Self-amplifying RNA encoding influenza virus antigen

Abstract

Self-amplifying RNA (saRNA) molecules encoding influenza virus antigens and methods of using the same are disclosed herein. 【Figure 1】 TIFF2025522946000067.tif83148 TIFF2025522946000068.tif83140

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 359,857, filed Jul. 10, 2022; U.S. Provisional Patent Application No. 63 / 431,462, filed Dec. 9, 2022; and U.S. Provisional Patent Application No. 63 / 484,745, filed Feb. 13, 2023, each of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to compositions and methods for the preparation, manufacture, and therapeutic use of ribonucleic acid vaccines comprising polynucleotide molecules encoding one or more influenza antigens, such as hemagglutinin antigens.

Background Art

[0003] Influenza viruses are members of the Orthomyxoviridae family and are classified into three types (A, B, and C) based on antigenic differences in their nucleoprotein (NP) and matrix (M) proteins.

[0004] The genome of influenza A virus contains eight (seven for influenza C virus) linear, negative - sense, single - stranded RNA molecules, which encode several polypeptides including RNA - dependent RNA polymerase proteins (PB2, PB1, and PA), the nucleoprotein (NP) that forms the nucleocapsid, matrix proteins (M1, M2, which is also a surface - exposed protein embedded in the viral membrane), two surface glycoproteins protruding from the lipoprotein envelope, hemagglutinin (HA) and neuraminidase (NA), and non - structural proteins (NS1 and NS2).

[0005] Hemagglutinin is the major envelope glycoprotein of influenza A and B viruses, and the hemagglutinin - esterase (HE) of influenza C virus is a protein homologous to HA.

[0006] Problems with the treatment and prevention using conventional vaccines against influenza and other infectious diseases are that the scope of the vaccines is limited and protection is provided only against closely related subtypes. Furthermore, the length of time required to complete the current standard influenza virus vaccine manufacturing process hinders the rapid development and production of a matching vaccine in a pandemic situation. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0007] There is a need for an improved composition, preferably an immunogenic composition, against influenza. MEANS FOR SOLVING THE PROBLEM

[0008] In particular, the present disclosure presents an as yet unaddressed need for an improved composition, preferably an immunogenic composition, against influenza. In one aspect, the present disclosure relates to a composition comprising a self-amplifying RNA (saRNA) comprising a 5' cap; a 5' untranslated region (5'UTR); a coding region for a non-structural protein derived from an alphavirus; a first subgenomic promoter derived from an alphavirus; a first open reading frame encoding a first gene of interest derived from influenza virus hemagglutinin (HA); a second subgenomic promoter derived from an alphavirus; a second open reading frame encoding a second gene of interest derived from an influenza virus; a 3' untranslated region (3'UTR); and a 3' polyA sequence.

[0009] In another aspect, the present disclosure relates to a composition comprising a self-amplifying RNA (saRNA) comprising a 5' cap; a 5' untranslated region (5'UTR); a coding region of a non-structural protein derived from an alphavirus; a subgenomic promoter derived from an alphavirus; an open reading frame encoding a gene of interest derived from an influenza virus; a 3' untranslated region (3'UTR); and a 3' polyA sequence, wherein at least 5% of the entire population of specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides.

[0010] In preferred embodiments, the saRNA polynucleotide has clinical-grade purity. In some embodiments, the purity of the RNA polynucleotide is between about 60% and about 100%. In some embodiments, the integrity of the purified RNA polynucleotide is determined by known methods such as capillary electrophoresis and is 60% or more, 70% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In some embodiments, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules in the composition, any one of these, at least any one of these, up to any one of these, or between any two of these, is a full-length RNA transcript. A "full-length" RNA molecule is one that includes a 5' cap and a polyA tail.

[0011] The following figures form a part of this specification and are included to further demonstrate certain aspects of the invention. The invention can be better understood by reference in combination with the detailed description of the specific embodiments presented herein with one or more of these figures.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] Embodiments of the present disclosure provide compositions comprising self-amplifying RNA (saRNA) polynucleotides encoding influenza virus antigens. Using the influenza virus RNA vaccines presented herein, a balanced immune response composed of both cellular and humoral immunity can be induced.

[0014] Any embodiment contemplated herein can be implemented with respect to any method or composition of the present disclosure, and vice versa is also intended. Further, the methods of the present disclosure can be realized using the compositions of the present disclosure.

[0015] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0016] Throughout this application, the term "about" is used to indicate a value that includes variations of error inherent in a measurement or quantification method.

[0017] The use of the words "a" or "an" when used in conjunction with the term "comprising" may mean "one", but is also consistent with the meanings of "one or more", "at least one", and "one or more than one".

[0018] The phrase "and / or" means "and" or "or". By way of example, A, B, and / or C includes only A, only B, only C, the combination of A and B, the combination of A and C, the combination of B and C, or the combination of A, B, and C. In other words, "and / or" functions as an inclusive "or".

[0019] The terms "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "includes" and "include") or "containing" (and any form of "containing", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] The phrase "substantially all" is defined as "at least 95%". When substantially all members of a group have a particular property, at least 95% of the members of that group have that property. In some cases, substantially all means any one of 95%, 96%, 97%, 98%, 99%, or 100% of the members of the group, at least any one of these, or between any two of these have that property.

[0021] Compositions and methods of using them may "comprise", "consist essentially of", or "consist of" any of the components or steps disclosed throughout this specification. Compositions and methods "consisting essentially of" any of the disclosed components or steps are limited in scope to those specified materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure.

[0022] A. Self-amplifying RNA (saRNA) In some embodiments, the RNA molecule, such as the first RNA molecule, is saRNA. "saRNA", "self-amplifying RNA", and "replicons" refer to RNAs that have the ability to replicate themselves. Self-amplifying RNA molecules can be produced by using replication elements derived from one or more viruses, such as alphaviruses, and replacing the structural viral polypeptides with nucleotide sequences encoding the polypeptide of interest. Self-amplifying RNA molecules are generally plus-strand molecules that can be directly translated after being delivered to a cell, and this translation results in an RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts of the delivered RNA. The production of a large number of daughter RNAs from the delivered RNA is thereby brought about. These daughter RNAs, as well as the subgenomic transcripts themselves on the same linear strand, can be translated to result in the in situ expression of the encoded gene of interest, such as a viral antigen, or can be transcribed to result in additional transcripts having the same sense as the delivered RNA, which are then translated to result in the in situ expression of the protein of interest, such as an antigen. As an overall result of this series of transcriptions, the number of introduced saRNAs is amplified, and thus the encoded gene of interest, such as a viral antigen, can become the major polypeptide product of the cell.

[0023] In some embodiments, the self-amplifying RNA comprises at least one or more genes selected from any one of a viral replicase, a viral protease, a viral helicase, and other viral non-structural proteins. In some embodiments, the self-amplifying RNA may also include 5'- and 3'-terminal tractive replication sequences, and optionally, a heterologous sequence encoding a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs the expression of the heterologous sequence can be included in the self-amplifying RNA. The heterologous sequence (e.g., an antigen of interest) can also be fused in-frame to another coding region within the self-amplifying RNA, and / or placed under the control of an internal ribosome entry site (IRES) within the sequence.

[0024] In some embodiments, the self-amplifying RNA molecule is not encapsulated in virus-like particles. The self-amplifying RNA molecules described herein can be designed such that the self-amplifying RNA molecules cannot induce the production of infectious virus particles. This can be achieved, for example, by removing one or more viral genes encoding structural proteins required for the production of viral particles in the self-amplifying RNA. For example, if the self-amplifying RNA molecule is based on an alphavirus, such as Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE), one or more genes encoding viral structural proteins, such as capsid and / or envelope glycoproteins, can be removed.

[0025] In some embodiments, the self-amplifying RNA molecules described herein encode (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, such as a viral antigen. In some embodiments, the polymerase may be, for example, an alphavirus replicase comprising any one of alphavirus proteins nsP1, nsP2, nsP3, nsP4, and any combination thereof. In some embodiments, the self-amplifying RNA molecules described herein may include one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine). In some embodiments, the self-amplifying RNA molecule does not include modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine).

[0026] The saRNA construct may encode at least one non-structural protein (NSP) located 5' or 3' of the sequence encoding at least one peptide or polypeptide of interest. In some embodiments, the sequence encoding at least one NSP is located 5' of the sequence encoding the peptide or polypeptide of interest. Thus, the sequence encoding at least one NSP may be located at the 5' end of the RNA construct. In some embodiments, at least one non-structural protein encoded by the RNA construct may be the viral polymerase nsP4. In some embodiments, the saRNA construct encodes nsP1, nsP2, nsP3, and nsP4. As is known in the art, nsP1 is the viral capping enzyme and the membrane anchor of the replication complex (RC). nsP2 is an RNA helicase and protease responsible for ns polyprotein processing. nsP3 can interact with several host proteins and modulate protein polyADP-ribosylation and monoADP-ribosylation. nsP4 is the central viral RNA-dependent RNA polymerase. In some embodiments, the polymerase may be an alphavirus replicase comprising, for example, one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4.

[0027] The native alphavirus genome encodes structural virion proteins in addition to non-structural replicase polypeptides, but in some embodiments, the self-amplifying RNA molecule does not encode alphavirus structural proteins. In some embodiments, the self-amplifying RNA can direct the production of its own genomic RNA copies in cells, but cannot direct the production of RNA containing virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike wild-type alphaviruses, self-amplifying RNA molecules cannot persist on their own as infectious forms. The alphavirus structural proteins required for persistence in wild-type viruses can be deleted from the self-amplifying RNA of the present disclosure, and a gene (s) encoding the immunogen of interest can be placed in their position, such that the subgenomic transcript encodes the immunogen rather than the alphavirus structural virion protein.

[0028] In some embodiments, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame may encode a replicase, and the second (3') open reading frame may encode a polypeptide containing the antigen of interest. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, for example, to encode additional antigens or to encode accessory polypeptides.

[0029] In some embodiments, the second RNA or saRNA molecule further comprises (1) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.

[0030] The self-amplifying RNA molecules described herein can also be designed to induce the production of attenuated or toxic infectious virus particles or to produce virus particles capable of subsequent rounds of infection.

[0031] In some embodiments, the saRNA molecule is alphavirus-based. Alphaviruses include a series of genetically, structurally, and serologically related arthropod-borne viruses of the family Togaviridae. Exemplary viruses and virus subtypes belonging to the genus Alphavirus include Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. Thus, an RNA replicase derived from any one of Semliki Forest virus (SFV), Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), Ross River virus (RRV), or other viruses belonging to the family Alphaviridae can be incorporated into the self-amplifying RNA described herein. In some embodiments, sequences derived from mutant or wild-type virus sequences can be incorporated into the self-amplifying RNA described herein. For example, the attenuated TC83 mutant of VEEV has been used in saRNA.

[0032] Alphavirus-based saRNA is a (+)-strand saRNA that can be translated after delivery to a cell, thereby resulting in the translation of a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein that self-cleaves to yield a replication complex that creates a genomic (-)-strand copy of the delivered (+)-strand RNA. These (-)-strand transcripts can themselves be transcribed such that additional copies of the (+)-strand parental RNA are produced and subgenomic transcripts encoding the desired gene product are also produced. Thus, translation of the subgenomic transcripts results in in situ expression of the desired gene product by the infected cell. Suitable alphavirus saRNAs can use replicases derived from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, or mutant variants thereof.

[0033] In some embodiments, the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, such as a plus-strand RNA virus, particularly a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus. Suitable wild-type alphavirus sequences are well known and are available from sequence depositories such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagash (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-1277), Middelburg (ATCC VR-370), Mukambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Trinidad (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250 ATCC VR-1249, ATCC VR-532), Western equine encephalitis (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Wataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375). In some aspects, one or more of the alphaviruses in this list can be excluded.

[0034] In some embodiments, the self-amplifying RNA molecules described herein are larger than other types of RNA (e.g., saRNA). Generally, the self-amplifying RNA molecules described herein comprise at least about 4 kb. For example, the self-amplifying RNA can be any one of 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, at least any one of these, at most any one of these, or between any two of these. In some cases, the self-amplifying RNA can comprise at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In certain examples, the self-amplifying RNA is from about 4 kb to about 12 kb, from about 5 kb to about 12 kb, from about 6 kb to about 12 kb, from about 7 kb to about 12 kb, from about 8 kb to about 12 kb, from about 9 kb to about 12 kb, from about 10 kb to about 12 kb, from about 11 kb to about 12 kb, from about 5 kb to about 11 kb, from about 5 kb to about 10 kb, from about 5 kb to about 9 kb, from about 5 kb to about 8 kb, from about 5 kb to about 7 kb, from about 5 kb to about 6 kb, from about 6 kb to about 12 kb, from about 6 kb to about 11 kb, from about 6 kb to about 10 kb, from about 6 kb to about 9 kb, from about 6 kb to about 8 kb, from about 6 kb to about 7 kb, from about 7 kb to about 11 kb, from about 7 kb to about 10 kb, from about 7 kb to about 9 kb, from about 7 kb to about 8 kb, from about 8 kb to about 11 kb, from about 8 kb to about 10 kb, from about 8 kb to about 9 kb, from about 9 kb to about 11 kb, from about 9 kb to about 10 kb, or from about 10 kb to about 11 kb.

[0035] In some embodiments, the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more polypeptide antigens linked such that when expressed as an amino acid sequence, each of the sequences retains its identity (e.g., is serially linked). Thus, the polypeptide produced from the self-amplifying RNA can be produced as a fusion polypeptide or engineered to result in separate polypeptides or peptide sequences. In some embodiments, the saRNA molecule can encode one or more polypeptides of interest, e.g., one or more antigens, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptides. Alternatively, or in addition, one saRNA molecule may encode more than one polypeptide of interest or more than one, e.g., one antigen, e.g., a bicistronic or tricistronic RNA molecule encoding different or identical antigens.

[0036] As used herein, the term "linked" refers to a first amino acid or polynucleotide sequence being joined to a second amino acid or polynucleotide sequence by a covalent or non-covalent bond. The first amino acid or polynucleotide sequence may be joined directly or in parallel to the second amino acid or polynucleotide sequence, or the first and second sequences may be joined covalently by an intervening sequence. The term "linked" not only means a fusion at the 5' or 3' end of a first RNA molecule and an RNA molecule, but also encompasses the insertion of an entire first RNA molecule into any two nucleotides of a second RNA molecule. The first RNA molecule may be linked to the second RNA molecule by a phosphodiester bond or a linker. The linker may be, for example, a polynucleotide.

[0037] In some embodiments, the self-amplifying RNAs described herein may encode one or more polypeptide antigens comprising various epitopes. In some embodiments, the self-amplifying RNAs described herein may encode epitopes capable of eliciting either or both a helper T cell response or a cytotoxic T cell response.

[0038] In some embodiments, the saRNA molecule can be purified by filtration, for example, by ultrafiltration, diafiltration, or, for example, tangential flow ultrafiltration / diafiltration.

[0039] Some embodiments of the present disclosure are directed to compositions comprising self-amplifying RNA molecules comprising a 5' cap, a 5' untranslated region, a sequence encoding an RNA-dependent RNA polymerase (also referred to as "replicase"), a subgenomic promoter such as those derived from alphavirus, a coding region comprising an open reading frame encoding a gene of interest (e.g., an antigen derived from influenza virus), a 3' untranslated region, and a 3' polyA sequence. In some embodiments, at least 5% of the entire population of specific nucleotides within the saRNA molecule are replaced by one or more modified or non-natural nucleotides.

[0040] In some embodiments, the saRNA molecule does not contain modified nucleotides, for example, does not contain modified nucleobases and all of the nucleotides within the RNA molecule are conventional standard ribonucleotides A, U, G, and C, with the exception of an optional 5' cap which may contain, for example, 7-methylguanosine, which is further described below. In some embodiments, the RNA may comprise a 5' cap comprising 7'-methylguanosine and the first one, two, or three 5' ribonucleotides may be methylated at the 2' position of the ribose.

[0041] The effectiveness of the product depends on the expression of the delivered saRNA, and fully intact RNA molecules are required. RNA integrity is one measure of RNA quality that quantifies intact RNA. It is also possible to detect potential degradation products with this method. RNA integrity is preferably determined by capillary gel electrophoresis. The initial specifications are set to ensure sufficient RNA integrity in the pharmaceutical product preparation. In some embodiments, the RNA polynucleotide has at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% integrity. In some embodiments, the RNA polynucleotide has about 95% or greater integrity. In some embodiments, the RNA polynucleotide has about 98% or greater integrity. In some embodiments, the RNA polynucleotide has about 99% or greater integrity.

[0042] In preferred embodiments, the saRNA polynucleotide has clinical-grade purity. In some embodiments, the purity of the RNA polynucleotide is between about 60% and about 100%. In some embodiments, the purity of the RNA polynucleotide is between about 80% and 99%. In some embodiments, the purity of the RNA polynucleotide is between about 90% and about 99%. In some embodiments, the purified mRNA has clinical-grade purity without further purification. In some embodiments, clinical-grade purity is achieved by methods including tangential flow filtration (TFF) purification. In some embodiments, clinical-grade purity is achieved without further purification selected from high performance liquid chromatography (HPLC) purification, ligand or binding-based purification, and / or ion exchange chromatography. In some embodiments, in the method of producing the RNA polynucleotide, long-chain incomplete RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual solvent, and / or residual salts are removed. In some embodiments, the short-chain incomplete transcript contaminants contain less than 15 bases. In some embodiments, the short-chain incomplete transcript contaminants contain about 8 - 12 bases. In some embodiments, in the method of the present invention, ribonuclease inhibitors are also removed.

[0043] In some embodiments, the purified saRNA polynucleotide, as determined by capillary electrophoresis, contains 5% or less, 4% or less, 3% or less, 2% or less, 1% or less protein contaminants, or is substantially free of protein contaminants. In some embodiments, the purified RNA polynucleotide, as determined by high performance liquid chromatography (HPLC), contains less than 5%, less than 4%, less than 3%, less than 2%, less than 1% salt contaminants, or is substantially free of salt contaminants. In some embodiments, the purified RNA polynucleotide, as determined by known methods such as high performance liquid chromatography (HPLC), contains 5% or less, 4% or less, 3% or less, 2% or less, 1% or less short chain incomplete transcript contaminants, or is substantially free of short chain incomplete transcript contaminants. In some embodiments, the purified RNA polynucleotide, as determined by known methods such as capillary electrophoresis, has a completeness of 60% or more, 70% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0044] B. Modified Nucleobases Modified nucleosides and nucleotides can be incorporated, and modified nucleobases that can be present within an RNA molecule include, for example, m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6 isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6-hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); m1I (1-methylinosine); m1Im (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); 5-forC (5-formylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4-acetyl-2'-O-methylcytidine); k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine);OHyWx (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2’-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2’-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2’-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethyl-1-aminomethyl-2-L-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2’-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine);rn62Am (N6,N6,0 - 2 - trimethyladenosine); m2,7G (N2,7 - dimethylguanosine); m2,2,7G (N2,N2,7 - trimethylguanosine); m3Um (3,2 - O - dimethyluridine); m5D (5 - methyldihydrouridine); f5Cm (5 - formyl - 2’ - O - methylcytidine); m1Gm (1,2’ - O - dimethylguanosine); m1Am (1,2 - O - dimethyladenosine) (irinomethyluridine); tm5s2U (S - taurolinomethyl - 2 - thiouridine)); imG - 14 (4 - demethylguanosine); imG2 (isoguanosine); ac6A (N6 - acetyladenosine), hypoxanthine, inosine, 8 - oxo - adenine, its 7 - substituted derivatives, dihydrouracil, pseudouracil, 2 - thiouracil, 4 - thiouracil, 5 - aminouracil, 5 - (C1 - C6) - alkyluracil, 5 - methyluracil, 5 - (C2 - C6) - alkenyluracil, 5 - (C2 - C6) - alkynyluracil, 5 - (hydroxymethyl)uracil, 5 - chlorouracil, 5 - fluorouracil, 5 - bromouracil, 5 - hydroxymethylcytosine, 5 - (C1 - C6) - alkylcytosine, 5 - methylcytosine, 5 - (C2 - C6) - alkenylcytosine, 5 - (C2 - C6) - alkynylcytosine, 5 - chlorocytosine, 5 - fluorocytosine, 5 - bromocytosine, N2 - dimethylguanine, 7 - deazaguanine, 8 - azaguanine, 7 - deaza - 7 - substituted guanine, 7 - deaza - 7 - (C2 - C6) alkynylguanine, 7 - deaza - 8 - substituted guanine, 8 - hydroxyguanine, 6 - thioguanine, 8 - oxoguanine, 2 - aminopurine, 2 - amino - 6 - chloropurine, 2,4 - diamino - purine, 2,6 - diamino - purine, 8 - azapurine, substituted 7 - deazapurine, 7 - deaza - 7 - substituted purine, 7 - deaza - 8 - substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2’ - O - methyl - U. In some embodiments, one or more of the modified nucleosides in the list can be excluded.;

[0045] Additional exemplary modified nucleotides include N-1-methylpseudouridine; any one of pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine. In some embodiments, the modified nucleotide is N-1-methylpseudouridine.

[0046] In some embodiments, the RNA molecule may comprise phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0047] In some embodiments, the RNA molecule comprises a modified nucleotide selected from any one of 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-methyluridine. In some embodiments, the modified or non-natural nucleotide is selected from the group consisting of 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-methyluridine. In some embodiments, the modified or non-natural nucleotide is selected from the group consisting of 5-methyluridine, N1-methylpseudouridine, 5-methoxyuridine, and 5-methylcytosine.

[0048] In some embodiments, at least 10% of the entire population of specific nucleotides within the saRNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the entire population of specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the entire population of specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 75% of the entire population of specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, substantially all of the specific nucleotide population within the molecule is replaced by one or more modified or unnatural nucleotides.

[0049] In some embodiments, at least a portion or all of a population of specific nucleotides within the saRNA molecule are replaced by two modified or non-natural nucleotides. In some embodiments, the two modified or non-natural nucleotides are provided in any one of 1:99; 2:98; 3:97; 4:96; 5:95; 6:94; 7:93; 8:92; 9:91; 10:90; 11:89; 12:88; 13:87; 14:86; 15:85; 16:84; 17:83; 18:82; 19:81; 20:80; 21:79; 22:78; 23:77; 24:76; 25:75; 26:74; 27:73; 28:72; 29:71; 30:70; 31:69; 32:68; 33:67; 34:66; 35:65; 36:64; 37:63; 38:62; 39:61; 40:60; 41:59; 42:58; 43:57; 44:56; 45:55; 46:54; 47:53; 48:52; 49:51; 50:50; 51:49; 52:48; 53:47; 54:46; 55:45; 56:44; 57:43; 58:42; 59:41; 60:40; 61:39; 62:38; 63:37; 64:36; 65:35; 66:34; 67:33; 68:32; 69:31; 70:30; 71:29; 72:28; 73:27; 74:26; 75:25; 76:24; 77:23; 78:22; 79:21; 80:20; 81:19; 82:18; 83:17; 84:16; 85:15; 86:14; 87:13; 88:12; 89:11; 90:10; 91:9; 92:8; 93:7; 94:6; 95:5; 96:4; 97:3; 98:2; and 99:1, or any one of 1:99 to 99:1 including any range derivable therefrom, at least any one of these, up to any one of these, or in a ratio between two of these.

[0050] In some embodiments, at least 10% of the entire population of a first specific nucleotide within the saRNA molecule disclosed herein is replaced by one or more modified or unnatural nucleotides, and at least 10% of the entire population of a second specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides, and at least 25% of the entire population of a second specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides, and at least 50% of the entire population of a second specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides, and at least 75% of the entire population of a second specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the entire population of a first specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides, and substantially all of the entire population of a second specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the entire population of a first specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides, and at least 25% of the entire population of a second specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides.In some embodiments, at least 25% of the entire population of the first specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides, and at least 50% of the entire population of the second specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the entire population of the first specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides, and at least 75% of the entire population of the second specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the entire population of the first specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides, and substantially all of the entire population of the second specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the entire population of the first specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides, and at least 50% of the entire population of the second specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the entire population of the first specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides, and at least 75% of the entire population of the second specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the entire population of the first specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides, and substantially all of the entire population of the second specific nucleotides within the molecule is replaced by one or more modified or unnatural nucleotides.In some embodiments, at least 75% of the entire population of a first specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides, and at least 75% of the entire population of a second specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 75% of the entire population of a first specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides, and substantially all of the entire population of a second specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, substantially all of the entire population of a first specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides, and substantially all of the entire population of a second specific nucleotide within the molecule is replaced by one or more modified or unnatural nucleotides.

