9 )N(R) 2 , and each n is independently selected from 1, 2, 3, 4, and 5;   each R 5  is independently selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H;   each R 6  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(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;   R 7  is selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H;   R 8  is selected from the group consisting of C 3-6  carbocycle and heterocycle;   R 9  is selected from the group consisting of H, CN, NO 2 , C 1-6  alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6  alkenyl, C 3-6  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, 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 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.   
     
     
         29 . The vaccine of  claim 24 , wherein subset of compounds of Formula (I) includes those in which
 R 1  is selected from the group consisting of C 5-30  alkyl, C 5-20  alkenyl, —R*YR″, —YR″, and —R″M′R′;   R 2  and R 3  are independently selected from the group consisting of H, C 2-14  alkyl, C 2-14  alkenyl, —R*YR″, —YR″, and —R*OR″, or R 2  and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;   R 4  is —(CH 2 ) n Q or —(CH 2 )·CHQR, where Q is —N(R) 2 , and n is selected from 3, 4, and 5;   each R 5  is independently selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H;   each R 6  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(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;   R 7  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 C 1-12  alkyl and C 1-12  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.   
     
     
         30 . The vaccine of  claim 24 , wherein a subset of compounds of Formula (I) includes those in which
 R 1  is selected from the group consisting of C 5-30  alkyl, C 5-20  alkenyl, —R*YR″, —YR″, and —R″M′R′;   R 2  and R 3  are independently selected from the group consisting of C 1-14  alkyl, C 2-14  alkenyl, —R*YR″, —YR″, and —R*OR″, or R 2  and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;   R 4  is selected from the group consisting of —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, and -CQ(R) 2 , where Q is —N(R) 2 , and n is selected from 1, 2, 3, 4, and 5;   each R 5  is independently selected from the group consisting of C 1-3  alkyl, C 2-3  alkenyl, and H;   each R 6  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(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;   R 7  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 C 1-12  alkyl and C 1-12  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.   
     
     
         31 . The vaccine of  claim 24 , wherein a subset of compounds of Formula (I) includes those of Formula (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; M 1  is a bond or M′; R 4  is unsubstituted C 1-3  alkyl, or —(CH 2 ) n Q, in which Q is OH, —NHC(S)N(R) 2 , —NHC(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)R 8 , —NHC(═NR9)N(R)2, —NHC(═CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and R 2  and R 3  are independently selected from the group consisting of H, C 1-14  alkyl, and C 2-14  alkenyl. 
     
     
         32 . The vaccine of  claim 24 , wherein the consensus hemagglutinin antigen is selected from the group consisting of influenza hemagglutinin 1 (HA1) and/or hemagglutinin 2 (HA2). 
     
     
         33 . The vaccine of  claim 24 , wherein the vaccine comprises at least two mRNA, each encoding at least one influenza virus antigenic polypeptide and wherein at least one influenza virus antigenic polypeptide is HA1, HA2, or a combination of HA1 and HA2, and at least one influenza virus antigenic polypeptide is selected from the group consisting of neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1) and non-structural protein 2 (NS2). 
     
     
         34 . The vaccine of  claim 33 , wherein at least one influenza virus antigenic polypeptide is HA1 and at least one influenza virus antigenic polypeptide is selected from the group consisting of NA, NP, M1, M2, NS1 and NS2. 
     
     
         35 . The vaccine of  claim 34 , wherein at least one influenza virus antigenic polypeptide is HA2 and at least one antigenic polypeptides is selected from the group consisting of NA, NP, M1, M2, NS1 and NS2. 
     
     
         36 . The vaccine of  claim 33 , wherein the at least one influenza virus antigenic polypeptide is from influenza virus strain H1/PuertoRico/8/1934, H1/New Caledonia/20/1999, H1/California/04/2009, H5/Vietnam/1194/2004, H2/Japan/305/1957, H9/Hong Kong/1073/99, H3/Aichi/2/1968, H3/Brisbane/10/2007, H7/Anhui/1/2013, H10/Jiangxi-Donghu/346/2013, H3/Wisconsin/67/2005, H1/Vietnam/850/2009, or a combination thereof. 
     
     
         37 . (canceled) 
     
     
         38 . The vaccine of  claim 24 , wherein the lipid nanoparticle further comprises a PEG-modified lipid, a sterol, and a non-cationic lipid. 
     
     
         39 . The vaccine of  claim 38 , wherein the lipid nanoparticle comprises a molar ratio of about 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and 5-25% non-cationic lipid. 
     
     
         40 . The vaccine of  claim 39 , wherein the non-cationic lipid is a neutral lipid and the sterol is a cholesterol. 
     
     
         41 . The vaccine of  claim 24 , wherein the nanoparticle has a polydispersity value of less than 0.4. 
     
     
         42 . The vaccine of  claim 24 , wherein the nanoparticle has a net neutral charge at a neutral pH value. 
     
     
         43 . The vaccine of  claim 24 , wherein the at least one RNA polynucleotide comprises at least one chemical modification. 
     
     
         44 . The vaccine of  claim 43 , wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. 
     
     
         45 . A method of inducing an immune response in a subject, the method comprising administering to the subject the vaccine of  claim 24  in an amount effective to produce an antigen-specific immune response in the subject. 
     
     
         46 . The method of  claim 45 , wherein the antigen specific immune response comprises a T cell response or a B cell response. 
     
     
         47 - 69 . (canceled) 
     
     
         70 . A method of inducing cross-reactivity against a variety of influenza strains in a mammal, the method comprising administering to the mammal in need thereof the vaccine of  claim 24 . 
     
     
         71 . The method of  claim 70 , wherein at least two ribonucleic acid (RNA) polynucleotides having an open reading frame each encoding a consensus hemagglutinin antigen are administered to the mammal separately. 
     
     
         72 . The method of  claim 70 , wherein at least two ribonucleic acid (RNA) polynucleotides having an open reading frame each encoding a consensus hemagglutinin antigen are administered to the mammal simultaneously. 
     
     
         73 - 75 . (canceled)