[0051] In some embodiments, at least 25% of the entire population of uridine nucleotides within the saRNA molecule are replaced by N1-methylpseudouridine. In some embodiments, at least 50% of the entire population of uridine nucleotides within the molecule are replaced by N1-methylpseudouridine. In some embodiments, at least 75% of the entire population of uridine nucleotides within the molecule are replaced by N1-methylpseudouridine. In some embodiments, substantially all of the uridine nucleotides within the molecule are replaced by N1-methylpseudouridine. In some embodiments, at least 50% of the entire population of uridine nucleotides within the molecule are replaced by 5-methoxyuridine. In some embodiments, substantially all of the uridine nucleotides within the molecule are replaced by 5-methoxyuridine. In some embodiments, at least 50% of the entire population of uridine nucleotides within the molecule are replaced by 5-methyluridine. In some embodiments, substantially all of the uridine nucleotides within the molecule are replaced by 5-methyluridine. In some embodiments, at least 50% of the entire population of cytosine nucleotides within the molecule are replaced by 5-methylcytosine. In some embodiments, substantially all of the cytosine nucleotides within the molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides within the molecule are replaced by 2-thiouridine. In some embodiments, substantially all of the uridine nucleotides within the molecule are replaced by 2-thiouridine.

[0052] In some embodiments, at least 50% of the entire population of uridine nucleotides within the molecule are replaced by N1-methylpseudouridine, and substantially all of the cytosine nucleotides within the molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides within the molecule are replaced by 5-methoxyuridine, and substantially all of the cytosine nucleotides within the molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides within the molecule are replaced by 5-methyluridine, and substantially all of the cytosine nucleotides within the molecule are replaced by 5-methylcytosine.

[0053] In some embodiments, substantially all of the uridine nucleotides within the molecule are replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudouridine. In some embodiments, substantially all of the uridine nucleotides within the molecule are replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseudouridine. In some embodiments, substantially all of the uridine nucleotides within the molecule are replaced by approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseudouridine.

[0054] C.UTR The 5' untranslated region (UTR) is a regulatory region of DNA located at the 5' end of the protein-coding sequence that is transcribed into mRNA but not translated into protein. The 5'UTR can contain various regulatory elements, such as a 5' cap structure, stem-loop structures, and internal ribosome entry sites (IRES) within the sequence, which can play a role in the control of translation initiation. The 3'UTR is located downstream of the protein-coding sequence and can be involved in regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization. In some embodiments, the UTR is derived from an mRNA that is naturally abundant in a particular tissue (e.g., lymphoid tissue) targeted for mRNA expression. In some embodiments, protein synthesis is increased by the UTR. Without being bound by mechanism or theory, the UTR can increase protein synthesis by increasing the time an mRNA remains on the polysomes where translation is occurring (message stability) and / or the rate at which ribosomes initiate translation on the message (message translation efficiency). Thus, the UTR sequence can specifically extend protein synthesis in a tissue. In some embodiments, the 5'UTR and 3'UTR sequences are computationally derived. In some embodiments, the 5'UTR and 3'UTR are derived from an mRNA that is naturally abundant in a tissue. The tissue can be, for example, liver, stem cells, or lymphoid tissue. Examples of lymphoid tissue can include any one of lymphocytes (e.g., B lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or natural killer cells), macrophages, monocytes, dendritic cells, neutrophils, eosinophils, and reticulocytes. In some embodiments, the 5'UTR and 3'UTR are derived from an alphavirus. In some embodiments, the 5'UTR and 3'UTR are derived from a wild-type alphavirus. Examples of alphaviruses are described below.

[0055] In some embodiments, the first RNA molecule comprises a 5' UTR and a 3' UTR derived from an mRNA that is naturally abundant in the tissue. In some embodiments, the first RNA molecule comprises a 5' UTR and a 3' UTR derived from an alphavirus. In some embodiments, the second RNA or saRNA molecule comprises a 5' UTR and a 3' UTR derived from an alphavirus. In some embodiments, the second RNA or saRNA molecule comprises a 5' UTR and a 3' UTR derived from a wild-type alphavirus. In some embodiments, the RNA molecule comprises a 5' cap.

[0056] D. Open Reading Frame (ORF) The 5' and 3' UTRs may be operably linked to an ORF that may be a sequence of codons capable of being translated into the polypeptide of interest. As described above, the RNA molecule may contain one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs).

[0057] In some embodiments, the ORF encodes a viral non-structural gene. In some embodiments, the ORF further comprises one or more subgenomic promoters. In some embodiments, the RNA molecule comprises a subgenomic promoter operably linked to the ORF. In some embodiments, the subgenomic promoter comprises a cis-acting regulatory element. In some embodiments, the cis-acting regulatory element is immediately downstream (5'-3') of B 2 In some embodiments, the cis-acting regulatory element is immediately downstream (5'-3') of B 2Immediately downstream of guanine, immediately downstream of guanine (5'-3'). In some embodiments, the cis-acting regulatory element is an AU-rich element. In some embodiments, the AU-rich element is au, auaaaagau, auaaaaagau, auag, auauauauau, auauauau, auauauauauau, augaugaugau, augau, auaaaagaua, or auaaaagaug. In some embodiments, the second RNA or saRNA molecule may comprise (i) an ORF encoding a replicase capable of transcribing RNA from the second RNA or saRNA molecule, and (ii) an ORF encoding at least one antigen or polypeptide of interest. The polymerase may be, for example, an alphavirus replicase including any one of alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4, or a combination thereof. In some embodiments, the RNA molecule comprises alphavirus nonstructural protein nsP1. In some embodiments, the RNA molecule comprises alphavirus nonstructural protein nsP2. In some embodiments, the RNA molecule comprises alphavirus nonstructural protein nsP3. In some embodiments, the RNA molecule comprises alphavirus nonstructural protein nsP4. In some embodiments, the RNA molecule comprises alphavirus nonstructural proteins nsP1, nsP2, and nsP3. In some embodiments, the RNA molecule comprises alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule comprises any combination of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule does not comprise nsP4.

[0058] In some embodiments, the open reading frames of the RNA (e.g., saRNA) composition are codon-optimized. In some embodiments, the open reading frame encoding an influenza polypeptide or a fragment thereof is codon-optimized.

[0059] E. Gene encoding an antigenic polypeptide In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or an immunogenic fragment thereof. In some embodiments, the hemagglutinin protein is H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18 or an immunogenic fragment thereof. In some embodiments, the hemagglutinin protein does not include the head domain. In some embodiments, the hemagglutinin protein includes a part of the head domain. In some embodiments, the hemagglutinin protein does not include the cytoplasmic domain. In some embodiments, the hemagglutinin protein includes a part of the cytoplasmic domain. In some embodiments, the truncated hemagglutinin protein includes a part of the transmembrane domain.

[0060] Some embodiments provide an influenza vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a hemagglutinin protein formulated within cationic lipid nanoparticles and a pharmaceutically acceptable carrier or excipient. In some embodiments, the hemagglutinin protein is selected from H1, H7, and H10. In some embodiments, the RNA polynucleotide further encodes a neuraminidase (NA) protein. In some embodiments, the hemagglutinin protein is derived from a strain of influenza A virus or a strain of influenza B virus or a combination thereof. In some embodiments, the influenza virus is selected from H1N1, H3N2, H7N9, and H10N8.

[0061] In some embodiments, the virus is an influenza A strain or an influenza B strain or a combination thereof. In some embodiments, the influenza A or influenza B strain is associated with birds, pigs, horses, dogs, humans, or non-human primates. In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or a fragment thereof. In some embodiments, the hemagglutinin protein is H7 or H10 or a fragment thereof. In some embodiments, the hemagglutinin protein comprises a part of the head domain (HA1). In some embodiments, the hemagglutinin protein comprises a part of the cytoplasmic domain. In some embodiments, a truncated hemagglutinin protein. In some embodiments, the protein is a truncated hemagglutinin protein and comprises a part of the transmembrane domain. In some embodiments, the virus is selected from the group consisting of H7N9 and H10N8. Protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of the polypeptide of interest. For example, any protein fragment of a reference protein of amino acid length 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 (meaning a polypeptide sequence of at least one amino acid residue that is shorter than the reference polypeptide sequence but identical in other respects) is presented herein.

[0062] In some embodiments, the at least one antigenic polypeptide is one of the defined antigenic subdomains of HA, designated HA1, HA2, or a combination of HA1 and HA2, and at least one antigenic polypeptide selected from neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), and non-structural protein 2 (NS2).

[0063] In some embodiments, the at least one antigenic polypeptide is HA or a derivative thereof comprising an antigenic sequence derived from HA1 and / or HA2, and at least one antigenic polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2.

[0064] In some embodiments, at least one antigenic polypeptide is an HA or a derivative thereof comprising an antigenic sequence derived from HA1 and / or HA2, and at least two antigenic polypeptides selected from HA, NA, NP, M1, M2, NS1, and NS2.

[0065] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an influenza virus protein or an immunogenic fragment thereof.

[0066] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding a plurality of influenza virus proteins or immunogenic fragments thereof.

[0067] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one of HA1, HA2, or a combination of both).

[0068] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one of HA1, HA2, or both, of any one or any or all combinations of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18), and at least one other RNA (e.g., saRNA) polynucleotide having an open reading frame encoding a protein selected from the group consisting of an HA protein, an NP protein, an NA protein, an M1 protein, an M2 protein, an NS1 protein, and an NS2 protein obtained from an influenza virus.

[0069] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one of any one or any or all combinations of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18), and at least two other RNA (e.g., saRNA) polynucleotides having two open reading frames encoding two proteins selected from the group consisting of an HA protein, an NP protein, an NA protein, an M1 protein, an M2 protein, an NS1 protein, and an NS2 protein obtained from an influenza virus.

[0070] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., any one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18 or at least one of any or all combinations thereof), and at least three other RNA (e.g., saRNA) polynucleotides having three open reading frames encoding three proteins selected from the group consisting of an HA protein, an NP protein, an NA protein, an M1 protein, an M2 protein, an NS1 protein, and an NS2 protein obtained from an influenza virus.

[0071] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., any one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18 or at least one of any or all combinations thereof), and at least four other RNA (e.g., saRNA) polynucleotides having four open reading frames encoding four proteins selected from the group consisting of an HA protein, an NP protein, an NA protein, an M1 protein, an M2 protein, an NS1 protein, and an NS2 protein obtained from an influenza virus.

[0072] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., any one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18 or at least one of any or all combinations thereof), and at least five other RNA (e.g., saRNA) polynucleotides having open reading frames encoding five proteins selected from the HA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, and NS2 protein obtained from influenza virus.

[0073] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., any one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18 or at least one of any or all combinations thereof), the HA protein, the NP protein or an immunogenic fragment thereof, the NA protein or an immunogenic fragment thereof, the M1 protein or an immunogenic fragment thereof, the M2 protein or an immunogenic fragment thereof, the NS1 protein or an immunogenic fragment thereof, and the NS2 protein or an immunogenic fragment thereof.

[0074] In some embodiments, the influenza RNA composition comprises saRNA encoding an antigenic fusion protein. Thus, the encoded antigen(s) can comprise two or more proteins (e.g., proteins and / or protein fragments) joined together. Alternatively, the protein fused to the protein antigen does not promote a strong immune response against itself but promotes a strong immune response against the influenza antigen. In some embodiments, the antigenic fusion protein retains the functional properties derived from each of the original proteins.

[0075] F. 5’ Cap In some embodiments, the saRNA molecules described herein comprise a 5’ cap. In some embodiments, the 5’ cap moiety is a natural 5’ cap.

[0076] The "natural 5' cap" is defined as a cap containing 7-methylguanosine connected to the 5' end of the mRNA molecule through a 5'-5' triphosphate linkage. In some embodiments, the 5' cap moiety is a 5' cap analog. In some embodiments, the 5' end of the RNA is capped with a modified ribonucleotide having the structure m7G(5')ppp(5')N (cap 0 structure) or a derivative thereof, and the cap structure can also be incorporated during RNA synthesis (e.g., co-transcriptional capping), or can be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping), where "N" is any ribonucleotide. In some embodiments, the 5' end molecule of the RNA is capped with a modified ribonucleotide by an enzymatic reaction after RNA transcription. In some embodiments, capping is performed after purification of the RNA molecule, e.g., tangential flow filtration. Exemplary enzymatic reactions for capping can include the use of vaccinia virus capping enzyme (VCE) including mRNA triphosphatase, guanylyl-transferase, and guanine-7-methyltransferase, which catalyze the construction of the N7-monomethylated cap 0 structure. The cap 0 structure can help maintain the stability and translational efficiency of the RNA molecule. The 5' cap of the RNA molecule can also be further modified by a 2'-O-methyltransferase that results in the production of the cap 1 structure (m7Gppp[m2'-O]N), thereby further increasing translational efficiency. In some embodiments, vaccinia guanylyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine are used to enzymatically cap the 5' end of the RNA molecule to obtain the cap 0 structure. The inverted 7-methylguanosine cap is added via a 5'-5' triphosphate bridge. Alternatively, by using 2'-O-methyltransferase and vaccinia guanylyltransferase, in addition to the cap 0 structure, a cap 1 structure in which the 2'OH group of the penultimate nucleotide is methylated can be obtained. S-adenosyl-L-methionine (SAM) is a cofactor utilized as a methyltransferase reagent.Non-limiting examples of 5’ cap structures include, inter alia, those having enhanced binding of cap-binding polypeptides, extended half-lives, reduced susceptibility to 5’ endonucleases, and / or reduced 5’ cap removal, as compared to synthetic 5’ cap structures (or wild-type, native or physiological 5’ cap structures) known in the art. For example, by recombinant vaccinia virus capping enzyme and recombinant 2’-O-methyltransferase enzyme, a canonical 5’-5’-triphosphate linkage can be created between the 5’ terminal nucleotide of an mRNA and a guanine cap nucleotide, where the cap guanine includes N7 methylation and the 5’ terminal nucleotide of the mRNA includes 2’-O-methyl. Such a structure is referred to as a cap 1 structure. This cap results in, for example, high translational fitness and reduced cellular instability and activation of pro-inflammatory cytokines in cells as compared to other 5’ cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5’)ppp(5’)N,pN2p (cap 0) and 7mG(5’)ppp(5’)N1mpNp (cap 1). Cap 0 is N7-methylguanosine connected to the 5’ nucleotide by a 5’-5’ triphosphate linkage and is generally referred to as the m7G cap or m7Gppp. In cells, the cap 0 structure can help to effect efficient translation of capped mRNAs. Cap 1 results from additional methylation at the 2’O position of the initiating nucleotide. Or cap 1 is referred to as m7GpppNm-, where Nm refers to any nucleotide having 2’O methylation. In some embodiments, the 5’ terminal cap includes a cap analog, for example, the 5’ terminal cap can include a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2’-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the cap addition region can include a single cap or a series of nucleotides that form a cap.In this embodiment, the cap addition region may be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or at least 2, or 10 or less nucleotides, at least any one of these, at most any one of these, or the length between any two of these. In some embodiments, there is no cap. In some embodiments, the first and second operable regions may be 3 to 40, such as 5 to 30, 10 to 20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or at least 4, or 30 or less nucleotides, at least any one of these, at most any one of these, or the length between any two of these, and may include one or more signals and / or restriction sequences in addition to start and / or stop codons.

[0077] In some embodiments, the 5' cap is of formula I:

[0078]

Chemical Formula

[0079] In some embodiments, the nucleotide immediately downstream of the 5' cap (in the 5' to 3' direction) contains guanine. In some embodiments, B 1 is adenine and B 2 is uracil. In some embodiments, B 1 is adenine and B 2 is uracil, R 1 is methyl, and R 2 is hydrogen. In some cases, the saRNA does not contain a 5' cap. In some cases, the 5' cap is not represented by Formula I. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') contains guanine, B 1 is adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, and this embodiment corresponds to CleanCap AU, and the uracil nucleotides downstream of B 2 may be substituted, but in some embodiments, it has been shown that including B 2 =uracil improves saRNA functionality. In some embodiments, the RNA molecule further comprises (1) an alphavirus 5' replication recognition sequence and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the RNA molecule encodes at least one antigen. In some embodiments, the RNA molecule comprises at least 7000 nucleotides. In some embodiments, the RNA molecule comprises at least 8000 nucleotides. In some embodiments, at least 80% of the entire RNA molecule is full-length. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus. In some embodiments, the alphavirus is Semliki Forest virus.

[0080] In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap contains guanine, B 1 is adenine, B 2 is uracil, R 1 is methyl, R 2 is hydrogen, and at least 50% of the entire population of uridine nucleotides within the molecule are replaced by N1-methylpseudouridine, and essentially all cytosine nucleotides within the molecule are replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap contains guanine, B 1 is adenine, B 2 is uracil, R 1 is methyl, R 2 is hydrogen, and at least 50% of the entire population of uridine nucleotides within the molecule are replaced by 5-methoxyuridine, and essentially all cytosine nucleotides within the molecule are replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap contains guanine, B 1 is adenine, B 2 is uracil, R 1 is methyl, R 2 is hydrogen, and at least 50% of the entire population of uridine nucleotides within the molecule are replaced by 5-methyluridine, and essentially all cytosine nucleotides within the molecule are replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap contains guanine, B 1 is adenine, B 2 is uracil, R 1 is methyl, R 2 is hydrogen, and essentially all uridine nucleotides within the molecule are replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudouridine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap contains guanine, B 1 is adenine, B 2is uracil, and R 1 is methyl, and R 2 is hydrogen, and essentially all uridine nucleotides within the molecule are replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseudouridine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap contains guanine, and B 1 is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen, and essentially all uridine nucleotides within the molecule are replaced by approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseudouridine.

[0081] In some embodiments, the 5' end cap is 7mG(5')ppp(5')NlmpNp. In some preferred embodiments, the 5' cap is

[0082] [Chemical formula] including. In some embodiments, the 5' cap is CLEANCAP® Reagent AG(3’OMe) for capping during mRNA transcription, m7(3’OMeG)(5’)ppp(5’)(2’OMeA)pG,

[0083] [Chemical formula] including. In alternative embodiments, the 5' cap is CLEANCAP® AU for self-amplifying mRNA, CLEANCAP® Reagent AU for capping during mRNA transcription, m7G(5’)ppp(5’)(2’OMeA)pU,

[0084] [Chemical formula] including.

[0085] G. PolyA Tail As used herein, "polyA tail" refers to a stretch of consecutive adenine residues that can be attached to the 3' end of an RNA molecule. The polyA tail can increase the half-life of the RNA molecule. The polyA tail can play an important regulatory role in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or high polyadenylation can signal RNA degradation. Exemplary designs include a polyA tail of about 40 adenine residues to about 80 adenine residues. In some embodiments, the RNA molecule further comprises an endonuclease recognition site sequence immediately downstream of the polyA tail sequence. In some embodiments, for example for a second RNA or saRNA molecule, the RNA molecule further comprises a polyA polymerase recognition sequence (e.g., AAUAAA) near the 3' end. A "full-length" RNA molecule is one that includes a 5' cap and a polyA tail.

[0086] In some embodiments, the polyA tail comprises a length of 5 to 400 nucleotides. The nucleotide length of the polyA tail can be any one of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400, at least any one of these, up to any one of these, or between any two of these. In some embodiments, the RNA molecule comprises a polyA tail comprising a sequence of about 25 to about 400 adenosine nucleotides, a sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides. In some embodiments, the RNA molecule comprises a polyA tail comprising a sequence of more than 30 adenosine nucleotides ("As"). In some embodiments, the RNA molecule comprises a polyA tail comprising about 40 As. In some embodiments, the RNA molecule comprises a polyA tail comprising about 80 As. As used herein, the term "about" refers to a deviation of ±10% of the value(s) to which it is attached. In some embodiments, the 3' polyA tail has a run of at least 10 consecutive adenosine residues and up to 300 consecutive adenosine residues. In some embodiments, the RNA molecule comprises at least 20 consecutive adenosine residues and up to 40 consecutive adenosine residues. In some embodiments, the RNA molecule comprises about 40 consecutive adenosine residues. In some embodiments, the RNA molecule comprises about 80 consecutive adenosine residues.

[0087] H. Compositions In some cases, the compositions described herein comprise at least one saRNA as described herein. Some embodiments of the present disclosure provide an influenza virus (influenza) vaccine (or composition or immunogenic composition) comprising at least one saRNA polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response against influenza).

[0088] In some embodiments, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules (capped and uncapped) in the composition, at least any one of these, at most any one of these, or between any two of these are capped.

[0089] In some embodiments, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total RNA molecules in the composition, any one of at least any one of these, at most any one of these, or between any two of these is the full-length RNA transcript. Purity can be determined as described herein, for example, by reverse-phase HPLC or electrophoresis based on a bioanalyzer chip, and, for example, by the peak area of the full-length RNA molecule relative to the entire peak. In some embodiments, a Fragment Analyzer (FA) can be used to quantify and purify the RNA. In the Fragment Analyzer, capillary electrophoresis and HPLC are performed automatically.

[0090] In some embodiments, the composition is substantially free of one or more impurities or contaminants, including linear DNA templates and / or reverse complementary transcripts, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, at least 98% pure, or at least 99% pure, any one of at least any one of these, at most any one of these, or between any two of these, and contains RNA molecules.

[0091] In some embodiments, the composition contains a first RNA molecule in an amount greater than the amount of a second RNA molecule. In some embodiments, the composition contains a first RNA molecule in an amount of at least about 1 to 2 times the amount of the second RNA molecule. In some embodiments, the composition contains a first RNA molecule in an amount of at least about 1 to 100 times the amount of the second RNA molecule.

[0092] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a pharmaceutically acceptable vehicle.

[0093] In some embodiments, the composition further comprises a lipid-based delivery system, whereby the RNA molecule is delivered into the interior of the cell, where the RNA molecule can replicate and / or express the polypeptide of interest that is encoded. The delivery system may have an adjuvant effect that enhances the immunogenicity of the encoded antigen. In some embodiments, the composition further comprises a neutral lipid, a cationic lipid, cholesterol, and polyethylene glycol (PEG) to form nanoparticles that encapsulate the RNA molecule. In some embodiments, the composition further comprises any one of a cationic lipid, liposome, lipid nanoparticle, polyplex, coacervate, virosome, immunostimulatory complex, microparticle, microsphere, nanosphere, unilamellar vesicle, multilamellar vesicle, water-in-oil emulsion, oil-in-water emulsion, emulsome, polycationic peptide, and cationic nanoemulsion. In some embodiments, the RNA molecule is encapsulated in, bound to, or adsorbed to any one of a cationic lipid, liposome, lipid nanoparticle, polyplex, coacervate, virosome, immunostimulatory complex, microparticle, microsphere, nanosphere, unilamellar vesicle, multilamellar vesicle, water-in-oil emulsion, oil-in-water emulsion, emulsome, polycationic peptide, and cationic nanoemulsion, or a combination thereof.

[0094] In some cases, the compositions described herein comprise at least two RNA molecules, a first saRNA molecule and a second RNA molecule as described herein. To provide protection against more than one strain of influenza, it contains RNA (e.g., saRNA) encoding at least one antigenic polypeptide protein (or an antigenic portion thereof) of a first influenza virus or organism, and further comprises a second RNA molecule encoding at least one antigenic polypeptide protein (or an antigenic portion thereof) of a second influenza virus or organism. A mixed vaccine composition can be administered. The RNA (e.g., saRNA) can be co-formulated, for example, in a single lipid nanoparticle (LNP), or formulated in separate LNPs for co-administration.

[0095] In some embodiments, the second RNA molecule comprises any one of, or any combination of, a 5' cap, 5' UTR, open reading frame, 3' UTR, and polyA sequence. In some embodiments, the second RNA molecule comprises a 5' cap portion. In some embodiments, the second RNA molecule comprises a 5' UTR and a 3' UTR. In some embodiments, the second RNA molecule comprises a 5' UTR, open reading frame, 3' UTR, and does not further comprise a 5' cap. In some embodiments, the second RNA molecule comprises a 5' cap portion, 5' UTR, coding region, 3' UTR, and 3' polyA sequence. In some embodiments, the second RNA molecule comprises a 5' cap portion, 5' UTR, non-coding region, 3' UTR, and 3' polyA sequence. In some embodiments, the second RNA molecule comprises a non-coding region and does not further comprise any one of a 5' cap portion, 5' UTR, 3' UTR, and 3' polyA sequence. In some embodiments, the second RNA molecule comprises a 5' cap portion, 5' untranslated region (5' UTR), modified nucleotides, open reading frame, 3' untranslated region (3' UTR), and 3' polyA sequence.

[0096] Some aspects of the present disclosure are directed to compositions comprising: (i) a first RNA molecule encoding a gene of interest derived from influenza, and (ii) a second RNA molecule comprising modified or unnatural nucleotides. In some cases, the first RNA molecule is any one of the saRNA molecules described herein. In some cases, the first RNA molecule comprises a 5' cap, a 5' untranslated region, a coding region for non-structural proteins including an RNA replicase, a subgenomic promoter, an open reading frame encoding the gene of interest, a 3' untranslated region, and a 3' polyA sequence. In some cases, at least 5% of the entire population of specific nucleotides within the first RNA molecule is replaced by one or more modified or unnatural nucleotides. In some cases, the saRNA molecule comprises natural unmodified nucleotides and does not comprise modified or unnatural nucleotides. In some cases, the 5' cap is represented by Formula I, wherein R 1 and R 2 are each independently H or Me, and B 1 and B 2 are each independently guanine, adenine, or uracil, a 5' untranslated region, a coding region for non-structural proteins derived from an alphavirus, a subgenomic promoter such as those derived from an alphavirus, an open reading frame encoding the gene of interest, a 3' untranslated region, and a 3' polyA sequence. In some embodiments, B 1 and B 2 are naturally occurring bases. In some embodiments, R 1 is methyl and R 2 is hydrogen. In some embodiments, B 1 is guanine. In some embodiments, B 1 is adenine. In some embodiments, B 2 is adenine. In some embodiments, B 2 is uracil. In some embodiments, the nucleotide immediately downstream of the 5' cap (in the 5' to 3' direction) comprises guanine.

[0097] In some embodiments, B 1is adenine, and B 2 is uracil. In some embodiments, B 1 is adenine, and B 2 is uracil, R 1 is methyl, and R 2 is hydrogen. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap contains guanine, B 1 is adenine, and B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, and this embodiment corresponds to CLEANCAP AU (Trilink), and B 2 the uracil nucleotide downstream of may be substituted, but B 2 including B = uracil has been shown in some embodiments to result in an increase in saRNA functionality.

[0098] In some embodiments, at least 10% of the entire population of specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the entire population of specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the entire population of specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 75% of the entire population of specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, substantially all of the population of specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, the one or more replaced modified or unnatural nucleotides include two modified or unnatural nucleotides provided in a ratio ranging from 1:99 to 99:1, or any derivable range therein. In some embodiments, at least 10% of the entire population of the first specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and at least 10% of the entire population of the second specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the entire population of the first specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and at least 25% of the entire population of the second specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides.In some embodiments, at least 10% of the entire population of the first specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and at least 50% of the entire population of the second specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the entire population of the first specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and at least 75% of the entire population of the second specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 10% of the entire population of the first specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and substantially all of the entire population of the second specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the entire population of the first specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and at least 25% of the entire population of the second specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the entire population of the first specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and at least 50% of the entire population of the second specific nucleotides in the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides.In some embodiments, at least 25% of the entire population of first specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and at least 75% of the entire population of second specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 25% of the entire population of first specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and substantially all of the entire population of second specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the entire population of first specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and at least 75% of the entire population of second specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 50% of the entire population of first specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and substantially all of the entire population of second specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides. In some embodiments, at least 75% of the entire population of first specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides, and substantially all of the entire population of second specific nucleotides within the first or second RNA molecule is replaced by one or more modified or unnatural nucleotides.

[0099] In some embodiments, at least 25% of the entire population of uridine nucleotides in the first RNA molecule is replaced by N1-methylpseudouridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the first RNA molecule is replaced by N1-methylpseudouridine. In some embodiments, at least 75% of the entire population of uridine nucleotides in the first RNA molecule is replaced by N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule are replaced by N1-methylpseudouridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the first RNA molecule is replaced by 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides in the molecule are replaced by 5-methoxyuridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the first RNA molecule is replaced by 5-methyluridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule are replaced by 5-methyluridine. In some embodiments, at least 50% of the entire population of cytosine nucleotides in the first RNA molecule is replaced by 5-methylcytosine. In some embodiments, substantially all cytosine nucleotides in the first RNA molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the first RNA molecule is replaced by 2-thiouridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule are replaced by 2-thiouridine.

[0100] In some embodiments, at least 25% of the entire population of uridine nucleotides in the second RNA molecule is replaced by N1-methylpseudouridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by N1-methylpseudouridine. In some embodiments, at least 75% of the entire population of uridine nucleotides in the second RNA molecule is replaced by N1-methylpseudouridine. In some embodiments, substantially all of the uridine nucleotides in the second RNA molecule are replaced by N1-methylpseudouridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by 5-methoxyuridine. In some embodiments, substantially all of the uridine nucleotides in the second RNA molecule are replaced by 5-methoxyuridine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by 5-methyluridine. In some embodiments, substantially all of the uridine nucleotides in the second RNA molecule are replaced by 5-methyluridine. In some embodiments, at least 50% of the entire population of cytosine nucleotides in the second RNA molecule is replaced by 5-methylcytosine. In some embodiments, substantially all of the cytosine nucleotides in the second RNA molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule is replaced by 2-thiouridine. In some embodiments, substantially all of the uridine nucleotides in the second RNA molecule are replaced by 2-thiouridine.

[0101] In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced by N1-methylpseudouridine, and substantially all of the cytosine nucleotides in the second RNA molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced by 5-methoxyuridine, and substantially all of the cytosine nucleotides in the second RNA molecule are replaced by 5-methylcytosine. In some embodiments, at least 50% of the entire population of uridine nucleotides in the second RNA molecule are replaced by 5-methyluridine, and substantially all of the cytosine nucleotides in the second RNA molecule are replaced by 5-methylcytosine.

[0102] In some embodiments, substantially all of the uridine nucleotides in the second RNA molecule are replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudouridine. In some embodiments, substantially all of the uridine nucleotides in the second RNA molecule are replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseudouridine. In some embodiments, substantially all of the uridine nucleotides in the second RNA molecule are replaced by approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseudouridine.

[0103] In some embodiments, substantially all uridine nucleotides within the first RNA molecule are replaced by N1-methylpseudouridine, and at least 50% of the entire population of uridine nucleotides within the second RNA molecule are replaced by N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides within the first RNA molecule are replaced by N1-methylpseudouridine, and substantially all uridine nucleotides within the second RNA molecule are replaced by N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides within the first RNA molecule are replaced by N1-methylpseudouridine, and at least 50% of the entire population of uridine nucleotides within the second RNA molecule are replaced by 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides within the first RNA molecule are replaced by N1-methylpseudouridine, and at least 50% of the entire population of uridine nucleotides within the second RNA molecule are replaced by 5-methyluridine, and substantially all cytosine nucleotides within the second RNA molecule are replaced by 5-methylcytosine. In some embodiments, substantially all uridine nucleotides within the first RNA molecule are replaced by N1-methylpseudouridine, and substantially all uridine nucleotides within the second RNA molecule are replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudouridine.

[0104] I. Method of Use Using the saRNA composition, influenza viruses of various genotypes, strains, and isolates can be treated and / or prevented. Some embodiments provide a method of preventing or treating influenza virus infection, the method comprising administering to a subject any of the saRNA compositions described herein. In some embodiments, the antigen-specific immune response includes a T cell response. In some embodiments, the antigen-specific immune response includes a B cell response. In some embodiments, the antigen-specific immune response includes both a T cell response and a B cell response. In some embodiments, the method of generating an antigen-specific immune response involves a single administration of the saRNA composition. In some embodiments, the saRNA composition is administered to a subject by intradermal, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration.

[0105] In some embodiments, the RNA (e.g., saRNA) polynucleotide or a portion thereof may encode, as an antigen, one or more polypeptides or fragments thereof of an influenza strain.

[0106] Some aspects of the present disclosure are directed to a method of inducing an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition disclosed herein. Some aspects of the present disclosure are directed to a method of vaccinating a subject, the method comprising administering to a subject in need thereof an effective amount of a composition disclosed herein. Some aspects of the present disclosure are directed to a method comprising administering to a subject in need thereof an effective amount of a composition disclosed herein. In some embodiments, an immune response including an antibody response is elicited by the composition disclosed herein. In some embodiments, an immune response including a T cell response is elicited by the composition disclosed herein.

[0107] Some embodiments of the present disclosure are methods of inducing an antigen-specific immune response in a subject, the method comprising administering to the subject any of the RNA (e.g., saRNA) compositions presented herein in an effective amount to elicit an antigen-specific immune response. In some embodiments, the RNA (e.g., saRNA) composition is an influenza vaccine. In some embodiments, the RNA (e.g., saRNA) composition is a mixed vaccine (broad-spectrum influenza vaccine) comprising a combination of influenza vaccines.

[0108] In some embodiments, the antigen-specific immune response comprises a T cell response or a B cell response. In some embodiments, the method of eliciting an antigen-specific immune response comprises administering to the subject a single dose (without a booster dose) of the influenza RNA (e.g., saRNA) composition of the present disclosure. In some embodiments, the method further comprises administering to the subject a second (booster) dose of the influenza RNA (e.g., saRNA) composition. Additional doses of the influenza RNA (e.g., saRNA) composition can be administered.

[0109] In some embodiments, the subject exhibits a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) after the first dose or the second (booster) dose of the vaccine. Seroconversion is the period during which specific antibodies are generated and become detectable in the blood. After seroconversion, the virus can be detected in a blood test for the antibody. During infection or immunization, the antigen enters the blood and the immune system responds by starting to produce antibodies. Before seroconversion, the antigen itself may or may not be detectable, but no antibodies are thought to be present. During seroconversion, antibodies are present but not yet detectable. After seroconversion, antibodies can be detected in the blood at any point in time, thereby indicating a previous or current infection.

[0110] In some embodiments, the influenza RNA (e.g., saRNA) composition is administered to a subject by intradermal injection, intramuscular injection, or by intranasal administration. In some embodiments, the influenza RNA (e.g., saRNA) composition is administered to a subject by intramuscular injection.

[0111] Some embodiments of the present disclosure provide a method of inducing an antigen-specific immune response in a subject, the method comprising administering to the subject an effective amount of an influenza RNA (e.g., saRNA) composition to generate an antigen-specific immune response in the subject. In some embodiments, the antigen-specific immune response in the subject can be determined by assaying the antibody titer (the titer of antibodies that bind to the influenza antigenic polypeptide) after administering any of the influenza RNA (e.g., saRNA) compositions of the present disclosure to the subject. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by at least 1 log as compared to the control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by 1 to 3 logs as compared to the control.

[0112] In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by at least 2-fold as compared to the control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by at least 5-fold as compared to the control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by at least 10-fold as compared to the control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject increases by at least 2 to 10-fold as compared to the control.

[0113] In some embodiments, the control is the anti-antigenic polypeptide antibody titer produced in a subject who has not received administration of an RNA (e.g., saRNA) composition of the present disclosure. In some embodiments, the control is the anti-antigenic polypeptide antibody titer produced in a subject who has received administration of a live-attenuated or inactivated influenza, or the control is the anti-antigenic polypeptide antibody titer produced in a subject who has received administration of a recombinant or purified influenza protein vaccine.

[0114] In some embodiments, the RNA (e.g., saRNA) composition is formulated in an effective amount to elicit an antigen-specific immune response in a subject.

[0115] In some embodiments, the effective amount is a total dose of 1 μg to 1000 μg, or 1 μg to 100 μg of saRNA. In some embodiments, the effective amount is a total dose of 30 μg. In some embodiments, the effective amount is a dose of 10 μg administered a total of 2 times to the subject. In some embodiments, the effective amount is a dose of 15 μg administered a total of 2 times to the subject. In some embodiments, the effective amount is a dose of 30 μg administered a total of 2 times to the subject.

[0116] In some embodiments, the method comprises administering to a subject the saRNA composition described herein at a dosage amount between 10 μg / kg and 400 μg / kg. In some embodiments, the dosage amount of the saRNA polynucleotide is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg, 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg per single dose. In some embodiments, the saRNA composition is administered to the subject by intradermal or intramuscular injection. In some embodiments, the saRNA composition is administered to the subject on day 0. In some embodiments, a second dose of the saRNA composition is administered to the subject on day 21.

[0117] In some embodiments, the subject is about 5 years of age or less. For example, the subject may be between about 1 and about 5 years old (e.g., about 1, 2, 3, 4, or 5 years old), or between about 6 months and about 1 year old (e.g., about 6, 7, 8, 9, 10, 11, or 12 months old). In some embodiments, the subject is about 12 months of age or less (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months old, or 1 month old). In some embodiments, the subject is about 6 months of age or less.

[0118] In some embodiments, the subject was full-term born (e.g., about 37 - 42 weeks). In some embodiments, the subject was preterm born at a time such as, for example, about 36 weeks of gestation or earlier (e.g., about 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, or 25 weeks). For example, the subject was born at about 32 weeks of gestation or earlier. In some embodiments, the subject was preterm born between about 32 weeks and about 36 weeks of gestation. For such subjects, an RNA (e.g., mRNA) vaccine can be administered later in life, e.g., at about 6 months of age to about 5 years of age, or later.

[0119] In some embodiments, the subject is a young adult between about 20 years old and about 50 years old (e.g., about 20, 25, 30, 35, 40, 45, or 50 years old).

[0120] In some embodiments, the subject is an elderly subject about 60 years old, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85, or 90 years old).

[0121] In some embodiments, the subject has already been exposed to influenza (e.g., Chlamydia trachomatis (C. trachomatis)), the subject is infected with influenza (e.g., Chlamydia trachomatis (C. trachomatis)), or the subject is at risk of infection with influenza (e.g., Chlamydia trachomatis (C. trachomatis)).

[0122] In some embodiments, the subject has already been exposed to beta coronavirus (e.g., SARS-CoV-2), the subject is infected with beta coronavirus (e.g., SARS-CoV-2), or the subject is at risk of infection with beta coronavirus (e.g., SARS-CoV-2).

[0123] In some embodiments, the subject has already received at least one dose of an immunogenic composition against a beta coronavirus (e.g., SARS-CoV-2), which is selected from, for example, any one of COMIRNATY (registered trademark), the Pfizer-BioNTech COVID-19 vaccine, the Moderna mRNA-1273 COVID-19 vaccine, and the Janssen COVID-19 vaccine; the subject has already received at least two doses of an immunogenic composition against a beta coronavirus (e.g., SARS-CoV-2); the subject has received at least one dose of an immunogenic composition against a beta coronavirus (e.g., SARS-CoV-2), which is selected from, for example, any one of COMIRNATY (registered trademark), the Pfizer-BioNTech COVID-19 vaccine, the Moderna mRNA-1273 COVID-19 vaccine, and the Janssen COVID-19 vaccine; or the subject is at risk of infection with a beta coronavirus (e.g., SARS-CoV-2), and administration of an immunogenic composition against a beta coronavirus (e.g., SARS-CoV-2), which is selected from, for example, any one of COMIRNATY (registered trademark), the Pfizer-BioNTech COVID-19 vaccine, the Moderna mRNA-1273 COVID-19 vaccine, and the Janssen COVID-19 vaccine, is received concurrently with, simultaneously with, or within 12 to 48 hours of any one of the immunogenic compositions against influenza disclosed herein.

[0124] In some embodiments, the subject is immunocompromised (has a dysfunction of the immune system, e.g., has an immunodeficiency or an autoimmune disorder).

[0125] Aspects of the present disclosure provide an saRNA composition comprising one or more saRNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the saRNA polynucleotide is present as a formulation for in vivo administration to a host that confers an antibody titer exceeding a criterion for antibody prevalence for a first antigen (e.g., HA) in an acceptable percentage of human subjects. In some embodiments, the antibody titer produced by the saRNA composition of the present disclosure is a neutralizing antibody titer. In some embodiments, the neutralizing antibody titer is higher than that by a protein vaccine. In other embodiments, the neutralizing antibody titer produced by the saRNA composition is higher than that by an adjuvant-added protein vaccine. In yet other embodiments, the neutralizing antibody titer produced by the saRNA composition is 1,000 to 10,000, 1,200 to 10,000, 1,400 to 10,000, 1,500 to 10,000, 1,000 to 5,000, 1,000 to 4,000, 1,800 to 10,000, 2000 to 10,000, 2,000 to 5,000, 2,000 to 3,000, 2,000 to 4,000, 3,000 to 5,000, 3,000 to 4,000, or 2,000 to 2,500. The neutralizing titer is generally expressed as the highest serum dilution required to achieve a 50% reduction in the number of plaques.

[0126] J. Nucleic Acids In certain embodiments, the nucleic acid sequence can be present in various situations, such as, for example, an isolated segment, as well as a recombinant vector incorporating the sequence, or a recombinant polynucleotide encoding a polypeptide such as an antigen or one or both of the antibody chains, or a fragment, derivative, mutant protein, or variant thereof, a hybridization probe, PCR primer or sequencing primer sufficient for use in identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, a polynucleotide as described above herein, an mRNA, saRNA, and an antisense nucleic acid for inhibiting the expression of the complementary sequence. The nucleic acid encodes an epitope to which an antibody can bind. Nucleic acids encoding fusion proteins containing these polypeptides are also provided. The nucleic acid can be single-stranded or double-stranded and can contain RNA and / or DNA nucleotides and their artificial analogs (e.g., peptide nucleic acids).

[0127] The term "polynucleotide" refers to a nucleic acid molecule that is recombinable or isolated from the whole genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of a length of 100 residues or less), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, etc. In certain embodiments, the polynucleotide includes regulatory sequences substantially isolated from their naturally occurring genes or protein-coding sequences. The polynucleotide can be single-stranded (coding or antisense) or double-stranded and can be RNA, DNA (genomic DNA, cDNA or synthetic DNA), an analog thereof, or a combination of these. Additional coding or non-coding sequences may or may not be present within the polynucleotide.

[0128] In this regard, the term "gene" is used to refer to a nucleic acid encoding a protein, polypeptide, or peptide (including any sequence necessary for accurate transcription, post-translational modification, or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that can express or be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. Nucleic acids encoding all or part of a polypeptide may contain a continuous nucleic acid sequence encoding all or part of such polypeptide. It is also contemplated that a particular polypeptide may be encoded by nucleic acids containing variations that have slightly different nucleic acid sequences but nevertheless encode the same or substantially similar polypeptides.

[0129] In certain embodiments, polynucleotide variants having substantial identity to the sequences disclosed herein; variants that contain sequence identity of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more, at least any one of these, at most any one of these, or between any two of these, as compared to the polynucleotide sequences presented herein, using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, an isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90% identity over the length of the sequence to the amino acid sequences described herein, or a nucleotide sequence complementary to the aforementioned isolated polynucleotide. In some embodiments, an isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 95% identity over the length of the sequence to the amino acid sequences described herein, or a nucleotide sequence complementary to the aforementioned isolated polynucleotide.

[0130] In some embodiments, the polynucleotide comprises a 5’UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 14. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 15. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 16. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 17. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 19. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 20. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 21.In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 22.

[0131] In some embodiments, the polynucleotide comprises a 5’UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 12; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 13; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 14; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 15; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 16; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 17; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 19; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 21; and a polyA tail comprising at least 20 consecutive adenines.

[0132] In some embodiments, the polynucleotide has a 5’UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 12; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 13; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 14; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 15; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 16; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 17; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 18; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 19; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 20; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 21; and a polyA tail comprising at least 20 consecutive adenines.

[0133] In some embodiments, the polynucleotide comprises a 5’UTR sequence having SEQ ID NO: 12; a polynucleotide sequence having SEQ ID NO: 13; a polynucleotide sequence having SEQ ID NO: 14; a sequence having SEQ ID NO: 15; a polynucleotide sequence having SEQ ID NO: 16; a polynucleotide sequence having SEQ ID NO: 17; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having SEQ ID NO: 19; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having SEQ ID NO: 21; and a polyA tail comprising at least 20 consecutive adenines.

[0134] In some embodiments, the polynucleotide comprises a 5’UTR sequence having SEQ ID NO: 12; a polynucleotide sequence having SEQ ID NO: 13; a polynucleotide sequence having SEQ ID NO: 14; a sequence having SEQ ID NO: 15; a polynucleotide sequence having SEQ ID NO: 16; a polynucleotide sequence having SEQ ID NO: 17; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 18; a polynucleotide sequence having SEQ ID NO: 19; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 20; a polynucleotide sequence having SEQ ID NO: 21; and a polyA tail comprising at least 20 consecutive adenines.

[0135] In some embodiments, the polynucleotide comprises a 5’UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 23. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 24. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 25. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 26. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 27. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 28. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 29. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 30. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 31. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 32.

[0136] In some embodiments, the polynucleotide has a 5’UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 23; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 24; a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 25; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 26; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 27; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 28; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 29; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 31; and a polyA tail containing at least 20 consecutive adenines.

[0137] In some embodiments, the polynucleotide comprises a 5’UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 23; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 24; a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 25; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 26; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 27; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 28; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 29; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 30; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 31; and a polyA tail comprising at least 20 consecutive adenines.

[0138] In some embodiments, the polynucleotide comprises a 5’UTR sequence having SEQ ID NO: 23; a polynucleotide sequence having SEQ ID NO: 24; a polynucleotide sequence having SEQ ID NO: 25; a polynucleotide sequence having SEQ ID NO: 26; a polynucleotide sequence having SEQ ID NO: 27; a polynucleotide sequence having SEQ ID NO: 28; a polynucleotide sequence having SEQ ID NO: 29; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1, and NS2; a polynucleotide sequence having SEQ ID NO: 31; and a polyA tail comprising at least 20 consecutive adenines.

[0139] In some embodiments, the polynucleotide comprises a 5’UTR sequence having SEQ ID NO: 23; a polynucleotide sequence having SEQ ID NO: 24; a polynucleotide sequence having SEQ ID NO: 25; a polynucleotide sequence having SEQ ID NO: 26; a polynucleotide sequence having SEQ ID NO: 27; a polynucleotide sequence having SEQ ID NO: 28; a polynucleotide sequence having SEQ ID NO: 29; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 30; a polynucleotide sequence having SEQ ID NO: 31; and a polyA tail comprising at least 20 consecutive adenines. The nucleic acid segment can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., regardless of the length of the coding sequence itself, and thus their overall length can vary significantly. The nucleic acid can be of any length. The nucleic acid can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length, any one of these, at least any one of these, at most any one of these, or between any two of these, and / or can include one or more additional sequences, such as regulatory sequences, and / or can be part of a larger nucleic acid, such as a vector. Thus, it is intended that nucleic acid fragments of almost any length can be used, and the overall length is limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can encode a polypeptide sequence having additional heterologous coding sequences to enable therapeutic benefits such as, for example, purification, transport, secretion, post-translational modification, or targeting or efficacy of the polypeptide.As described above, a tag or other heterologous polypeptide can be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0140] K. Lipid Delivery In some embodiments, the saRNA composition comprises a lipid. The lipid and the saRNA can be combined to form nanoparticles. The lipid can encapsulate the mRNA to form lipid nanoparticles (LNPs), which can assist in the entry and stability of the RNA / lipid nanoparticles into cells.

[0141] The lipid nanoparticles can comprise lipid components and one or more additional components, such as a therapeutic agent and / or a prophylactic agent. The LNPs can be designed for one or more specific applications or targets. The components of the LNPs can be selected based on a specific application or target and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more of the components. Similarly, a specific formulation of the LNPs can be selected for a specific application or target, for example, depending on the efficacy and toxicity of a specific combination of components. The stability of the formulation can also affect the efficacy and tolerability of the LNP formulation.

[0142] The lipid nanoparticles can be designed for one or more specific applications or targets. For example, the LNPs can be designed such that a therapeutic agent and / or a prophylactic agent, such as an RNA, is delivered to specific cells, tissues, organs, or systems or groups thereof in a mammalian body.

[0143] The physicochemical properties of lipid nanoparticles can be modified to increase their selectivity for specific body targets. For example, the particle size can be adjusted based on the fenestration sizes of different organs. The therapeutic and / or prophylactic agents included in the LNP can also be selected based on the desired delivery target(s). For example, the therapeutic and / or prophylactic agent can be selected for a specific indication, condition, disease, or disorder and / or for delivery (e.g., localization or specific delivery) to a specific cell, tissue, organ, or system or group thereof. In certain embodiments, the LNP can include mRNA encoding a polypeptide of interest that can be translated intracellularly to produce the polypeptide of interest. Such compositions can be designed to be specifically delivered to a particular organ. In some embodiments, the composition can be designed to be specifically delivered to the mammalian liver. In some embodiments, the composition can be designed to be specifically delivered to lymph nodes. In some embodiments, the composition can be designed to be specifically delivered to the mammalian spleen.

[0144] The LNP can include one or more of the components described herein. In some embodiments, the LNP formulations of the present disclosure include at least one lipid nanoparticle component. The lipid nanoparticles can include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic agent, e.g., a nucleic acid. The LNP can be designed for one or more specific applications or targets. The components of the LNP can be selected based on a specific application or target and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more of the components. Similarly, a specific formulation of the LNP can be selected for a specific application or target, e.g., depending on the efficacy and toxicity of a specific combination of components. The stability of the formulation can also affect the efficacy and tolerability of the LNP formulation.

[0145] In some embodiments, for example, the polymer can be included in the LNP and / or used to encapsulate or partially encapsulate the LNP. The polymer may be biodegradable and / or biocompatible. The polymer can be selected from, but not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, as the polymer, poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(ester amide), polyamide, poly(ester ether), polycarbonate, polyalkylene, such as polyethylene and polypropylene, polyalkylene glycol, such as poly(ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene terephthalate, such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl ester, such as poly(vinyl acetate), polyhalogenated vinyl, such as poly(vinyl chloride) (PVC), polyvinyl pyrrolidone (PVP), polysiloxane, polystyrene, polyurethane, derivatized cellulose, such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers of acrylic acid, such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and their copolymers and mixtures, polydioxanone and its copolymers, polyhydroxyalkanoic acid, polypropylene fumarate, polyoxymethylene, poloxamer, poloxamine, poly(ortho)ester, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), its derivatives, and polyglycerol can be mentioned.,

[0146] Examples of surface modifiers include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, mugwort, bromelain, papain, plantain, bromhexine, carbocysteine, epratrizone, mesna, ambroxol, sobrerol, domiodol, restelin, streptonin, tiopronin, gelsolin, thymosin β4, dornase alfa, neruthenexin, and erdostein), and DNA degrading enzymes (e.g., rhDNase). The surface modifier can be disposed inside the nanoparticles and / or on the surface of the LNP (e.g., by coating, adsorption, covalent linkage, or other processes).

[0147] The LNP may also contain one or more functionalized lipids. For example, the lipid can be functionalized with an alkyne group that can undergo a cycloaddition reaction when exposed to azide under appropriate reaction conditions. In particular, the lipid bilayer can be functionalized in this manner using one or more groups useful for facilitating membrane permeation, cell recognition, or imaging. One or more useful antibodies can also be conjugated to the surface of the LNP. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0148] In addition to these components, the lipid nanoparticles can contain any substance useful in a pharmaceutical composition. For example, the lipid nanoparticles can contain one or more pharmaceutically acceptable excipients or adjuncts such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, surfactants, buffers, preservatives, and other species.

[0149] Surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, and / or combinations thereof.

[0150] Examples of preservatives include, but are not limited to, antioxidants, chelating agents, free radical scavengers, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antibacterial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoic acid, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Examples of other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deferoxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium bisulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™, KATHON™, and / or EUXYL®. Exemplary free radical scavengers include butylated hydroxytoluene (BHT or butylhydroxytoluene) or deferoxamine.

[0151] Examples of buffers include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium glyceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium phosphate, potassium dihydrogen phosphate, mixture of potassium phosphates, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium hydrogen phosphate, sodium dihydrogen phosphate, mixture of sodium phosphates, tromethamine, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic physiological saline, Ringer's solution, ethyl alcohol, Tris buffer, and / or combinations thereof.

[0152] In some embodiments, formulations containing LNPs may further contain salts such as chloride salts. In some embodiments, formulations containing LNPs may further contain sugars such as disaccharides. In some embodiments, the formulation further contains sugar but does not contain salts such as chloride salts. In some embodiments, the LNP may further contain one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols. Carbohydrates can include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0153] The characteristics of LNPs can depend on their components. For example, an LNP containing cholesterol as a structural lipid can have different characteristics from an LNP containing a different structural lipid. As used herein, the term "structural lipid" refers to sterols and also refers to lipids containing a sterol moiety. "Sterol" as defined herein is a subgroup of steroids consisting of steroid alcohols. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is alpha-tocopherol.

[0154] In some embodiments, the characteristics of LNPs can depend on the absolute or relative amounts of their components. For example, an LNP containing a higher molar concentration fraction of phospholipid can have different characteristics from an LNP containing a lower molar concentration fraction of phospholipid. The characteristics can vary depending on the method and conditions for preparing the lipid nanoparticles. Generally, phospholipids contain a phospholipid moiety and one or more fatty acid moieties.

[0155] The phospholipid moiety can be selected from the non-limiting group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin. The fatty acid moiety can be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Certain phospholipids can facilitate fusion with membranes. In some embodiments, the cationic lipid can interact with one or more negatively charged lipids of a membrane (e.g., cell membrane or intracellular membrane). Fusion of the phospholipid with the membrane can enable one or more components of the lipid-containing composition (e.g., LNP) (e.g., therapeutic agent) to pass through the membrane, thereby enabling, for example, delivery of one or more components to a target tissue. Non-natural phospholipid species are also contemplated, including those with modifications and substitutions including branching, oxidation, cyclization, and alkynes, including natural species. In some embodiments, the phospholipid can be functionalized or cross-linked using one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, the alkyne group can undergo copper-catalyzed cycloaddition upon exposure to azide. Such reactions can be useful for functionalizing the lipid bilayer of the nanoparticle composition to facilitate membrane permeation or cell recognition, or for conjugating the nanoparticle composition with useful components such as targeting or imaging moieties (e.g., dyes). Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Sphingophospholipids such as sphingomyelin are also included as phospholipids.In some embodiments, the phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC.

[0156] Lipid nanoparticles can be characterized by various methods. For example, a microscope (e.g., a transmission electron microscope or a scanning electron microscope) can be used to investigate the morphology and size distribution of the LNP. Zeta potential can be measured using dynamic light scattering or potentiometry (e.g., potentiometric titration). Particle size can also be determined using dynamic light scattering. Instruments such as a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple LNP characteristics such as particle size, polydispersity index, and zeta potential.

[0157] The average size of the LNP can be in the range of tens of nm to hundreds of nm, for example, measured by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the LNP can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the average size of the LNP can be from about 70 nm to about 100 nm. In a particular embodiment, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0158] The LNPs can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the LNPs, such as the particle size distribution of the lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The LNPs can have a polydispersity index of from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the LNPs can be from about 0.10 to about 0.20.

[0159] The zeta potential of the LNPs can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can explain the surface charge of the LNPs. Lipid nanoparticles with relatively low positive or negative charges are generally desirable because more highly charged species may undesirably interact with cells, tissues, and other components in the body. In some embodiments, the zeta potential of the LNPs can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0160] The encapsulation efficiency of a therapeutic and / or prophylactic agent describes the amount of the therapeutic and / or prophylactic agent encapsulated in or otherwise associated with the LNP after preparation, compared to the initially prepared amount. It is desirable for the encapsulation efficiency to be high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the therapeutic and / or prophylactic agent in a solution containing lipid nanoparticles before and after disruption of the lipid nanoparticles by one or more organic solvents or surfactants. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. For the lipid nanoparticles described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0161] The LNP may include one or more coatings. For example, the LNP can be formulated as a capsule, film, or tablet having a coating. The capsule, film, or tablet containing the composition described herein may have any useful size, tensile strength, hardness, or density.

[0162] Formulations comprising amphiphilic polymers and lipid nanoparticles can be formulated, in whole or in part, as pharmaceutical compositions. The pharmaceutical composition can comprise one or more amphiphilic polymers and one or more lipid nanoparticles. For example, the pharmaceutical composition can comprise one or more amphiphilic polymers and one or more lipid nanoparticles comprising one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition can further comprise one or more pharmaceutically acceptable excipients or adjuncts, such as those described herein. General guidelines regarding the formulation and manufacture of pharmaceutical compositions and medicaments are available, for example, in Remington’s The Science and Practice of Pharmacy, 21st Edition, A.R.Gennaro; Lippincott, Williams&Wilkins, Baltimore, MD, 2006. Conventional excipients and adjuncts can be used in any pharmaceutical composition, provided that any conventional excipient or adjunct is not incompatible with one or more of the components of the LNP or one or more of the amphiphilic polymers in the formulations of the present disclosure. An excipient or adjunct can be incompatible with the components of the LNP or amphiphilic polymers of the formulation if the combination of the excipient or adjunct with the components or amphiphilic polymers results in any undesirable biological effects or other adverse effects.

[0163] In some embodiments, one or more excipients or ancillary components may constitute more than 50% of the total mass or volume of the pharmaceutical composition comprising the LNPs. For example, one or more excipients or ancillary components may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable excipients are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipients are approved for human and animal use. In some embodiments, the excipients are approved by the Food and Drug Administration. In some embodiments, the excipients are of pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and / or International Pharmacopeia. The relative amounts of one or more amphiphilic polymers, one or more lipid nanoparticles, one or more pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical compositions according to the present disclosure vary depending on the identity, size, and / or condition of the subject to be treated and further depend on the route of administration of the composition. By way of example, the pharmaceutical composition may comprise 0.1% to 100% (wt / wt) of one or more lipid nanoparticles. As another example, the pharmaceutical composition may comprise 0.1% to 15% (wt / vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% w / v).

[0164] In certain embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen (e.g., at a temperature of 4°C or lower, e.g., between about -150°C and about 0°C or between about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C)) for storage and / or transportation. For example, a pharmaceutical composition comprising one or more amphiphilic polymers and one or more lipid nanoparticles is a solution or solid (e.g., by lyophilization) refrigerated at, for example, about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C for storage and / or transportation. In certain embodiments, the present disclosure also relates to a method of increasing the stability of lipid nanoparticles by adding an effective amount of an amphiphilic polymer and storing the lipid nanoparticles and / or its pharmaceutical composition at a temperature of 4°C or lower, e.g., between about -150°C and about 0°C or between about -80°C and about -20°C, e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C).

[0165] In some embodiments, the lipid components of the LNP include cationic lipids, phospholipids, PEG lipids, and structural lipids. In certain embodiments, the lipid components of the lipid nanoparticles include about 30 mol% to about 60 mol% cationic lipid, about 0 mol% to about 30 mol% phospholipid, about 18.5 mol% to about 48.5 mol% structural lipid, and about 0 mol% to about 10 mol% PEG lipid, provided that the total mol% does not exceed 100%. In some embodiments, the lipid components of the lipid nanoparticles include about 35 mol% to about 55 mol% of a cationic lipid compound, about 5 mol% to about 25 mol% phospholipid, about 30 mol% to about 40 mol% structural lipid, and about 0 mol% to about 10 mol% PEG lipid. In a particular embodiment, the lipid components include about 50 mol% of the aforementioned cationic lipid, about 10 mol% phospholipid, about 38.5 mol% structural lipid, and about 1.5 mol% PEG lipid. In another embodiment, the lipid components include about 40 mol% of the aforementioned cationic lipid, about 20 mol% phospholipid, about 38.5 mol% structural lipid, and about 1.5 mol% PEG lipid. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-DMG and / or the structural lipid can be cholesterol.

[0166] In some embodiments, the ionizable lipid is of formula (I):

[0167] [Chemical formula] a compound thereof or its N-oxide, or a salt or isomer thereof, wherein R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R * YR”, -YR”, and -R”M’R’; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR”, -YR”, and -R *selected from the group consisting of "OR", or R2 and R3, together with the atoms to which they are attached, form a heterocyclic or carbocyclic compound; R4 is hydrogen, a C3-6 carbocyclic compound, -(CH2) n Q, -(CH2) n selected from the group consisting of CHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, wherein Q is a carbocyclic compound, a heterocycle, -OR, -0(CH2) n N(R)2, -C(0)0R, -0C(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)Re, N(R)S(0)2R8, -0(CH2) nSelected from 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 -C(R)N(R)2C(0)OR; each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(0)0-, -OC(O)-, -0C(0)-M”-C(0)0-, -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-, aryl group, and heteroaryl group; M” is a bond, C1-13 alkyl or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocyclic compounds and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(0)2R, -S(0)2N(R)2, C2-6 alkenyl, C3-6 carbocyclic compounds and heterocycles; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently C1-13 alkyl, C2-13 alkenyl, -R * Selected from the group consisting of YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; each R *is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocyclic compound; 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, where R4 is (CH2)nQ, -(CH2) n CHQR, -CHQR, or -CQ(R)2, (i) Q is not -N(R)2 and n is 1, 2, 3, 4, or 5, or (ii) Q is not a 5, 6, or 7-membered heterocycloalkyl and n is 1 or 2. In some embodiments, the ionizable lipid is

[0168] [Chemical formula] is.

[0169] In some embodiments, the compound has the following structure (I):

[0170] [Chemical formula] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein one of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond; G 1 and G 2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G 3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; R 1 and R 2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R 3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C1-C12 alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1, or 2. In a preferred embodiment, the ionizable lipid is

[0171]

Chemical formula

[0172] The lipid components of the lipid nanoparticle composition may include one or more molecules containing polyethylene glycol such as PEG or PEG-modified lipids. Such species may alternatively be referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. PEG lipids can be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid. As used herein, the term "PEG lipid" refers to a polyethylene glycol (PEG)-modified lipid. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, and PEG-modified 1,2-diacyl-oxypropane-3-amine. Such lipids are also referred to as PEGylated lipids. In some embodiments, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid. In some embodiments, the PEG-modified lipid is a modified form of PEG DMG. In some embodiments, the PEG-modified lipid has the formula (IV):

[0173] [Chemical formula] (wherein R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain may be interrupted by one or more ester bonds: and w has an average value ranging from 30 to 60) is a PEG lipid having the formula.

[0174] L. formulation In one aspect, the present disclosure provides an immunogenic composition comprising: (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen, wherein the antigen comprises at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, and (ii) a second RNA polynucleotide having an open reading frame encoding a second antigen, wherein the second antigen comprises at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the first and second RNA polynucleotides are formulated in lipid nanoparticles (LNP). In some embodiments, the first and second antigens comprise hemagglutinin (HA) or an immunogenic fragment or variant thereof. In some embodiments, the first antigen comprises HA from an influenza virus subtype different from the influenza virus antigenic polypeptide or an immunogenic fragment thereof of the second antigen. In some embodiments, the composition further comprises (iii) a third antigen comprising at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the third antigen is derived from an influenza virus but from a strain different from either the first or second antigen. In some embodiments, the first, second, and third RNA polynucleotides are formulated in lipid nanoparticles.

[0175] In some embodiments, the composition further comprises (iv) a fourth RNA polynucleotide having an open reading frame encoding a fourth antigen, wherein the antigen comprises at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, and the fourth antigen is derived from an influenza virus but from a strain different from the first, second, and third antigens. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in lipid nanoparticles.

[0176] In some embodiments, the RNA polynucleotides are mixed at a desired ratio in a single container and then formulated into lipid nanoparticles. In some embodiments, by first inputting different RNA polynucleotides in known ratios to be formulated in a single LNP process, an LNP is provided in which different RNA polynucleotides are encapsulated in a ratio that is approximately the same as the input ratio. Such embodiments may be referred to herein as "pre-mixing."

[0177] Thus, in some embodiments, a first and a second RNA polynucleotide are formulated into a single lipid nanoparticle. In some embodiments, a first, second, third, and fourth RNA polynucleotides are formulated into a single LNP. In some embodiments, a first, second, third, fourth, and fifth RNA polynucleotides are formulated into a single LNP. In some embodiments, a first, second, third, fourth, fifth, and sixth RNA polynucleotides are formulated into a single LNP. In some embodiments, a first, second, third, fourth, fifth, sixth, and seventh RNA polynucleotides are formulated into a single LNP. In some embodiments, a first, second, third, fourth, fifth, sixth, seventh, and eighth RNA polynucleotides are formulated into a single LNP.

[0178] In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide in the mixture of RNA polynucleotides before formulation into the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide is greater than 1:1.

[0179] In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide in the mixture of RNA polynucleotides before formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide is greater than 1:1.

[0180] In some embodiments, the molar ratio of the first RNA polynucleotide to the fourth RNA polynucleotide in the mixture of RNA polynucleotides before formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the fourth RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the fifth RNA polynucleotide in the mixture of RNA polynucleotides before formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the fifth RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the sixth RNA polynucleotide in the mixture of RNA polynucleotides before formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the sixth RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the seventh RNA polynucleotide in the mixture of RNA polynucleotides before formulation in the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the seventh RNA polynucleotide is greater than 1:1.In some embodiments, the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide in the mixture of RNA polynucleotides before formulation into the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide is greater than 1:1.

[0181] In alternative embodiments, each RNA polynucleotide encoding a particular antigen is formulated into an individual LNP, and thus each LNP encapsulates an RNA polynucleotide encoding the same antigen. Such embodiments may be referred to herein as "post-mixing." Thus, in some embodiments, the first RNA polynucleotide is formulated into the first LNP, the second RNA polynucleotide is formulated into the second LNP, the third RNA polynucleotide is formulated into the third LNP, the fourth RNA polynucleotide is formulated into the fourth LNP, the fifth RNA polynucleotide is formulated into the fifth LNP, the sixth RNA polynucleotide is formulated into the sixth LNP, the seventh RNA polynucleotide is formulated into the seventh LNP, and the eighth RNA polynucleotide is formulated into the eighth LNP.

[0182] In some embodiments, the molar ratio of the first LNP to the second LNP in the mixture of LNPs before formulation into the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first LNP to the second LNP is greater than 1:1.

[0183] In some embodiments, the molar ratio of the first LNP to the third LNP in the mixture of LNPs before formulation into the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first LNP to the third LNP is greater than 1:1.

[0184] In some embodiments, the molar ratio of the first LNP to the fourth LNP in the mixture of LNPs before formulation into the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first LNP to the fourth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the fifth LNP in the mixture of LNPs before formulation into the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first LNP to the fifth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the sixth LNP in the mixture of LNPs before formulation into the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first LNP to the sixth LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the seventh LNP in the mixture of LNPs before formulation into the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first LNP to the seventh LNP is greater than 1:1. In some embodiments, the molar ratio of the first LNP to the eighth LNP in the mixture of LNPs before formulation into the LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first LNP to the eighth LNP is greater than 1:1.

[0185] In some embodiments, the relative amount of RNA encoding an antigen of influenza B virus can be increased compared to the RNA encoding influenza A virus (e.g., a higher neutralizing titer against influenza B virus (e.g., higher than a composition containing RNA encoding an equal amount of influenza A antigen and RNA encoding influenza B antigen (e.g., as determined by the pseudovirus neutralization assay described herein))). The present disclosure also provides exemplary dosages of RNA that can generate a strong immune response (e.g., clinically significant levels of neutralizing titer and / or seroconversion rate (e.g., (i) a neutralizing titer equal to or exceeding that previously shown to prevent influenza symptoms, and / or (ii) a neutralizing titer and / or seroconversion rate equal to or exceeding that induced by a relevant comparator (e.g., a commercially approved influenza vaccine or an influenza RNA vaccine)) against both types of influenza virus. In some embodiments, a composition comprising an amount of RNA encoding influenza B antigen that is greater than the amount of RNA encoding influenza A antigen generates an immune response equal to or exceeding that induced by a non-RNA influenza vaccine (e.g., an approved vaccine) and / or an RNA vaccine comprising an equal amount of RNA encoding influenza A antigen and RNA encoding influenza B antigen against each of influenza B virus and influenza A virus.

[0186] In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 to 0.2 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.12 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.14 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.16 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.18 mg / ml. In some embodiments, by administering about 200 μL of the RNA preparation, about 30 μg of RNA is administered. In some embodiments, the RNA in the pharmaceutical RNA preparation is diluted before administration (for example, diluted to a concentration of about 0.05 mg / ml). In some embodiments, the volume of administration is between about 200 μl and about 300 μl. In some embodiments, the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer and about 10% sucrose.

[0187] In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.1 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.12 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.14 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.16 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation contains RNA at a concentration of about 0.18 mg / ml and is formulated in about 10 mM Tris buffer and about 10% sucrose. Such formulations can be diluted prior to administration as needed to administer different doses of RNA while keeping the total injection volume relatively constant. For example, a dose of about 10 μg of RNA can be administered by diluting such a pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0188] In some embodiments, the vaccine is formulated in a vial (e.g., a glass vial). In some embodiments, the glass vial is sealed with a bromobutyl elastomer stopper and an aluminum seal with a flip-off plastic cap.

[0189] In some embodiments, the composition comprises RNA encoding an influenza virus antigen (e.g., HA protein) recommended by the relevant health authorities for inclusion in a seasonally adapted vaccine (e.g., cell-based, recombinant, or live attenuated virus). In some embodiments, the composition comprises multiple RNAs encoding the antigen (e.g., HA protein) of each influenza virus recommended by the relevant health authorities for inclusion in a seasonally adapted vaccine (e.g., cell-based, recombinant, or live attenuated virus). In some embodiments, the influenza virus is an influenza A, B, or C virus. In some embodiments, the influenza A virus is an H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7, or H10N8 virus. In some embodiments, the influenza A virus is an H1N1, H3N2, H5N1, or H5N8 virus. In some embodiments, the influenza A virus is an H1N1 virus (e.g., A / Wisconsin / 588 / 2019 or A / Sydney / 5 / 2021). In some embodiments, the influenza A virus is an H3N2 virus. In some embodiments, the H3N2 virus is A / Cambodia / e0826360 / 2020 or A / Darwin / 6 / 2021. In some embodiments, the influenza B virus is of the B / Yamagata or B / Victoria lineage. In some embodiments, the B / Victoria lineage influenza virus is B / Washington / 02 / 2019. In some embodiments, the B / Victoria lineage virus is B / Austria / 1359417 / 2021. In some embodiments, the B / Yamagata lineage influenza virus is B / Phuket / 3073 / 2013.

[0190] In some embodiments, the compositions described herein include a multivalent influenza vaccine. In some embodiments, the multivalent influenza vaccine includes 2 to 50 distinct RNA molecules (e.g., 2 to 40, 2 to 30, or 2 to 20 RNA molecules), each of which, in some embodiments, can encode different antigenic polypeptides (or different versions of a particular antigenic polypeptide) related to influenza, as described, for example, in Arevalo, Claudia P. et al., "A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes.", Science 378.6622 (2022): 899 - 904. In some embodiments, the compositions described herein include a trivalent influenza vaccine. In some embodiments, the trivalent influenza vaccine includes RNA encoding antigenic polypeptides related to two A viruses and one B virus predicted to spread within the relevant jurisdiction. In some embodiments, the compositions described herein include a quadrivalent influenza vaccine. In some embodiments, the quadrivalent influenza vaccine includes RNA encoding antigenic polypeptides related to two A viruses and two B viruses predicted to spread within the relevant jurisdiction. In some embodiments, the compositions described herein include an octavalent influenza vaccine. In some embodiments, the octavalent influenza vaccine includes RNA encoding two antigenic polypeptides (e.g., the HA protein and the NA protein or immunogenic fragments thereof related to each virus) related to each of two A viruses and two B viruses predicted to spread within the relevant jurisdiction.In some embodiments, the composition disclosed herein comprises RNA comprising a nucleotide sequence encoding an HA protein associated with the H1N1 virus (e.g., A / Wisconsin / 588 / 2019), RNA comprising a nucleotide sequence encoding an HA protein associated with the H3N2 virus (e.g., A / Cambodia / e0826360 / 2020), RNA comprising a nucleotide sequence encoding an HA protein associated with the B / Victoria lineage influenza virus (e.g., B / Washington / 02 / 2019), and a quadrivalent influenza vaccine comprising an HA protein associated with the B / Yamagata lineage influenza virus (e.g., B / Phuket / 3073 / 2013).

[0191] In some embodiments, a composition comprising a quadrivalent influenza vaccine comprises RNA encoding antigenic polypeptides associated with two A-type viruses and two B-type viruses predicted to spread within the relevant jurisdiction. In some embodiments, the quadrivalent influenza vaccine comprises RNA encoding an antigenic polypeptide associated with the H1N1 influenza virus, RNA encoding an antigenic polypeptide associated with the H3N2 influenza virus, RNA encoding an antigenic polypeptide associated with the Victoria lineage influenza virus, and RNA encoding an antigenic polypeptide associated with the Yamagata lineage influenza virus. In some embodiments, the quadrivalent influenza vaccine comprises RNA associated with an influenza type predicted to spread within the relevant jurisdiction (e.g., an HA polypeptide associated with the H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses predicted to spread within the relevant jurisdiction).

[0192] In some embodiments, a composition comprising an octavalent influenza vaccine comprises RNA encoding antigenic polypeptides related to two A-type viruses and two B-type viruses predicted to spread within the relevant jurisdiction. In some embodiments, the octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide derived from an HA from an A-type influenza virus, RNA encoding an antigenic polypeptide derived from an HA from an A-type influenza virus, RNA encoding an antigenic polypeptide derived from an HA from a B-type influenza virus, RNA encoding an antigenic polypeptide derived from an HA from a B-type influenza virus, RNA encoding an antigenic polypeptide derived from an antigenic polypeptide selected from NA, NP, M1, M2, NS1, and NS2 from an A-type influenza virus, RNA encoding an antigenic polypeptide derived from an antigenic polypeptide selected from NA, NP, M1, M2, NS1, and NS2 from an A-type influenza virus, RNA encoding an antigenic polypeptide derived from an antigenic polypeptide selected from NA, NP, M1, M2, NS1, and NS2 from a B-type influenza virus, and RNA encoding an antigenic polypeptide derived from an antigenic polypeptide selected from NA, NP, M1, M2, NS1, and NS2 from a B-type influenza virus. In some embodiments, the octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide derived from an HA from an A-type influenza virus, RNA encoding an antigenic polypeptide derived from an HA from an A-type influenza virus, RNA encoding an antigenic polypeptide derived from an HA from a B-type influenza virus, RNA encoding an antigenic polypeptide derived from an HA from a B-type influenza virus, RNA encoding an antigenic polypeptide derived from an NA from an A-type influenza virus, RNA encoding an antigenic polypeptide derived from an NA from an A-type influenza virus, RNA encoding an antigenic polypeptide derived from an NA from a B-type influenza virus, and RNA encoding an antigenic polypeptide derived from an NA from a B-type influenza virus.In some embodiments, the octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide associated with an H1N1 influenza virus, RNA encoding an antigenic polypeptide associated with an H3N2 influenza virus, RNA encoding an antigenic polypeptide associated with a Victoria lineage influenza virus, and RNA encoding an antigenic polypeptide associated with a Yamagata lineage influenza virus. In some embodiments, the octavalent influenza vaccine comprises RNA associated with an influenza type predicted to spread within the relevant jurisdiction (e.g., HA polypeptides associated with H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses predicted to spread within the relevant jurisdiction).

[0193] In some embodiments, the RNAs in the compositions disclosed herein each encode an antigenic polypeptide associated with an infectious agent predicted to spread within the relevant jurisdiction. Such compositions can reduce the number of required vaccinations.

[0194] In some embodiments, the nucleic acid-containing particle comprises two or more RNA molecules, each comprising a nucleotide sequence encoding an antigen (e.g., an HA protein) associated with a different influenza virus. In some embodiments, the nucleic acid-containing particle comprises three or more RNA molecules, each comprising a nucleotide sequence encoding an antigen (e.g., an HA protein) associated with a different influenza virus. In some embodiments, the nucleic acid-containing particle comprises four or more RNA molecules, each comprising a nucleotide sequence encoding an antigen (e.g., an HA protein) associated with a different influenza virus. In some embodiments, the nucleic acid-containing particle comprises an RNA molecule comprising a nucleotide sequence encoding an antigenic polypeptide associated with an H1N1 influenza virus, an RNA molecule comprising a nucleotide sequence encoding an antigenic polypeptide associated with an H3N2 influenza virus, an RNA molecule comprising a nucleotide sequence encoding an antigenic polypeptide associated with an influenza B / Victoria lineage virus, and an RNA molecule comprising a nucleotide sequence encoding an antigenic polypeptide associated with an influenza B / Yamagata virus. In some embodiments, each RNA in the composition that comprises a nucleotide sequence encoding an antigenic polypeptide associated with an influenza virus is formulated in the same nucleic acid-containing particle. In some embodiments, each RNA in the composition that comprises a nucleotide sequence encoding an antigenic polypeptide associated with an influenza virus is formulated in a separate nucleic acid-containing particle.

[0195] In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP described herein) comprising two or more RNA molecules comprises each RNA molecule in the same amount (i.e., in a 1:1 ratio).

[0196] In some embodiments, a nucleic acid-containing particle comprising two or more RNA molecules (e.g., in some embodiments, the LNPs described herein) contains each RNA molecule in a different amount. For example, in some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, where the first RNA molecule is present in an amount that is 0.01 to 100 times the amount of the second RNA molecule (e.g., the amount of the first RNA molecule is 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to 4, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 to 1.5 times that of the second RNA molecule). In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, where the concentration of the first RNA molecule is 1 to 10 times the concentration of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, where the concentration of the first RNA molecule is 1 to 5 times the concentration of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, where the concentration of the first RNA molecule is 1 to 3 times the concentration of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, where the concentration of the first RNA molecule is 2 times the concentration of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, where the concentration of the first RNA molecule is 3 times the concentration of the second RNA molecule.

[0197] In some embodiments, a nucleic acid-containing particle comprising three RNA molecules (e.g., in some embodiments, the LNPs described herein) contains each RNA molecule in the same amount (i.e., in a 1:1:1 ratio).

[0198] In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP described herein) comprising three RNA molecules comprises each RNA molecule in a different amount. For example, in some embodiments, the ratio of the first RNA molecule: the second RNA molecule: the third RNA molecule is 1: 0.01-100: 0.01-100 (e.g., 1: 0.01-50: 0.01-50; 1: 0.01-40: 0.01-40; 1: 0.01-30: 0.01-25; 1: 0.01-25: 0.01-25; 1: 0.01-20: 0.01-20; 1: 0.01-15: 0.01-15; 1: 0.01-10: 0.01-9; 1: 0.01-9: 0.01-9; 1: 0.01-8: 0.01-8; 1: 0.01-7: 0.01-7; 1: 0.01-6: 0.01-6; 1: 0.01-5: 0.01-5; 1: 0.01-4: 0.01-4; 1: 0.01-3: 0.01-3; 1: 0.01-2: 0.01-2, or 1: 0.01-1.5: 0.01-1.5). In some embodiments, the ratio of the first RNA molecule: the second RNA molecule: the third RNA molecule is 1:1:3. In some embodiments, the ratio of the first RNA molecule: the second RNA molecule: the third RNA molecule is 1:3:3.

[0199] As used herein, the term "dose" generally refers to the "amount of dose", i.e., the amount of RNA administered per administration, i.e., per dose.

[0200] In some embodiments, administration of the immunogenic composition or vaccine of the present disclosure can be effected by a single administration or boosted by multiple administrations.

[0201] In some embodiments, the regimen described herein includes at least one dose. In some embodiments, the regimen includes a first dose and at least one subsequent dose. In some embodiments, the amount of the first dose is the same as at least one subsequent dose. In some embodiments, the amount of the first dose is the same as all subsequent doses. In some embodiments, the amount of the first dose is different from at least one subsequent dose. In some embodiments, the amount of the first dose is different from all subsequent doses. In some embodiments, the regimen includes two doses. In some embodiments, the provided regimen consists of two doses. In some embodiments, the regimen includes three doses.

[0202] In one embodiment, in the present disclosure, administration of a single dose is contemplated. In one embodiment, in the present disclosure, administration of a priming dose followed by one or more booster doses is contemplated. The booster dose or the first booster dose can be administered 7 to 28 days or 14 to 24 days after administration of the priming dose. In some embodiments, the first booster dose can be administered 1 week to 3 months after administration of the priming dose (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks later). In some embodiments, subsequent booster doses can be administered at least 1 week or more after the preceding booster dose, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks later, or more. In some embodiments, subsequent booster doses can be administered at intervals of about 5 to 9 weeks or 6 to 8 weeks. In some embodiments, at least one subsequent booster dose (e.g., after the first booster dose) can be administered at least 3 months or more after the preceding dose, for example, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or more.

[0203] In some embodiments, the dose comprises a total amount of RNA of 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg, such as, for example, about 1 μg, about 2 μg, about 3 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, or about 100 μg. In some embodiments, the dose comprises a total amount of RNA (e.g., modRNA) of up to about 100 μg. In some embodiments, the dose comprises one or more first RNAs of 0.1 μg to 100 μg and one or more second RNAs of 0.1 μg to 100 μg, wherein each of the one or more first RNAs comprises a nucleotide sequence encoding an antigenic polypeptide associated with a first infectious agent (e.g., a coronavirus), and each of the one or more second RNAs comprises a nucleotide sequence encoding an antigenic polypeptide associated with a second infectious agent (e.g., influenza). In some embodiments, the dose comprises one or more first RNAs of 3 μg to 60 μg and one or more second RNAs of 3 μg to 90 μg. In some embodiments, the dose comprises one or more first RNAs of 3 μg to 60 μg and one or more second RNAs of 3 μg to 90 μg, and the total RNA contained in the dose is at most 100 μg. In some embodiments, the dose comprises one or more first RNAs of 3 μg to 30 μg and one or more second RNAs of 3 μg to 60 μg, and the total RNA contained in the dose is at most 100 μg. In some embodiments, the dose comprises one or more first RNAs of 3 μg and one or more second RNAs of 3 μg. In some embodiments, the dose comprises one or more first RNAs of 3 μg and one or more second RNAs of 6 μg. In some embodiments, the dose comprises one or more first RNAs of 10 μg and one or more second RNAs of 10 μg. In some embodiments, the dose comprises one or more first RNAs of 10 μg and one or more second RNAs of 20 μg. In some embodiments, the dose comprises one or more first RNAs of 30 μg and one or more second RNAs of 30 μg.In some embodiments, the dosage comprises 30 μg of one or more first RNAs and 60 μg of one or more second RNAs. In some embodiments, the dosage comprises 60 μg of one or more first RNAs and 30 μg of one or more second RNAs.

[0204] In some embodiments, the amount of RNA in a subsequent dose (e.g., as part of a primary regimen or booster regimen) administered to an individual can be the same as that previously administered to that individual. In some embodiments, the amount of RNA in a subsequent dose (e.g., as part of a primary regimen or booster regimen) administered to an individual can be different compared to the amount previously administered to that individual. For example, in some embodiments, based on consideration of various factors including, for example, immunogenicity and / or reactogenicity induced by a previous dose, disease prevalence, etc., the subsequent dose can be more or less than the previous dose. In some embodiments, the subsequent dose can be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than the previous dose. In some embodiments, the subsequent dose can be at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, or more than the previous dose. In some embodiments, the subsequent dose can be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than the previous dose. In some embodiments, the subsequent dose can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or less than the previous dose. In some embodiments, the RNA described herein can be administered in an amount of 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg per dose (e.g., in a given single dose), for example, about 1 μg, about 2 μg, about 3 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, or about 100 μg.

[0205] In some embodiments, the RNA described herein can be administered in an amount of 60 μg or less, 55 μg or less, 50 μg or less, 45 μg or less, 40 μg or less, 35 μg or less, 30 μg or less, 25 μg or less, 20 μg or less, 15 μg or less, 10 μg or less, 5 μg or less, 3 μg or less, 2.5 μg or less, or 1 μg or less per dose (e.g., in a given single dose).

[0206] In some embodiments, at least 0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 10 μg, at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 40 μg, at least 50 μg, or at least 60 μg of the RNA described herein can be administered per dose (e.g., in a given single dose). In some embodiments, at least 3 μg of the RNA described herein can be administered at least once in a given dose. In some embodiments, at least 10 μg of the RNA described herein can be administered at least once in a given dose. In some embodiments, at least 15 μg of the RNA described herein can be administered at least once in a given dose. In some embodiments, at least 20 μg of the RNA described herein can be administered at least once in a given dose. In some embodiments, at least 25 μg of the RNA described herein can be administered at least once in a given dose. In some embodiments, at least 30 μg of the RNA described herein can be administered at least once in a given dose. In some embodiments, at least 50 μg of the RNA described herein can be administered at least once in a given dose. In some embodiments, at least 60 μg of the RNA described herein can be administered at least once in a given dose. In some embodiments, combinations of the above amounts can be administered in a regimen comprising two or more doses (e.g., the amounts of the previous and subsequent doses can be different as described herein). In some embodiments, combinations of the above amounts can be administered in a primary regimen and a booster regimen (e.g., different doses can be given in the primary and booster regimens).

[0207] In some embodiments, the RNA described herein can be administered in an amount of 0.25 μg to 60 μg, 0.5 μg to 55 μg, 1 μg to 50 μg, 5 μg to 40 μg, or 10 μg to 30 μg per dose. In some embodiments, the RNA described herein can be administered in an amount of 3 μg to 30 μg in at least one of a given dose. In some embodiments, the RNA described herein can be administered in an amount of 3 μg to 20 μg in at least one of a given dose. In some embodiments, the RNA described herein can be administered in an amount of 3 μg to 15 μg in at least one of a given dose. In some embodiments, the RNA described herein can be administered in an amount of 3 μg to 10 μg in at least one of a given dose. In some embodiments, the RNA described herein can be administered in an amount of 10 μg to 30 μg in at least one of a given dose.

[0208] In some embodiments, the regimen administered to the subject may include multiple doses (e.g., at least 2 doses, at least 3 doses, or more). In some embodiments, the regimen administered to the subject may include a first dose and a second dose, which are administered at an interval of at least 2 weeks, at least 3 weeks, at least 4 weeks, or more. In some embodiments, such multiple doses can be spaced at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more apart. In some embodiments, the multiple doses can be administered at intervals of several days, e.g., at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 days or more. In some embodiments, the multiple doses can be administered at intervals of about 1 to about 3 weeks, or about 1 to about 4 weeks, or about 1 to about 5 weeks, or about 1 to about 6 weeks, or an interval of more than 1 week to more than 6 weeks. In some embodiments, the multiple doses can be spaced at intervals of about 7 to about 60 days, e.g., about 14 to about 48 days. In some embodiments, the minimum number of days between multiple doses can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more. In some embodiments, the maximum number of days between multiple doses can be about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or less.In some embodiments, multiple doses can be spaced about 21 to about 28 days apart. In some embodiments, multiple doses can be spaced about 19 to about 42 days apart. In some embodiments, multiple doses can be spaced about 7 to about 28 days apart. In some embodiments, multiple doses can be spaced about 14 to about 24 days apart. In some embodiments, multiple doses can be spaced about 21 to about 42 days apart.

[0209] In some embodiments, the vaccination regimen includes a first dose and a second dose. In some embodiments, the first dose and the second dose are administered at least 21 days apart. In some embodiments, the first dose and the second dose are administered at least 28 days apart.

[0210] In some embodiments, the vaccination regimen includes a first dose and a second dose, wherein the amount of RNA administered in the first dose is the same as the amount of RNA administered in the second dose. In some embodiments, the vaccination regimen includes a first dose and a second dose, wherein the amount of RNA administered in the first dose is different from the amount of RNA administered in the second dose.

[0211] In some embodiments, the vaccination regimen includes a first dose and a second dose, where the amount of RNA administered in the first dose is less than the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10% - 90% of the second dose. In some embodiments, the amount of RNA administered in the first dose is 10% - 50% of the second dose. In some embodiments, the amount of RNA administered in the first dose is 10% - 20% of the second dose. In some embodiments, administering the first dose and the second dose includes administering them at an interval of at least 2 weeks, including at an interval of at least 3 weeks, at an interval of at least 4 weeks, at an interval of at least 5 weeks, at an interval of at least 6 weeks or more. In some embodiments, the first dose and the second dose are administered at an interval of at least 3 weeks.

[0212] In some embodiments, the first dose contains less than about 30 μg of RNA and the second dose contains at least about 30 μg of RNA. In some embodiments, the first dose contains from about 1 to less than about 30 μg of RNA (e.g., less than about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or about 30 μg of RNA) and the second dose contains from about 30 to about 100 μg of RNA (e.g., about 30, about 40, about 50, or about 60 μg of RNA). In some embodiments, the first dose contains from about 1 to about 20 μg of RNA, from about 1 to about 10 μg of RNA, or from about 1 to about 5 μg of RNA, and the second dose contains from about 30 to about 60 μg of RNA.

[0213] In some embodiments, the first dose contains from about 1 to about 10 μg of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 μg of RNA) and the second dose contains from about 30 to about 60 μg of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 μg of RNA).

[0214] In some embodiments, the first dose contains about 1 μg of RNA and the second dose contains about 30 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 30 μg of RNA. In some embodiments, the first dose contains about 5 μg of RNA and the second dose contains about 30 μg of RNA. In some embodiments, the first dose contains about 10 μg of RNA and the second dose contains about 30 μg of RNA. In some embodiments, the first dose contains about 15 μg of RNA and the second dose contains about 30 μg of RNA.

[0215] In some embodiments, the first dose contains about 1 μg of RNA and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 5 μg of RNA and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 6 μg of RNA and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 10 μg of RNA and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 15 μg of RNA and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 20 μg of RNA and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 25 μg of RNA and the second dose contains about 60 μg of RNA. In some embodiments, the first dose contains about 30 μg of RNA and the second dose contains about 60 μg of RNA.

[0216] In some embodiments, the first dose comprises less than about 10 μg of RNA and the second dose comprises at least about 10 μg of RNA. In some embodiments, the first dose comprises from about 0.1 to less than about 10 μg of RNA (e.g., from about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about 10 μg of RNA), and the second dose comprises from about 10 to about 30 μg of RNA (e.g., from about 10, about 15, about 20, about 25, or about 30 μg of RNA). In some embodiments, the first dose comprises from about 0.1 to about 10 μg of RNA, from about 1 to about 5 μg of RNA, or from about 0.1 to about 3 μg of RNA, and the second dose comprises from about 10 to about 30 μg of RNA.

[0217] In some embodiments, the first dose comprises from about 0.1 to about 5 μg of RNA (e.g., from about 0.1, about 0.5, about 1, about 2, about 3, about 4, or about 5 μg of RNA), and the second dose comprises from about 10 to about 20 μg of RNA (e.g., from about 10, about 12, about 14, about 16, about 18, or about 20 μg of RNA).

[0218] In some embodiments, the first dose comprises about 0.1 μg of RNA and the second dose comprises about 10 μg of RNA. In some embodiments, the first dose comprises about 0.3 μg of RNA and the second dose comprises about 10 μg of RNA. In some embodiments, the first dose comprises about 1 μg of RNA and the second dose comprises about 10 μg of RNA. In some embodiments, the first dose comprises about 3 μg of RNA and the second dose comprises about 10 μg of RNA.

[0219] In some embodiments, the first dose comprises less than about 3 μg of RNA and the second dose comprises at least about 3 μg of RNA. In some embodiments, the first dose comprises from about 0.1 to less than about 3 μg of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5 μg of RNA) and the second dose comprises from about 3 to about 10 μg of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10 μg of RNA). In some embodiments, the first dose comprises from about 0.1 to about 3 μg of RNA, from about 0.1 to about 1 μg of RNA, or from about 0.1 to about 0.5 μg of RNA and the second dose comprises from about 3 to about 10 μg of RNA.

[0220] In some embodiments, the first dose comprises from about 0.1 to about 1.0 μg of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 μg of RNA) and the second dose comprises from about 1 to about 3 μg of RNA (e.g., about 1.0, about 1.5, about 2.0, about 2.5, or about 3.0 μg of RNA).

[0221] In some embodiments, the first dose comprises about 0.1 μg of RNA and the second dose comprises about 3 μg of RNA. In some embodiments, the first dose comprises about 0.3 μg of RNA and the second dose comprises about 3 μg of RNA. In some embodiments, the first dose comprises about 0.5 μg of RNA and the second dose comprises about 3 μg of RNA. In some embodiments, the first dose comprises about 1 μg of RNA and the second dose comprises about 3 μg of RNA.

[0222] In some embodiments, the vaccination regimen includes a first dose and a second dose, where the amount of RNA administered in the first dose is greater than the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the second dose is 10% to 90% of the amount of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10% to 50% of the amount of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10% to 20% of the amount of the first dose. In some embodiments, administering the first dose and the second dose at an interval of at least 2 weeks, including at an interval of at least 3 weeks, at an interval of at least 4 weeks, at an interval of at least 5 weeks, at an interval of at least 6 weeks or more. In some embodiments, the first dose and the second dose are administered at an interval of at least 3 weeks.

[0223] In some embodiments, the first dose includes at least about 30 μg of RNA and the second dose includes less than about 30 μg of RNA. In some embodiments, the first dose includes about 30 to about 100 μg of RNA (e.g., about 30, about 40, about 50, or about 60 μg of RNA) and the second dose includes about 1 to about 30 μg of RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or about 30 μg of RNA). In some embodiments, the second dose includes about 1 to about 20 μg of RNA, about 1 to about 10 μg of RNA, or about 1 to 5 μg of RNA. In some embodiments, the first dose includes about 30 to about 60 μg of RNA and the second dose includes about 1 to about 20 μg of RNA, about 1 to about 10 μg of RNA, or about 0.1 to about 3 μg of RNA.

[0224] In some embodiments, the first dose includes about 30 to about 60 μg of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 μg of RNA) and the second dose includes about 1 to about 10 μg of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 μg of RNA).

[0225] In some embodiments, the first dose comprises about 30 μg of RNA and the second dose comprises about 1 μg of RNA. In some embodiments, the first dose comprises about 30 μg of RNA and the second dose comprises about 3 μg of RNA. In some embodiments, the first dose comprises about 30 μg of RNA and the second dose comprises about 5 μg of RNA. In some embodiments, the first dose comprises about 30 μg of RNA and the second dose comprises about 10 μg of RNA. In some embodiments, the first dose comprises about 30 μg of RNA and the second dose comprises about 15 μg of RNA.

[0226] In some embodiments, the first dose comprises about 60 μg of RNA and the second dose comprises about 1 μg of RNA. In some embodiments, the first dose comprises about 60 μg of RNA and the second dose comprises about 3 μg of RNA. In some embodiments, the first dose comprises about 60 μg of RNA and the second dose comprises about 5 μg of RNA. In some embodiments, the first dose comprises about 60 μg of RNA and the second dose comprises about 6 μg of RNA. In some embodiments, the first dose comprises about 60 μg of RNA and the second dose comprises about 10 μg of RNA. In some embodiments, the first dose comprises about 60 μg of RNA and the second dose comprises about 15 μg of RNA. In some embodiments, the first dose comprises about 60 μg of RNA and the second dose comprises about 20 μg of RNA. In some embodiments, the first dose comprises about 60 μg of RNA and the second dose comprises about 25 μg of RNA. In some embodiments, the first dose comprises about 60 μg of RNA and the second dose comprises about 30 μg of RNA.

[0227] In some embodiments, the first dose comprises at least about 10 μg of RNA and the second dose comprises less than about 10 μg of RNA. In some embodiments, the first dose comprises about 10 to about 30 μg of RNA (e.g., about 10, about 15, about 20, about 25, or about 30 μg of RNA), and the second dose comprises about 0.1 to less than about 10 μg of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about 10 μg of RNA). In some embodiments, the first dose comprises about 10 to about 30 μg of RNA, or about 0.1 to about 3 μg of RNA, and the second dose comprises about 1 to about 10 μg of RNA, or about 1 to about 5 μg of RNA.

[0228] In some embodiments, the first dose comprises about 10 to about 20 μg of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20 μg of RNA), and the second dose comprises about 0.1 to about 5 μg of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, or about 5 μg of RNA).

[0229] In some embodiments, the first dose comprises about 10 μg of RNA and the second dose comprises about 0.1 μg of RNA. In some embodiments, the first dose comprises about 10 μg of RNA and the second dose comprises about 0.3 μg of RNA. In some embodiments, the first dose comprises about 10 μg of RNA and the second dose comprises about 1 μg of RNA. In some embodiments, the first dose comprises about 10 μg of RNA and the second dose comprises about 3 μg of RNA.

[0230] In some embodiments, the first dose comprises at least about 3 μg of RNA and the second dose comprises less than about 3 μg of RNA. In some embodiments, the first dose comprises about 3 to about 10 μg of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10 μg of RNA), and the second dose comprises 0.1 to less than about 3 μg of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5 It contains about 2.0, or about 2.5 μg of RNA). In some embodiments, the first dose contains about 3 to about 10 μg of RNA, and the second dose contains about 0.1 to about 3 μg of RNA, about 0.1 to about 1 μg of RNA, or about 0.1 to about 0.5 μg of RNA.

[0231] In some embodiments, the first dose contains about 1 to about 3 μg of RNA (e.g., about 1, about 1.5, about 2.0, about 2.5, or about 3.0 μg of RNA), and the second dose contains about 0.1 to 0.3 μg of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 μg of RNA).

[0232] In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 0.1 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 0.3 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 0.6 μg of RNA. In some embodiments, the first dose contains about 3 μg of RNA and the second dose contains about 1 μg of RNA.

[0233] In some embodiments, the vaccination regimen includes at least two doses, such as at least three doses, at least four doses or more doses. In some embodiments, the vaccination regimen includes three doses. In some embodiments, the time interval between the first dose and the second dose may be the same as the time interval between the second dose and the third dose. In some embodiments, the time interval between the first dose and the second dose may be longer than the time interval between the second dose and the third dose by, for example, several days or weeks (e.g., including at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or more). In some embodiments, the time interval between the first dose and the second dose may be shorter than the time interval between the second dose and the third dose by, for example, several days or weeks (e.g., including at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or more). In some embodiments, the time interval between the first dose and the second dose may be shorter than the time interval between the second dose and the third dose by, for example, at least 1 month (e.g., including at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more).

[0234] In some embodiments, the last dose of the primary regimen and the first dose of the booster regimen are given with an interval of at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more. In some embodiments, the primary regimen may include two doses. In some embodiments, the primary regimen may include three doses.

[0235] In some embodiments, the first dose and the second dose (and / or any other subsequent doses) can be administered by intramuscular injection. In some embodiments, the first dose and the second dose (and / or any other subsequent doses) can be administered into the deltoid muscle. In some embodiments, the first dose and the second dose (and / or any other subsequent doses) can be administered into the same arm.

[0236] In some embodiments, the mRNA compositions described herein are administered (e.g., by intramuscular injection) as a series of two doses (e.g., 0.3 mL each) at an interval of 21 days. In some embodiments, the mRNA compositions described herein are administered (e.g., by intramuscular injection) as a series of two doses (e.g., 0.2 mL each) at an interval of 21 days. In some embodiments, the mRNA compositions described herein are administered (e.g., by intramuscular injection) as a series of three doses (e.g., 0.3 mL or less, e.g., including 0.2 mL), where the doses are given at intervals of at least three weeks. In some embodiments, the first dose and the second dose can be administered at an interval of three weeks, while the second dose and the third dose can be administered at a longer time interval than between the first dose and the second dose, e.g., at an interval of at least four weeks or more (including at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, or more). In some embodiments, each dose is about 60 μg. In some embodiments, each dose is about 50 μg. In some embodiments, each dose is about 30 μg. In some embodiments, each dose is about 25 μg. In some embodiments, each dose is about 20 μg. In some embodiments, each dose is about 15 μg. In some embodiments, each dose is about 10 μg. In some embodiments, each dose is about 3 μg.

[0237] In some embodiments, at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 60 μg. In some embodiments, at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 50 μg. In some embodiments, at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 30 μg. In some embodiments, at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 25 μg. In some embodiments, at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 20 μg. In some embodiments, at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 15 μg. In some embodiments, at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 10 μg. In some embodiments, at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3 μg.

[0238] In one embodiment, the RNA described herein is administered in an amount of about 60 μg per single dose. In one embodiment, the RNA described herein is administered in an amount of about 50 μg per single dose. In one embodiment, the RNA described herein is administered in an amount of about 30 μg per single dose. In one embodiment, the RNA described herein is administered in an amount of about 25 μg per single dose. In one embodiment, the RNA described herein is administered in an amount of about 20 μg per single dose. In one embodiment, the RNA described herein is administered in an amount of about 15 μg per single dose. In one embodiment, the RNA described herein is administered in an amount of about 10 μg per single dose. In one embodiment, the RNA described herein is administered in an amount of about 5 μg per single dose. In one embodiment, the RNA described herein is administered in an amount of about 3 μg per single dose. In one embodiment, at least two such doses are administered. For example, the second dose can be administered about 21 days after the administration of the first dose.

[0239] In some embodiments, the effectiveness of the RNA vaccines described herein (e.g., administered in two doses, where the second dose may be administered approximately 21 days after the administration of the first dose and, for example, may be administered in an amount of about 30 μg per dose) is at least 70%, at least 80%, at least 90, or at least 95% starting 7 days after the administration of the second dose (e.g., starting 28 days after the administration of the first dose if the second dose is administered 21 days after the administration of the first dose). In some embodiments, such effectiveness is observed in a population of at least 50 years of age, at least 55 years of age, at least 60 years of age, at least 65 years of age, at least 70 years of age, or older. In some embodiments, the effectiveness of the RNA vaccines described herein (e.g., administered in two doses, where the second dose may be administered approximately 21 days after the administration of the first dose and, for example, may be administered in an amount of about 30 μg per dose) is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% in a population of at least 65 years of age, e.g., 65 - 80 years of age, 65 - 75 years of age, or 65 - 70 years of age starting 7 days after the administration of the second dose (e.g., starting 28 days after the administration of the first dose if the second dose is administered 21 days after the administration of the first dose). Such effectiveness can be observed over a period of up to 1 month, 2 months, 3 months, 6 months or more.

[0240] In one embodiment, vaccine effectiveness is defined as the percent reduction in the number of subjects with signs of infection (vaccinated subjects versus non-vaccinated subjects).

[0241] In one embodiment, the methods and agents described herein are administered to a pediatric population. In various embodiments, the pediatric population includes or consists of subjects under 18 years old, such as, for example, between 5 years old and less than 18 years old, between 12 years old and less than 18 years old, between 16 years old and less than 18 years old, between 12 years old and 16 years old, between 5 years old and 12 years old, or between 6 months old and 12 years old. In various embodiments, the pediatric population includes or consists of subjects under 5 years old, such as, for example, between 2 years old and less than 5 years old, between 12 months old and less than 24 months old, between 7 months old and 12 months old, or less than 6 months old. In some such embodiments, the mRNA compositions described herein are administered to subjects less than 2 years old, such as, for example, between 6 months old and less than 2 years old. In some such embodiments, the mRNA compositions described herein are administered to subjects less than 6 months old, such as, for example, between 1 month old and less than 4 months old. In some embodiments, the dosing regimen (e.g., dose and / or dosing schedule) for the pediatric population can vary for different age groups. For example, in some embodiments, administration to subjects between 6 months old and 4 years old includes at least 3 doses, with the first 2 doses administered at an interval of at least 3 weeks (e.g., including at least 4 weeks, at least 5 weeks, at least 6 weeks, or more), followed by administration of the third dose at least 8 weeks (e.g., including at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or more) after the second dose, according to a primary regimen. In some such embodiments, at least one dose administered is 3 μg of the RNA described herein. In some embodiments, administration to subjects 5 years old and above can be according to a primary regimen that includes at least 2 doses, with the 2 doses administered at an interval of at least 3 weeks (e.g., including at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or more). In some such embodiments, at least one dose administered is 10 μg of the RNA described herein.In some embodiments, administration to a subject who is immunocompromised and 5 years of age or older (e.g., in some embodiments, a subject who has received a solid organ transplant or a subject diagnosed with a condition equivalent to immunocompromise) can be performed according to a primary regimen that includes at least three doses, with the first two doses administered at an interval of at least three weeks (e.g., including at least three weeks, at least four weeks, at least five weeks, at least six weeks, or more), and subsequently, the third dose is administered at least four weeks after the second dose (e.g., at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, or more later).

[0242] In some embodiments, the mRNA composition described herein is administered to a subject 12 years of age or older, and each dose is about 30 μg. In some embodiments, the mRNA composition described herein is administered to a subject 12 years of age or older (e.g., including 18 years of age or older), and each dose is greater than 30 μg, including, for example, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, or more. In some such embodiments, the mRNA composition described herein is administered to a subject 12 years of age or older, and each dose is about 60 μg. In some such embodiments, the mRNA composition described herein is administered to a subject 12 years of age or older, and each dose is about 50 μg. In one embodiment, the pediatric population includes or consists of subjects 12 years of age to less than 18 years of age, including subjects 16 years of age to less than 18 years of age and / or subjects 12 years of age to less than 16 years of age. In this embodiment, the treatment may include two vaccinations at an interval of 21 days. In one embodiment, the vaccine is administered, for example, intramuscularly at a dose of 30 μg of RNA per dose. In some embodiments, higher doses are administered to older pediatric patients and adults, e.g., patients 12 years of age or older, compared to younger pediatric patients or infants, e.g., less than 2 to 5 years of age, less than 6 months to 2 years of age, or less than 6 months of age. In some embodiments, higher doses are administered to pediatric patients 2 to 5 years of age compared to infants and / or neonates, e.g., less than 6 months to 2 years of age, or less than 6 months of age.

[0243] In one embodiment, the pediatric population includes or consists of subjects aged 5 to less than 18 years, including subjects aged 12 to less than 18 years and / or subjects aged 5 to less than 12 years. In this embodiment, the treatment may include two vaccinations at an interval of 21 days. In various embodiments, the vaccine is administered at a dose of 10 μg, 20 μg, or 30 μg of RNA per single dose, for example, by intramuscular administration. In some such embodiments, the mRNA composition described herein is administered to subjects aged 5 to 11 years, and each dose is about 10 μg.

[0244] In one embodiment, the pediatric population includes or consists of subjects less than 5 years old, including subjects aged 2 to less than 5 years, subjects aged 12 to less than 24 months, subjects aged 7 to less than 12 months, subjects aged 6 to less than 12 months, and / or subjects less than 6 months old. In this embodiment, the treatment may include two vaccinations, for example, at an interval of 21 to 42 days, such as at an interval of 21 days. In various embodiments, the vaccine is administered at a dose of 3 μg, 10 μg, 20 μg, or 30 μg of RNA per single dose, for example, by intramuscular administration. In some such embodiments, the mRNA composition described herein is administered to subjects aged 2 to less than 5 years, and each dose is about 3 μg. In some such embodiments, the mRNA composition described herein is administered to subjects aged about 6 months to less than about 5 years, and each dose is about 3 μg.

[0245] In some embodiments, the mRNA composition described herein is administered to a subject 12 years of age or older, and at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 60 μg. In some embodiments, the mRNA composition described herein is administered to a subject 12 years of age or older, and at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 30 μg. In some embodiments, the mRNA composition described herein is administered to a subject 12 years of age or older, and at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 15 μg. In some embodiments, the mRNA composition described herein is administered to a subject 5 to less than 12 years of age, and at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 10 μg. In some embodiments, the mRNA composition described herein is administered to a subject 2 to less than 5 years of age, and at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3 μg. In some embodiments, the mRNA composition described herein is administered to a subject 6 months to less than 2 years of age, and at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3 μg or less, including, for example, 2 μg, 1 μg, or less. In some embodiments, the mRNA composition described herein is administered to an infant less than 6 months of age, and at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3 μg or less, including, for example, 2 μg, 1 μg, 0.5 μg, or less.

[0246] In some embodiments, the dose administered to a subject in need thereof may include administration of a single mRNA composition described herein.

[0247] In some embodiments, the dosage administered to a subject in need thereof can include the administration of at least two or more (e.g., at least three or more) different pharmaceutical products / formulations. For example, in some embodiments, at least two or more different pharmaceutical products / formulations can include at least two different mRNA compositions described herein (e.g., in some embodiments, each containing a different RNA construct).

[0248] In some embodiments, two or more RNAs are administered to a subject (e.g., as part of either a primary regimen or a booster regimen), where the two or more RNAs are administered on the same day or at the same visit. In some embodiments, the two or more RNAs are administered as separate compositions, e.g., by administering each RNA to a different part of the subject (e.g., by intramuscular administration to different arms of the subject or different sites on the same arm of the subject). In some embodiments, the two or more RNAs are mixed prior to administration (e.g., immediately prior to administration, e.g., by the practitioner administering the dose). In some embodiments, the two or more RNAs are formulated together (e.g., (a) by mixing separate populations of LNPs, each population containing a different RNA, or (b) by mixing two or more RNAs prior to LNP formulation such that each LNP contains two or more RNAs).

[0249] In some embodiments, one or more first RNAs and one or more second RNAs are each administered to a subject in the same amount (i.e., in a 1:1 ratio), or the composition includes one or more first RNAs and one or more second RNAs each in the same amount (i.e., in a 1:1 ratio).

[0250] In some embodiments, one or more first RNAs and one or more second RNAs are administered to a subject in different amounts, or the composition comprises one or more first RNAs and one or more second RNAs in different amounts. For example, in some embodiments, one or more first RNAs are administered to a subject in an amount that is 0.01 to 100 times the amount of one or more second RNAs, or the composition comprises one or more first RNAs in an amount that is 0.01 to 100 times the amount of one or more second RNAs (e.g., the amount of one or more first RNAs is 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to 4, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 to 1.5 times the amount of one or more second RNAs). In some embodiments, one or more first RNAs and one or more second RNAs are administered to a subject, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the concentration of one or more first RNAs is 1 to 10 times the concentration of one or more second RNAs. In some embodiments, one or more first RNAs and one or more second RNAs are administered to a subject, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the amount of one or more first RNAs is 1 to 5 times the amount of one or more second RNAs. In some embodiments, one or more first RNAs and one or more second RNAs are administered to a subject, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the concentration of one or more first RNAs is 1 to 3 times the concentration of one or more second RNAs.In some embodiments, one or more first RNAs and one or more second RNAs are administered to a subject, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the amount of the one or more first RNAs is twice the amount of the one or more second RNAs. In some embodiments, one or more first RNAs and one or more second RNAs are administered to a subject, or the composition comprises one or more first RNAs and one or more second RNAs, wherein the concentration of the one or more first RNAs is three times the concentration of the one or more second RNAs.

[0251] In some embodiments, two first RNAs, each encoding an antigen derived from an influenza strain or variant, are administered to a subject, or the composition comprises two first RNAs, each encoding an antigen derived from an influenza strain or variant, wherein the amount of each RNA is not the same. For example, in some embodiments, the ratio of the two first RNAs is from 1:0.01 to 100 (such as 1:0.01 to 50; 1:0.01 to 40; 1:0.01 to 30; 1:0.01 to 25; 1:0.01 to 20; 1:0.01 to 15; 1:0.01 to 10; 1:0.01 to 9; 1:0.01 to 8; 1:0.01 to 7; 1:0.01 to 6; 1:0.01 to 5; 1:0.01 to 4; 1:0.01 to 3; 1:0.01 to 2; 1:0.01 to 1.5, 1:0.1 to 10, 1:0.1 to 5, 1:0.1 to 3, 1:2 to 10, 1:2 to 5, or 1:2 to 3). In some embodiments, the two first RNAs are administered to the subject at a ratio of 1:3, or the composition comprises the two first RNAs at a ratio of 1:3. In some embodiments, the two first RNAs are administered to the subject at a ratio of 1:2, or the composition comprises the two first RNAs at a ratio of 1:2.

[0252] For example, in some embodiments, the ratio of the three first RNAs is 1:0.01 to 100:0.01 to 100 (e.g., 1:0.01 to 50:0.01 to 50; 1:0.01 to 40:0.01 to 40; 1:0.01 to 30:0.01 to 30; 1:0.01 to 25:0.01 to 25; 1:0.01 to 20:0.01 to 20; 1:0.01 to 15:0.01 to 15; 1:0.01 to 10:0.01 to 10; 1:0.01 to 9:0.01 to 9; 1:0.01 to 8:0.01 to 8; 1:0.01 to 7:0.01 to 7; 1:0.01 to 6:0.01 to 6; 1:0.01 to 5:0.01 to 5; 1:0.01 to 4:0.01 to 4; 1:0.01 to 3:0.01 to 3; 1:0.01 to 2:0.01 to 2; 1:0.01 to 1.5:0.01 to 1.5; 1:0.1 to 10:0.1 to 10, 1:0.1 to 5:0.1 to 5, 1:0.1 to 3:0.1 to 3, 1:2 to 10:2 to 10, 1:2 to 5:2 to 5, or 1:2 to 3:2 to 3). In some embodiments, the three first RNAs are administered to a subject in a ratio of 1:1:3, or the composition comprises the three first RNAs in a ratio of 1:1:3. In some embodiments, the three first RNAs are administered to a subject in a ratio of 1:3:3, or the composition comprises the three first RNAs in a ratio of 1:3:3.

[0253] In some embodiments, two or more second RNAs are administered to a subject, or the composition comprises two or more second RNAs, one or more of which encode the HA protein of influenza A virus and one or more of which encode the HA protein of influenza B virus. In some embodiments, one or more second RNAs encoding the HA protein of influenza A virus and one or more second RNAs encoding the HA protein of influenza B virus are present or administered in the same amount (i.e., in a 1:1 ratio). In some embodiments, one or more second RNAs encoding the HA protein of influenza A virus and one or more second RNAs encoding the HA protein of influenza B virus are administered in different amounts (e.g., in a ratio between 1:10 and 10:1, or in a ratio of 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1 (total RNA encoding A antigen:total RNA encoding B antigen)).

[0254] In some embodiments, two second RNAs encoding the HA protein of different influenza virus types (e.g., a second RNA encoding the HA protein of influenza A virus and a second RNA encoding the HA protein of influenza B virus) are administered to a subject, or the composition comprises two second RNAs encoding the HA protein of different influenza virus types (e.g., a second RNA encoding the HA protein of influenza A virus and a second RNA encoding the HA protein of influenza B virus). In some embodiments, the second RNAs are administered or present in the same amount (i.e., in a 1:1 ratio). In some embodiments, the second RNAs are administered or present in different amounts (e.g., in a ratio between 1:10 and 10:1, or in a ratio of 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1 (A:B)).

[0255] In some embodiments, three second RNAs encoding HA proteins of different influenza virus subtypes (e.g., HA proteins of A / Wisconsin (H1N1) virus, A / Darwin (H3N2) virus, and B / Austria (Victoria) virus) are administered to a subject, or the composition comprises three second RNAs encoding HA proteins of different influenza virus subtypes (e.g., HA proteins of A / Wisconsin (H1N1) virus, A / Darwin (H3N2) virus, and B / Austria (Victoria) virus). In some embodiments, each of the three second RNAs is administered to the subject in the same amount (i.e., in a 1:1:1 ratio), or the composition comprises each of the three second RNAs in the same amount (i.e., in a 1:1:1 ratio). In some embodiments, one or more of the three second RNAs are administered to the subject in different amounts (e.g., in a ratio between 1:1:2 and 1:1:10 (e.g., in a ratio of 1:1:2, 1:1:3, 1:1:4, or 1:1:5), or in a ratio between 2:2:1 and 2:2:10 (e.g., in a ratio of 2:2:1, 3:3:1, 4:4:1, or 5:5:1)), or the composition comprises one or more of the three second RNAs in different amounts (e.g., in a ratio between 1:1:2 and 1:1:10 (e.g., in a ratio of 1:1:2, 1:1:3, 1:1:4, or 1:1:5), or in a ratio between 2:2:1 and 2:2:10 (e.g., in a ratio of 2:2:1, 3:3:1, 4:4:1, or 5:5:1)).

[0256] In some embodiments, three second RNAs, two of which encode HA proteins of different A-type influenza viruses and one of which encodes an HA protein of a B-type influenza virus, are administered to a subject or the composition comprises three second RNAs, two of which encode HA proteins of different A-type influenza viruses and one of which encodes an HA protein of a B-type influenza virus. In some such embodiments, the second RNA encoding the HA protein of the B-type influenza virus is present or administered in a greater amount compared to any of the second RNAs encoding HA proteins derived from A-type viruses (e.g., in some embodiments, the ratio of the two second RNAs encoding HA proteins derived from A-type influenza viruses to the second RNA encoding the HA protein derived from B-type influenza virus is 1:1:1 to 10, 1:1:2, 1:1:3, 1:1:4, or 1:1:5 (A:A:B)). In some embodiments, three second RNAs, two encoding HA proteins of A-type influenza virus and one encoding an HA protein of B-type influenza virus, are administered to a subject or the composition comprises three second RNAs, two encoding HA proteins of A-type influenza virus and one encoding an HA protein of B-type influenza virus, wherein the ratio of the three second RNAs is 1:1:4 (A:A:B). In some embodiments, the two second RNAs encoding HA proteins of A-type influenza virus are each present in a greater amount or each administered in a greater amount compared to the second RNA encoding the HA protein derived from B-type virus (e.g., in some embodiments, the ratio of the two second RNAs encoding HA proteins derived from A-type influenza viruses to the second RNA encoding the HA protein derived from B-type influenza virus is 1 to 10:1 to 10:1, 2:2:1, 3:3:1, 4:4:1, or 5:5:1 (A:A:B)).

[0257] In some embodiments, four second RNAs encoding HA proteins of different influenza virus subtypes are administered to a subject, or the composition comprises four second RNAs encoding HA proteins of different influenza virus subtypes. In some such embodiments, the four second RNAs comprise two second RNAs encoding HA proteins of different influenza A viruses and two second RNAs encoding HA proteins of different influenza B viruses (e.g., the HA protein of the H1N1 virus, the HA protein of the H3N2 virus, the HA protein of the B / Victoria lineage virus, and the HA protein of the B / Yamagata lineage virus). In some embodiments, each of the two second RNAs encoding HA proteins of influenza A viruses and each of the second RNAs encoding HA proteins of two influenza B viruses are present in the same amount (i.e., the ratio of the four second RNAs is 1:1:1:1). In some embodiments, the second RNAs encoding HA proteins of two influenza B viruses are administered in a greater amount or are present in a greater amount compared to any of the second RNAs encoding HA proteins of influenza A viruses (e.g., in some embodiments, the ratio of the two second RNAs encoding HA proteins of influenza A viruses to the second RNAs encoding HA proteins of two influenza B viruses is 1:1:2-10:2-10, 1:1:2-5:2-5, 1:1:2:2, 1:1:3:3, 1:1:4:4, 1:1:5:5, 1:1:6:6, 1:1:7:7, 1:1:8:8, 1:1:9:9, 1:1:10:10 (A:A:B:B)).In some embodiments, two second RNAs encoding the HA protein of influenza A virus and two second RNAs encoding the HA protein of influenza B virus are administered to the subject, or the composition comprises two second RNAs encoding the HA protein of influenza A virus and two second RNAs encoding the HA protein of influenza B virus, wherein the ratio of the four second RNAs is 1:1:5:5 (A:A:B:B). In some embodiments, the two second RNAs encoding the HA protein of influenza A virus are administered in an amount greater than or present in an amount greater than any of the second RNAs encoding the HA protein from a B virus, when compared to any of the second RNAs encoding the HA protein from a B virus (e.g., in some embodiments, the ratio of the two second RNAs encoding the HA protein from an influenza A virus to the two second RNAs encoding the HA protein from an influenza B virus is 2-10:2-10:1:1, 2-5:2-5:1:1, 2:2:1:1, 3:3:1:1, 4:4:1:1, 5:5:1:1, 6:6:1:1, 7:7:1:1, 8:8:1:1, 9:9:1:1, 10:10:1:1 (A:A:B:B)).

[0258] In some embodiments, the composition comprises or is administered to a subject four second RNAs encoding HA proteins of different influenza A viruses and one second RNA encoding an HA protein of an influenza B virus (e.g., A / Wisconsin (H1N1), A / Darwin (H3N2), A / Cambodia (H3N2), and B / Austria (Victoria)). In some such embodiments, each of the four second RNAs is administered or present in the same amount (i.e., in a 1:1:1:1 ratio). In some embodiments, the amount of the second RNA encoding the HA protein of the influenza B virus is higher than any one of the second RNAs encoding the HA proteins of the influenza A viruses (e.g., in some embodiments, the ratio of the second RNAs is 1:1:1:1 to 10, 1:1:1:1 to 5, 1:1:1:2, 1:1:1:3, 1:1:1:4, or 1:1:1:5 (A:A:A:B)). In some embodiments, the ratio of the second RNAs administered or present in the composition is 1:1:1:5 (A:A:A:B). In some embodiments, the amount of each of the second RNAs encoding the HA protein of the influenza A virus is greater than the amount of the second RNA encoding the HA protein of the influenza B virus (e.g., in some embodiments, the ratio of the second RNAs is 1 to 10:1 to 10:1 to 10:1, 1 to 5:1 to 5:1 to 5:1, 2:2:2:1, 3:3:3:1, 4:4:4:1, or 5:5:5:1 (A:A:A:B)).

[0259] In certain embodiments, one or more second RNAs encoding HA protein(s) of influenza virus (e.g., two second RNAs, three second RNAs, or four second RNAs, each encoding an HA protein of a different influenza virus) are administered to a subject in a total amount of 0.1 to 100 μg (e.g., 1 to 90 μg, 3 to 90 μg, 1 to 60 μg, 3 to 60 μg, 5 to 60 μg, 10 to 60 μg, 30 to 60 μg, 3 to 30 μg), or the composition comprises one or more second RNAs encoding HA protein(s) of influenza virus (e.g., two second RNAs, three second RNAs, or four second RNAs, each encoding an HA protein of a different influenza virus) in a total amount of 0.1 to 100 μg (e.g., 1 to 90 μg, 3 to 90 μg, 1 to 60 μg, 3 to 60 μg, 5 to 60 μg, 10 to 60 μg, 30 to 60 μg, 3 to 30 μg). In certain embodiments, one or more second RNAs encoding HA protein(s) of influenza virus are administered to a subject in a total amount of 3 μg, 5 μg, 6 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 45 μg, 60 μg, 75 μg, or 90 μg, or the composition comprises one or more second RNAs encoding HA protein(s) of influenza virus in a total amount of 3 μg, 5 μg, 6 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 45 μg, 60 μg, 75 μg, or 90 μg.

[0260] In certain embodiments, three or four second RNAs, each encoding an HA antigen of a different influenza strain, are administered to a subject in one of the amounts listed in Table C below, or the composition comprises three or four second RNAs, each encoding an HA antigen of a different influenza strain, in one of the amounts listed in Table C below (each “influenza component” corresponds to a second RNA encoding an HA antigen (e.g., a second RNA as described herein).

[0261] In some embodiments, the compositions described herein produce an influenza neutralizing antibody titer within at least 2-fold of the influenza neutralizing antibody titer produced by a reference vaccine against each influenza virus encoding an antigen (e.g., the reference vaccine is a quadrivalent influenza RNA vaccine administered alone or an approved (non-RNA) influenza vaccine).

[0262] In some embodiments, the influenza vaccine is an alpha influenza virus, beta influenza virus, gamma influenza virus or delta influenza virus vaccine. In some embodiments, the vaccine is an influenza A virus, influenza B virus, influenza C virus, or influenza D virus vaccine. In some embodiments, the influenza A virus vaccine comprises a hemagglutinin selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18 or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any one of them. In some embodiments, the influenza A virus vaccine comprises or encodes a neuraminidase (NA) selected from N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11, or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any one of them. In some embodiments, the influenza vaccine comprises at least one influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, and / or polymerase It contains basic protein 2 (PB2), or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any one of them.

Example

[0263] (Example 1) Description of the manufacturing process This section presents the description of the manufacturing process and process control of the influenza saRNA vaccine drug substance. The manufacturing process includes RNA synthesis via an in vitro transcription (IVT) step and a purification step by ultrafiltration / diafiltration (UFDF-1). The RNA is then enzymatically capped, purified by chromatography and final UFDF-2, and subsequently subjected to final filtration and formulation.

[0264] For the production of clinical materials, the process was scaled up to a starting IVT volume of 1.5 L. There were no significant changes to the non-clinical toxicology / development process other than what was necessary to scale up the process to 1.5 L.

[0265] RT-ddPCR (identity of the encoded RNA sequence) The identity of the influenza saRNA is confirmed by performing a one-step reverse transcription (RT)-ddPCR assay on the RNA in the sample, provided that the tested sample is positive for the replicase sequence (confirmation of self-amplification of the RNA construct) and the target sequence (confirmation of the encoded influenza sequence). Digital droplet polymerase chain reaction (ddPCR) technology is a digital form of polymerase chain reaction (PCR) that uses a water-oil emulsion system to quantify the target nucleic acid. The RNA sample is diluted to a final theoretical input concentration that falls within the linear range of the ddPCR assay. A reaction mixture containing reverse transcriptase, DNA polymerase, as well as sequence-specific primers and probes is dispensed into droplets, and the PCR reaction is carried out individually in each dispense. The results are calculated by counting the number of amplified target sequences (positive droplets measured by fluorescence amplitude above background) and the dispenses where no amplification is present (negative droplets). After investigating the positive and negative controls and determining that they are valid and acceptable, the identity is confirmed if the positive droplet count exceeds the established threshold.

[0266] Reverse-phase HPLC (presence of pseudouridine) The presence of pseudouridine is determined by reverse-phase high-performance liquid chromatography (RP-HPLC) after complete digestion of the mRNA. The individual nucleosides obtained have characteristic elution patterns that include the separation of uridine and pseudouridine. The presence of pseudouridine is confirmed by comparison with uridine and pseudouridine references as well as limit standards.

[0267] Capillary gel electrophoresis (RNA integrity) RNA integrity is determined by capillary gel electrophoresis (CGE) based on the differential migration of RNAs of different molecular weights in an applied electric field. The RNA is subjected to a denaturing agent that unfolds the RNA and dissociates non-covalent complexes. The denatured RNA species, when subjected to an electric field, migrate towards the anode through the gel matrix according to their length and size. During migration, an intercalating dye binds to the RNA and associated fragments, thereby enabling fluorescence detection. Intact RNA is separated from any fragmented species, thereby allowing quantification of RNA integrity by determining the relative percent time-corrected area for the intact (major) peak.

[0268] qPCR (residual DNA template) The level of residual DNA template is determined by quantitative polymerase chain reaction (qPCR) using fluorescence technology. A qPCR master mix containing target-specific primers and a fluorescent qPCR quantification reagent is added to all sample wells. Samples are prepared at a series of dilutions and analyzed in real-time by qPCR. The measured fluorescence signal is proportional to the amount of PCR product. Quantification of DNA is performed at the cycle threshold (Ct) at which the signal exceeds a signal threshold at which amplification of the target sequence is established during the logarithmic phase of the reaction. This Ct point depends on the amount of DNA originally present in the sample. The concentration of DNA in the test sample is interpolated from the linear regression of the calibration curve, taking into account the dilution factor. The results are reported as ng of DNA per mg of RNA.

[0269] For one batch of regulatory-based toxicity assessment material and one GMP batch of the drug substance used in clinical trials, the batch details and batch analysis summary data are presented in Table 1.

[0270]

Table 1

[0271] (Example 2) S.4.1 Description and Composition of the Pharmaceutical Product The PF-07867246 (Structure 6 (TC83-delkozak-HA-SGP-NA-80A) (SEQ ID NO: 1)) pharmaceutical product is a sterile dispersion of liquid nanoparticles (LNP) in an aqueous cryoprotective substance buffer without preservatives for intramuscular administration. The pharmaceutical product is formulated with 0.06 mg / mL of RNA in 10 mM Tris buffer, 10% sucrose, and optionally 20 mM glutamic acid, pH 7.4. The pharmaceutical product is supplied in a 2 mL glass vial sealed with an aluminum seal with a chlorobutyl elastomer stopper and a flip-off plastic cap (nominal volume 0.5 mL). Table 2 presents the composition of the pharmaceutical product, including the unit dosage amounts applicable to each component, the amount per vial, the function, and the quality standards.

[0272]

Table 2

[0273]

Table 3-1

[0274]

Table 3-2

[0275]

Table 3-3

[0276]

Table 3-4

[0277]

Table 3-5

[0278]

Table 3-6

[0279] (Example 3) S.4.1. Description and Composition of Pharmaceutical Product The PF-07871987 (Construct 7 TC83-HA-40A 50U-50pU (SEQ ID NO: 2)) pharmaceutical product is a sterile dispersion of liquid nanoparticles (LNP) in an aqueous cryoprotective substance buffer without preservatives for intramuscular administration. This pharmaceutical product is formulated with 0.06 mg / mL of RNA in 10 mM Tris buffer, 10% sucrose, and optionally 20 mM glutamic acid, pH 7.4.

[0280] This pharmaceutical product is supplied in a 2 mL glass vial sealed with a stopper made of chlorobutyl elastomer and an aluminum seal with a flip-off plastic cap (nominal volume 0.5 mL).

[0281] Table 4 presents the composition of the pharmaceutical product, including the unit dosage amounts applicable to each component, the amount per vial, the function, and the quality standards.

[0282] [Table 4]

[0283] [Table 5]

[0284] [Table 6-1]

[0285] [Table 6-2]

[0286] [Table 6-3]

[0287]

Table 6-4

[0288]

Table 6-5

[0289] (Example 4) S.4.4.1 Structure 7: TC83-HA-40A 50U-50pU (PF-07871987) The batch details and batch analysis summary data are presented in Table 7 for one batch of materials for regulatory-based toxicity assessment and one GMP batch of the drug substance used in clinical trials.

[0290]

Table 7

[0291] (Example 5) Assay Hemagglutination inhibition assay The main serological assay used to measure the immune response induced by vaccines against influenza is the hemagglutination inhibition assay (HAI). In the HAI, functional antibodies in serum that prevent HA-mediated agglutination of red blood cells in a reaction containing a serum sample pretreated with receptor-destroying enzyme, influenza virus, and red blood cells derived from chickens or guinea pigs are quantitatively measured. The HAI titer is the reciprocal of the highest serum dilution that results in the loss of HA activity visualized as a teardrop shape when the microtiter plate is tilted. Titers from multiple determinations per sample are reported as the geometric mean titer (GMT). An HAI titer of 1:40 or greater is generally considered protective in humans.

[0292] Influenza microneutralization assay The influenza virus micro-neutralization assay (MNT) quantitatively measures functional antibodies in serum that neutralize influenza virus activity and thereby prevent productive infection of a host cell monolayer. A neutralization reaction occurs when influenza virus is incubated with a serum sample. The reaction mixture is then applied to a monolayer of Madin-Darby canine kidney (MDCK) cells to measure the degree of neutralization. The MNT titer is reported as the reciprocal of the dilution that results in a 50% or 90% reduction in infection compared to a serum-free control. Anti-HA neutralizing antibodies are measured in the 1-Day MNT, and both anti-HA and anti-NA neutralizing antibodies are measured in the 3-Day MNT.

[0293] Neuraminidase inhibition assay The neuraminidase inhibition assay (NAI) quantitatively measures functional antibodies in serum that prevent NA-mediated cleavage of sialic acid in an enzyme-linked lectin assay. Briefly, serum containing antibodies is incubated with influenza virus, and the mixture is transferred to a plate coated with fetuin-lectin. Cleavage of sialic acid from fetuin is monitored by binding peanut agglutinin conjugated to horseradish peroxidase to the exposed galactose moiety and a colorimetric quantification reaction after addition of substrate. The NAI titer is the reciprocal of the highest serum dilution that results in a 50% decrease in NA activity compared to a serum-free control. Titers from multiple determinations per sample are reported as the geometric mean titer (GMT).

[0294] (Example 6) Evaluation of an influenza bicistronic HA-NA saRNA vaccine design in mice This study was conducted to compare the immunogenicity of bicistronic saRNA vaccine candidates encoding influenza hemagglutinin (HA) and neuraminidase (NA) and to determine the optimal bicistronic HA-NA saRNA vaccine design. All saRNA vectors used in this study were based on the TC-83 backbone, but this study was designed to evaluate the immunogenicity and the effect of the length of the polyA tail (40A or 80A) when the exogenous Kozak sequence was present or absent upstream of the first target gene.

[0295] The key bicistronic design elements evaluated in this study included the regulatory elements (subgenomic promoter (SGP) and the corresponding internal ribosomal entry site (IRES)) used to drive the expression of the second target gene, and the order of positioning the antigens on the vector (HA-NA or NA-HA).

[0296] Functional and neutralizing antibody responses were induced by intramuscular immunization of Balb / c mice with LNP-formulated saRNA vaccines encoding the A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA antigens.

[0297] Overall, similar titers were elicited with all bicistronic saRNA vaccines tested, and these titers were also similar to those of saRNA vaccines composed of individually formulated saRNA-HA + saRNA-NA. These results confirmed that the bicistronic saRNA approach is capable of eliciting titers and is feasible.

[0298] All the saRNA vectors used in this study were based on the TC-83 backbone. This study was designed to evaluate the immunogenicity and the effect of the length of the polyA tail (40A or 80A) when the exogenous Kozak sequence was present or absent upstream of the first target gene. The key bicistronic design elements evaluated in this study included the regulatory elements necessary to drive the expression of the second target gene and the order of positioning the antigen on the vector (HA-NA or NA-HA). The regulatory elements selected for comparison were the native VEEV subgenomic promoter (SGP; 61 nucleotides) and the internal ribosomal entry site (IRES, 587 nucleotides) derived from encephalomyocarditis virus. As a comparison, a modRNA vaccine encoding influenza HA or NA was also included in this study.

[0299] Mice were immunized with saRNA or modRNA LNP formulations on days 0 and 28, and sera were collected 21 days after priming and 14 days after boosting. On days 21 and 42, neutralizing and functional antibodies were measured to determine immunogenicity.

[0300] This study was designed using 15 groups as shown in Table 8, with a total of 10 female mice in each group (mouse strain: BALB / c). The mRNA drug products were evaluated at a dose volume of 0.05 mL.

[0301]

Table 8

[0302]

Table 9

[0303]

Table 10-1

[0304]

Table 10-2

[0305] M. Analytical test results of the test article

[0306]

Table 11

[0307] 2. Results and discussion As measured by HAI, 1-Day MNT, 3-Day MNT, and NAI (Figures 1, 2, 3, and 4, respectively), intramuscular immunization of Balb / c mice with LNP-formulated saRNA vaccines encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA antigens induced functional and neutralizing antibody responses, with a distinct boosting effect 2 weeks after the second immunization. The results of the 3-Day MNT on day 42 showed that the contribution of NA to neutralization was minimal compared to HA in this assay. Overall, similar titers were achieved with all bicistronic saRNA vaccine designs evaluated, and these titers were also similar to those of saRNA vaccines composed of individually formulated HA and NA monicistronic saRNAs (HA / NA Post-Mix) as well as modRNA. The titers of saRNA vaccines expressing two antigens were similar to or slightly lower than those of the saRNA-HA or saRNA-NA only controls. These data confirmed that the bicistronic saRNA approach is feasible.

[0308] Deletion of the Kozak sequence, the length of the polyA tail, the regulatory element used to drive the second gene of interest (whether IRES or SGP), nor the order of antigen positioning (HA-NA or NA-HA) had a significant impact on the titers elicited.

[0309] Regarding Figure 1, female Balb / c mice were immunized intramuscularly (IM) on days 0 and 28 with various LNP-formulated influenza saRNA vaccine constructs and with an influenza modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA. The HA / NA post-mix preparation consisted of a 1:1 mixture of individually formulated saRNA-HA and saRNA-NA. The functional antibody response against A / Wisconsin / 588 / 2019 was measured by hemagglutination inhibition (HAI) on day 21 (3 weeks after prime) and day 42 (2 weeks after boost).

[0310] Regarding Figure 2, female Balb / c mice were immunized intramuscularly (IM) on days 0 and 28 with various LNP-formulated influenza saRNA vaccine constructs and with an influenza modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA. The HA / NA post-mix preparation consisted of a 1:1 mixture of individually formulated saRNA-HA and saRNA-NA. The functional antibody response against A / Wisconsin / 588 / 2019 was measured by a 1-Day MNT assay on day 21 (3 weeks after prime) and day 42 (2 weeks after boost). The 50% neutralization titer is reported.

[0311] Regarding Figure 3, female Balb / c mice were immunized intramuscularly (IM) on days 0 and 28 with various LNP-formulated influenza saRNA vaccine constructs and with an influenza modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA. The HA / NA post-mix preparation consisted of a 1:1 mixture of individually formulated saRNA-HA and saRNA-NA. The functional antibody response against A / Wisconsin / 588 / 2019 was measured by a 3-Day MNT assay on day 42 (2 weeks after boost). The 50% neutralization titer is reported.

[0312] Regarding Figure 4, female Balb / c mice were immunized intramuscularly on days 0 and 28 with various LNP-formulated influenza saRNA vaccine constructs and with an influenza modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA. The HA / NA post-mixture preparation consisted of a 1:1 mixture of individually formulated saRNA-HA and saRNA-NA. The functional antibody response against A / Wisconsin / 588 / 2019 was measured by NAI on day 21 (3 weeks after prime) and day 42 (2 weeks after boost).

[0313] 3. Conclusion The seasonal influenza saRNA vaccine is intended to express four different HA proteins and four different NA proteins to match the influenza strains that mainly prevail in each season. This has the potential to be achieved by using eight individual saRNA components or four bicistronic saRNA components. Towards this end, the feasibility of the bicistronic saRNA approach was evaluated in a mouse immunogenicity study. A bicistronic saRNA vaccine candidate (TC83-delkozak-HA-SGP-NA-80A) encoding both HA antigen and NA antigen was compared with monocistronic saRNA-HA or saRNA-NA controls (TC83-HA-40A or TC83-NA-40A) encoding a single antigen, and also with a 1:1 mixture of individually formulated saRNA-HA + saRNA-NA components. A modRNA vaccine candidate encoding the same A / Wisconsin / 588 / 2019 (H1N1) HA or NA antigen was also included as an additional comparison. Balb / c mice were immunized IM on day 0 with 20 ng of the bicistronic saRNA vaccine preparation, 20 ng of the monocistronic saRNA vaccine preparation, a total of 40 ng (20 ng each) of a 1:1 mixture of saRNA-HA + saRNA-NA, and 200 ng of the modRNA comparator. All saRNA LNPs were formulated in a 10 mM Tris / 10% sucrose + 20 mM glutamic acid, pH 7.4 base selected for clinical use. On day 21 (3 weeks after the first immunization), an anti-HA antibody response was induced as measured by HAI and MNT (Figures 1 and 2), and an anti-NA antibody response was also induced as measured by NAI (Figure 4). The bicistronic saRNA vaccine candidate achieved titers similar to those of the saRNA vaccine composed of a 1:1 mixture of individually formulated monocistronic saRNA HA + saRNA-NA. The titers of the saRNA vaccine formulation expressing two antigens were similar to or slightly lower than those brought about by the saRNA controls expressing only a single antigen.From these results, it was confirmed that the bicistronic saRNA method is feasible, and in Balb / c mice, it was shown that using the saRNA vaccine results in dose savings compared to modRNA based on antibody titers after a single immunization.

[0314] Overall, the evaluated bicistronic saRNA constructs resulted in functional and neutralizing antibody titers similar to those induced by saRNA vaccines composed of individually formulated HA and NA monocistronic saRNAs. The titers of the saRNA vaccine expressing the two antigens were the same as or slightly lower than those of the saRNA-HA or saRNA-NA only controls. From these preliminary results, it was confirmed that the bicistronic saRNA method is feasible regardless of the regulatory element (either IRES or SGP) used to drive the second target gene, the order of antigen positioning (HA-NA or NA-HA), the deletion of the Kozak sequence, and the length of the polyA tail (either 40A or 80A).

[0315] (Example 7) Immunogenicity of saRNA Influenza Vaccines Containing Modified Nucleosides To determine whether incorporation of modified bases can produce more tolerable and potent saRNA vaccines, saRNA preparations expressing influenza HA were generated by replacing uridine with various amounts of N1-methylpseudouridine from 0% to 100%. The effect of increasing the percentage of modified bases on in vitro antigen expression was cell type dependent. Specifically, in immunocompetent human cell lines such as HeLa, saRNAs containing 25 - 75% N1-methylpseudouridine resulted in higher in vitro antigen expression than unmodified (0%) saRNA controls. However, saRNAs with 100% modified bases consistently produced low levels of antigen regardless of cell type. This may be due to impaired replicase function. Increasing the percentage of modified nucleosides incorporated into saRNA also correlated with decreased levels of activation of various PRRs or RNA sensors, such as TLR3, TLR7, and RIG-1, in reporter cell lines (data not shown).

[0316] To assess immunogenicity, Balb / c mice were immunized IM on day 0 with 200 ng of an saRNA vaccine preparation containing various amounts of N1-methylpseudouridine. Incorporation of higher amounts of modified nucleosides correlated with lower innate immune activation as measured by cytokine and chemokine secretion in serum on day 1 post-vaccination (Figure 5), whereby vaccine tolerability could be improved. However, the presence of higher amounts of modified nucleosides in saRNA also correlated with a decrease in neutralizing antibody titers 3 weeks post-vaccination (Figure 6), which potentially reflects an effect on replicase activity. These results were confirmed in different C57BL6 / J mouse strains (Figures 7 and 8). Based on the mouse data, an saRNA construct with 50% incorporation of modified nucleosides substantially reduced cytokine and chemokine secretion compared to an unmodified control and also had a more moderate decrease in antibody titers to levels similar to or higher than the modRNA-HA benchmark. Overall, the data suggest that partial incorporation of modified bases can be tolerated by saRNA while reducing initial innate immune stimulation yet still eliciting a strong adaptive humoral response.

[0317] The use of 50% modified bases may partially affect replicase function, but for humans, the enhanced tolerability may potentially lead to an improved saRNA vaccine.

[0318] Regarding Figure 5, female Balb / c mice were immunized intramuscularly (IM) on day 0 with 200 ng of an LNP-formulated influenza saRNA vaccine preparation containing different amounts (0% - 100%) of N1-methylpseudouridine, or with an influenza modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA. Serum cytokines and chemokines were measured 24 hours after the first immunization using the Mouse Anti-Virus Response panel LEGENDplex assay. Data are reported as the median with the interquartile range.

[0319] Regarding Figure 6, female Balb / c mice were immunized IM on day 0 with an LNP-formulated influenza saRNA vaccine preparation containing different amounts (0% - 100%) of N1-methylpseudouridine or a modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA. Antibody responses against A / Wisconsin / 588 / 2019 were measured by HAI or 1-Day MNT assay on day 21 (3 weeks after immunization). HAI and 50% neutralization titers are reported (geometric mean and geometric SD).

[0320] Regarding Figure 7, female C57BL6 / J mice were immunized IM on day 0 with 200 ng of an LNP-formulated influenza saRNA vaccine preparation containing different amounts (0% or 50%) of N1-methylpseudouridine, or with an influenza modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA. Serum cytokines and chemokines were measured 24 hours after the first immunization using the Mouse Anti-Virus Response panel LEGENDplex assay. Data are reported as the median with the interquartile range.

[0321] Regarding Figure 8, female C57BL6 / J mice were immunized intramuscularly on day 0 with 200 LNP-formulated influenza saRNA vaccine preparations containing different amounts (0% or 50%) of N1-methylpseudouridine or a modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA. On day 21 (3 weeks after immunization), the antibody response against A / Wisconsin / 588 / 2019 was measured by HAI or 1-Day MNT assay. HAI and 50% neutralization titers are reported (geometric mean and geometric SD).

[0322] (Example 8) Immunogenicity of a Quadrivalent Bicistronic saRNA Influenza Vaccine Encoding HA and NA from Four Seasonal Influenza Strains The major pharmacological properties of the influenza saRNA vaccine were evaluated in in vitro and in vivo non-clinical studies. In vitro and in vivo studies demonstrated that in mice, the influenza saRNA vaccine induces potent functional and neutralizing antibody responses as well as robust CD4+ and CD8+ T cell responses and enables influenza HA and / or NA proteins to be encoded at significantly lower doses compared to modRNA. Replication of saRNA also results in innate immune activation, which can enhance the adaptive immune response to the expressed antigen(s). Efficient in vitro expression of HA and NA glycoproteins from the influenza saRNA vaccine was demonstrated in cultured cells. Mouse, rat, and ferret immunogenicity studies demonstrated that various influenza saRNA vaccine preparations elicit potent functional and neutralizing antibody responses as well as T cell responses. Innate immune activation, measured by serum cytokine and chemokine release 24 hours after immunization, was also demonstrated in mice and rats. Mouse immunogenicity studies using the influenza modRNA vaccine as a benchmark also support the use of a bicistronic influenza saRNA construct expressing two separate influenza antigens (HA and NA) from the same saRNA vector. Mouse immunogenicity studies also show that saRNA can tolerate partial incorporation of modified nucleosides to reduce initial innate immune stimulation while still eliciting a strong adaptive humoral response. Finally, mouse immunogenicity studies support the combination of four bicistronic influenza saRNA constructs, each encoding different HAs and NAs, for targeting four seasonal influenza strains.

[0323] The influenza saRNA vaccine candidates selected for the initial POC tests contain codon-optimized coding sequences of the full-length HA or NA glycoproteins derived from the A / Wisconsin / 588 / 2019 (H1N1) cell-based virus strain recommended for use in the Northern Hemisphere during the 2021, 2022, and 2022-2023 influenza seasons and in the Southern Hemisphere during the 2022 season.

[0324]

Table 12

[0325] Seasonal influenza saRNA vaccines expressing four different HA proteins and four different NA proteins to match the influenza strains predominantly circulating in each season can be achieved using four bicistronic saRNA components. The feasibility of the quadrivalent bicistronic saRNA approach was evaluated in a mouse immunogenicity study benchmarked against the licensed adjuvanted seasonal quadrivalent influenza vaccine (QIV; FluAd) for the Northern Hemisphere 2021-22 season. BALB / c mice were immunized IM on days 0 and 28 with a total dose of 0.8 μg of the quadrivalent saRNA vaccine (0.2 μg per component) or 2.4 μg of the licensed QIV comparator. On day 42 (2 weeks after the second immunization), an anti-HA antibody response was induced as measured by HAI and MNT for each of the four components (Figure 9), and an anti-NA antibody response was also induced as measured by NAI (Figure 10). The quadrivalent bicistronic saRNA vaccine candidate achieved HA and NA titers similar to or higher than those of the QIV comparator.

[0326] Regarding Figure 9, female Balb / c mice were immunized intramuscularly on day 0 with 20 ng of LNP-formulated 4-valent saRNA composed of four bicistronic constructs encoding HA and NA derived from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage), or with 2.4 μg of licensed adjuvant-containing 4-valent inactivated vaccine (QIV; FluAd). On day 42 (2 weeks after the second dose), the antibody responses to each vaccine component were measured by HAI or 1-Day MNT assay. HAI and 50% neutralization titers are reported (geometric mean and geometric SD).

[0327] Regarding Figure 10, female Balb / c mice were immunized intramuscularly on day 0 with 20 ng of LNP-formulated 4-valent saRNA composed of four bicistronic constructs encoding HA and NA derived from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage), or with 2.4 μg of licensed adjuvant-containing 4-valent inactivated vaccine (QIV; FluAd). On day 42 (2 weeks after the second dose), the antibody responses to each vaccine component were measured by NAI. NAI titers are reported for three of the four strains (geometric mean and geometric SD). The H3N2 NAI titer could not be reported for either saRNA or QIV due to technical problems associated with the NAI assay for this strain.

[0328] (Example 9) Effect in humans C4861001 is an ongoing Phase 1 FIH study evaluating the safety, tolerability, and immunogenicity of PF-07845104. As of the data cut-off date of November 15, 2022, 253 participants were randomly assigned and 248 participants received vaccination. A total of 5 participants did not receive vaccination (1 participant in vaccine preparation 4 of the 2.5 μg group, 2 participants in vaccine preparation 5 of the 2.5 μg group, 1 participant in vaccine preparation 5 of the 10 μg group, and 1 participant in vaccine preparation 6 of the 2.5 μg group). Approved QIV was used as the comparator.

[0329] Safety and Efficacy - C4861001 Study C4861001 is an ongoing, first-in-human (FIH), Phase 1, randomized, placebo-controlled, evaluator-blinded, sponsor-unblinded, dose-ranging, vaccine composition / formulation selection study in healthy adults. In this study, the safety, tolerability, and immunogenicity of single doses of various monosistronic and ultimately bicistronic saRNA vaccine preparations against influenza are evaluated. Participants aged 18 - 49 years are randomly assigned 4:1 to receive either an saRNA vaccine preparation or placebo. An additional group of participants who receive approved QIV as a control is independently enrolled.

[0330] Details of the saRNA vaccine preparations are presented in Table 13.

[0331]

Table 13

[0332] Vaccine Preparations 1, 2 and Control Group - Randomized Participants A total of 36 participants were randomly assigned to each of the vaccine preparation 1 and vaccine preparation 2 groups, 63 participants were randomly assigned to the placebo group, and 33 participants were randomly assigned to the control group.

[0333] Vaccine Preparation 1 For this group, 11 participants received a 1 μg vaccination, 13 participants received a 2.5 μg vaccination, and 12 participants received a 10 μg vaccination. One participant was randomly assigned to 2.5 μg but mistakenly received a 1 μg dose and was thus included in the 1 μg group for safety analysis. Five participants (1 from the 1 μg group and 2 each from the 2.5 μg and 10 μg groups) discontinued their participation in the trial after vaccination.

[0334] Vaccine Preparation 2 For this group, 12 participants each received 1 μg, 2.5 μg, and 10 μg vaccinations. One participant in the 1 μg group was lost to follow-up after vaccination.

[0335] Vaccine Preparations 3, 4, 7 and Control Group - Randomly Assigned Participants A total of 36 participants each were randomly assigned to the Vaccine Preparation 3, Vaccine Preparation 4, and Vaccine Preparation 7 groups, and 63 participants were randomly assigned to the placebo. One participant in the 2.5 μg group of Vaccine Preparation 4 did not receive the vaccination.

[0336] Vaccine Preparation 3 For this group, 12 participants each received 1 μg, 2.5 μg, and 10 μg vaccinations.

[0337] Two participants in the 1 μg group discontinued their participation in the trial after vaccination.

[0338] Vaccine Preparation 4 For this group, 12 participants each received 1 μg and 10 μg vaccinations, and 11 participants received a 2.5 μg vaccination.

[0339] Two participants (1 each from the 1 μg and 10 μg groups) discontinued their participation in the trial after vaccination.

[0340] Vaccine Preparation 7 For this group, 12 participants each received vaccinations of 1 μg, 2.5 μg, and 10 μg.

[0341] Vaccine Preparations 5, 6 and Control Group - Randomly Assigned Participants A total of 38 participants were randomly assigned to Vaccine Preparation 5, 35 participants were randomly assigned to Vaccine Preparation 6, 63 participants were randomly assigned to the placebo, and 33 participants were randomly assigned to the control group. Three participants in Vaccine Preparation 5 (2 participants in the 2.5 μg group and 1 participant in the 10 μg group) and 1 participant in the 2.5 μg of Vaccine Preparation 6 did not receive the vaccination.

[0342] Vaccine Preparation 5 For this group, 12 participants each received vaccinations of 1 μg and 2.5 μg, and 11 participants received a vaccination of 10 μg. Two participants in the 2.5 μg group and 1 participant in the 10 μg group did not receive the vaccination.

[0343] Two participants in the 1 μg group discontinued their participation in the trial after vaccination.

[0344] Vaccine Preparation 6 For this group, 11 participants each received vaccinations of 1 μg and 2.5 μg, and 12 participants received a vaccination of 10 μg. One participant in the 2.5 μg group did not receive the vaccination.

[0345] Five participants (1 participant in the 1 μg group and 4 participants in the 2.5 μg group) discontinued their participation in the trial after vaccination.

[0346] Immunogenicity A total of 253 participants were randomly assigned to receive the vaccination, of which 196 participants were evaluable for immunogenicity.

[0347] A dose-dependent increase in HAI GMT was observed 4 weeks after vaccination. HAI GMT on day 1 (before vaccination), and 1, 2, and 4 weeks after vaccination are shown in Figures 11, 12, and 13.

[0348] Vaccination preparations 1 and 2 The percentage of participants achieving seroconversion 4 weeks after 10 μg was higher than the percentage of participants achieving seroconversion after 1 μg and 2.5 μg (Table 14).

[0349] Vaccination preparations 3, 4, and 7 For vaccination preparation 3, the percentage of participants achieving seroconversion 4 weeks after 10 μg was higher than the percentage of participants achieving seroconversion after 1 μg and 2.5 μg (Table 15).

[0350]

Table 14-1

[0351]

Table 14-2

[0352]

Table 15

[0353]

Table 16

[0354] For vaccination preparation 4, the percentage of participants achieving seroconversion 4 weeks after 1 μg was higher than the percentage of participants achieving seroconversion after 2.5 μg and 10 μg (Table 15).

[0355] For the vaccination preparation 7, the percentage of participants achieving seroconversion 4 weeks after 1 μg was equal to the percentage of participants achieving seroconversion after 2.5 μg (Table 15).

[0356] Vaccination preparations 5 and 6 For vaccination preparation 5, no participants achieved seroconversion after 4 weeks. For vaccination preparation 6, the percentage of participants achieving seroconversion 4 weeks after 10 μg was higher than the percentage of participants achieving seroconversion after 1 μg and 2.5 μg (Table 16).

[0357] (Example 10) Immunogenicity of an 8-valent HA / NA saRNA influenza vaccine in mice This study was conducted to test the immunogenicity of an 8-valent saRNA-LNP vaccine encoding HA and NA antigens from four influenza virus strains in a mouse model. The saRNA constructs used in this study encoded hemagglutinin (HA) and / or neuraminidase (NA) proteins from influenza virus strains A / Wisconsin / 588 / 2019, A / Cambodia / e0926360 / 2020, B / Phuket / 3073 / 2013, or B / Washington / 02 / 2019. Compared to the approved comparator FluAd, the 8-valent saRNA-LNP vaccine elicited equivalent or higher levels of functional anti-HA, anti-NA, and virus-neutralizing antibodies.

[0358] The octavalent vaccine formulation composed of either a monocistronic saRNA construct or a bicistronic saRNA construct had equivalent immunogenicity in mice after two doses. Multivalent saRNA vaccines mixed either before formulation using LNP ( "pre - mixed") or after formulation ( "post - mixed") had equivalent immunogenicity in mice. In the octavalent saRNA - LNP vaccine, moderate interference occurred with the immunogenicity of some virus strains, particularly influenza B virus strains, compared to mice vaccinated with a monocistronic single - antigen control. Overall, these data provide support for the continued evaluation of multivalent saRNA vaccines encoding influenza virus antigens.

[0359] The primary objective of this study was to evaluate the immunogenicity of an octavalent saRNA vaccine encoding hemagglutinin (HA) or neuraminidase (NA) proteins derived from influenza virus in mice, along with monocistronic and bicistronic saRNA vaccine controls. The octavalent vaccine was composed of either eight monocistronic HA or NA saRNAs or four bicistronic HA - NA saRNA constructs. The octavalent saRNA vaccine was also compared to an approved quadrivalent inactivated influenza virus vaccine comparator (containing eight HA / NA antigens from four strains) and a quadrivalent modified RNA (modRNA) - LNP vaccine encoding four HA proteins. The secondary objective of this study was to compare the immunogenicity in a mouse model of octavalent vaccine constructs combined either before or after formulation using lipid nanoparticles (LNP).

[0360] In this study, we tested saRNA (TC83-delkozak-80A) constructs that either encode HA or NA alone (monocistronic) or both HA and NA proteins (bicistronic) derived from H1N1 A / Wisconsin / 588 / 2019, H3N2 A / Cambodia / e0926360 / 2020, B / Yam B / Phuket / 3073 / 2013, or B / Vic B / Washington / 02 / 2019. An octavalent formulation containing any of eight monocistronic (TC83-delkozak-HA-80A or TC83-delkozak-NA-80A) or four bicistronic (TC83-delkozak-HA-SGP-NA-80A) saRNAs formulated in LNP was tested in mice. The octavalent saRNA vaccine was either pre-mixed (before formulation into LNP) or post-mixed (after formulating each saRNA construct into LNP). Two alternative quadrivalent vaccine comparators were included in this study: a nucleoside-modified RNA (modRNA)-LNP vaccine encoding four HA proteins, and FluAd, an approved comparator composed of four inactivated influenza viruses. Monocistronic and bicistronic saRNA-LNP vaccines against each strain were also included as controls to assess any potential interference with the antibody responses observed for the octavalent vaccine formulations.

[0361] This study was designed using 20 groups as shown in Table 17, with a total of 10 female mice (mouse strain: Balb / c) in each group. The assay schedule used in this study is described below.

[0362]

Table 17-1

[0363]

Table 17-2

[0364] Assay of this test: HAI (Groups 1, 3, 4, 6, 8, 10 to 20); NAI (Groups 1, 2, 5, 7, 9, 10 to 17, 19, 20); 1D neutralization D21, 42

[0365] One 0.3 mL syringe was filled up to 0.05 mL, and the vaccine was administered to each animal via the intramuscular route. The procedure was repeated on the 28th day as a booster vaccination.

[0366]

Table 18-1

[0367]

Table 18-2

[0368]

Table 18-3

[0369]

Table 19-1

[0370]

Table 19-2

[0371]

Table 19-3

[0372]

Table 19-4

[0373]

Table 19-5

[0374]

Table 19-6

[0375] When an 8-valent saRNA-LNP (i.e., “4×saRNA bicistronic”) vaccine encoding HA and NA antigens from 4 influenza virus strains was intramuscularly injected into mice as a single dose, robust functional anti-HA (Figure 14), anti-NA (Figure 15), and virus-neutralizing antibodies (Figure 16) were elicited. Generally, the HAI antibody levels elicited against IAV strains after a single dose were higher than those elicited against IBV strains (Figure 14). The 8-valent saRNA-LNP vaccine elicited equivalent or slightly higher HAI and neutralizing titers compared to a 4-valent modRNA vaccine or FluAd (either 12 μg or 2.4 μg doses). The neuraminidase inhibition (NAI) antibody levels elicited by the 8-valent saRNA-LNP vaccine on day 21 tended to be slightly lower than those of the single antigen control but tended to be higher than the NAI titers elicited by FluAd (either 12 μg or 2.4 μg doses) (Figure 15). Generally, the virus-neutralizing titers elicited after a single dose of the 8-valent saRNA vaccine were equivalent to or exceeded those of a single dose of a 4-valent modRNA vaccine or FluAd (either 12 μg or 2.4 μg doses) (Figure 16).

[0376] All groups received the second dose of the vaccine on day 28, and blood was collected on day 42, 14 days later. After the second dose, functional anti-HA (Figure 17), anti-NA (Figure 18), and virus-neutralizing antibodies (Figure 19) were boosted in all vaccine groups. The impact of the multivalent formulation on the HAI and virus-neutralizing titers elicited against A / Cambodia / e0926360 / 2020 was minimal, and similar titers were elicited by the 8-valent vaccine and the single-antigen vaccine against this virus (Figure 17, Figure 19). After two doses of the vaccine, the NAI titers elicited by the 8-valent saRNA-LNP vaccine were equivalent to or exceeded those elicited by FluAd at either the 12 μg or 2.4 μg dose (Figure 18). Except for the virus-neutralizing titers elicited against A / Cambodia / e0926360 / 2020, after two doses of the vaccine, the 8-valent saRNA-LNP vaccine elicited neutralizing titers equivalent to or exceeding those of the modRNA-LNP vaccine encoding 4-valent HA or FluAd (12 μg or 2.4 μg dose) (Figure 19).

[0377] To evaluate whether the 8-valent vaccine is better prepared by first premixing all saRNA constructs and then formulating with LNP (premixing), or by mixing after formulating the saRNA-LNP vaccine (postmixing), the immunogenicity of each process was compared. For the 8-valent vaccine composed of 8 monosistronic HA or NA saRNAs, as well as for the 8-valent vaccine composed of 4 bicistronic saRNAs (HA-SGP-NA), premixed and postmixed formulations were evaluated. Similar levels of functional anti-HA (Figure 14, Figure 17), anti-NA (Figure 15, Figure 18), and virus-neutralizing antibodies (Figure 16, Figure 19) were elicited by either preparation method, either after one dose (Figure 14, Figure 15, Figure 16) or two doses (Figure 17, Figure 18, Figure 19) of the vaccine. These data suggest that co-formulated multivalent saRNA vaccines and multivalent saRNA vaccines pooled after formulation have equivalent immunogenicity.

[0378] The immunogenicity of an octavalent saRNA-LNP vaccine composed of either 8 monosistronic saRNA constructs or 4 bisistronic (HA-SGP-NA) saRNA constructs was also evaluated in this study. At this point, no difference was observed in the HAI titers against the IBV strain for the monosistronic or bisistronic octavalent vaccines. In mice administered with either the monosistronic saRNA construct or the bisistronic saRNA construct octavalent vaccine, the HAI titers against both the IAV and IBV strains were equivalent until day 42 (2 weeks after the second dose) (Figure 17). Three weeks after the first dose, the neutralizing antibody levels elicited by the monosistronic or bisistronic octavalent vaccines were generally equivalent (Figure 16), with the exception that, regarding the titers elicited against A / Cambodia / e0926360 / 2020, the monosistronic octavalent vaccine formulation elicited approximately 10-fold higher titers than the bisistronic octavalent vaccine. In mice that received either the monosistronic or bisistronic octavalent saRNA vaccine, after the second dose, the neutralizing antibody levels were equivalent against all 4 viruses (Figure 19).

[0379] The objective of this study was to evaluate the immunogenicity of an octavalent saRNA-LNP vaccine encoding HA and NA antigens from four influenza virus strains in a mouse model. After two doses of the octavalent vaccine formulation, functional anti-HA antibodies measured by the HAI assay, functional anti-NA antibodies measured by the NAI assay, and total virus neutralizing antibodies by 1-Day MNT were elicited. When comparing octavalent vaccines composed of either eight monocistronic or four bicistronic saRNA constructs, the monocistronic octavalent vaccine was slightly superior after a single dose, but after two doses of the vaccine, the monocistronic and bicistronic octavalent vaccines had comparable immunogenicity. Similar immunogenicity was observed between co-formulated (pre-mixed) octavalent saRNA vaccines and pooled (post-mixed) octavalent saRNA vaccines after formulation. Compared to a 4-valent modRNA-LNP vaccine encoding HA and FluAd, the octavalent saRNA vaccine was generally able to elicit immunogenicity equal to or exceeding these comparators.

[0380] Figure 14 Mice were vaccinated intramuscularly on day 0 with either saRNA encoding HA or both HA / NA formulated in LNP, a 4-valent modRNA-LNP vaccine encoding HA, or the licensed comparator FluAd. Serum was collected 3 weeks after vaccination (day 21, 3 wks PD1). Functional anti-HA antibody titers against A / Wisconsin / 588 / 2019 (upper left), A / Cambodia / e0926360 / 2020 (upper right), B / Phuket / 3073 / 2013 (lower left), or B / Washington / 02 / 2019 (lower right) in serum collected from mice (n = 5) were measured by hemagglutination inhibition (HAI) assay. The geometric mean titer (GMT) at each time point is shown at the top of each graph and is also indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by a dotted line.

[0381] On day 0, mice were vaccinated intramuscularly with either saRNA encoding HA or both HA / NA formulated in LNP, or the approved comparator FluAd. Serum was collected 3 weeks after vaccination (day 21, 3wks PD1). Functional anti-NA antibody titers against A / Wisconsin / 588 / 2019 (upper left), B / Phuket / 3073 / 2013 (lower left), or B / Washington / 02 / 2019 (lower right) in sera collected from mice (n = 5) were measured by enzyme-linked lectin assay (ELLA). The NAI titer against A / Cambodia / e0926360 / 2020 was not determined due to technical issues associated with the virus. The geometric mean titer (GMT) at each time point is shown at the top of each graph and is also indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by a dotted line.

[0382] On day 0, mice were vaccinated intramuscularly with either saRNA encoding HA or both HA / NA formulated in LNP, a 4-valent modRNA-LNP vaccine encoding HA, or the approved comparator FluAd. Serum was collected 3 weeks after vaccination (day 21, 3wks PD1). Virus neutralization antibody titers against A / Wisconsin / 588 / 2019 (upper left), A / Cambodia / e0926360 / 2020 (upper right), B / Phuket / 3073 / 2013 (lower left), or B / Washington / 02 / 2019 (lower right) in sera collected from mice (n = 5) were measured by 1-Day micro-neutralization test (MNT). The geometric mean titer (GMT) at each time point is shown at the top of each graph and is also indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by a dotted line.

[0383] On days 0 and 28, mice were vaccinated intramuscularly with either saRNA encoding HA or both HA / NA formulated in LNP, a 4-valent modRNA-LNP vaccine encoding HA, or the approved comparator FluAd. Serum was collected 2 weeks after the second vaccination (day 42, 2 wks PD2). Functional anti-HA antibody titers against A / Wisconsin / 588 / 2019 (upper left), A / Cambodia / e0926360 / 2020 (upper right), B / Phuket / 3073 / 2013 (lower left), or B / Washington / 02 / 2019 (lower right) in serum collected from mice (n = 5) were measured by hemagglutination inhibition (HAI) assay. The geometric mean titer (GMT) at each time point is shown at the top of each graph and is also indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by a dotted line.

[0384] On days 0 and 28, mice were vaccinated intramuscularly with either saRNA encoding HA or both HA / NA formulated in LNP, or the approved comparator FluAd. Serum was collected 2 weeks after the second vaccination (day 42, 2 wks PD2). Functional anti-NA antibody titers against A / Wisconsin / 588 / 2019 (upper left), B / Phuket / 3073 / 2013 (lower left), or B / Washington / 02 / 2019 (lower right) in serum collected from mice (n = 5) were measured by enzyme-linked lectin assay (ELLA). The NAI titer against A / Cambodia / e0926360 / 2020 was not determined due to technical issues associated with the virus. The 50% inhibitory titer is plotted, and the geometric mean titer (GMT) at each time point is shown at the top of each graph and is also indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by a dotted line.

[0385] On day 0 and day 28, mice were intramuscularly vaccinated with either saRNA encoding HA formulated in LNP or both HA / NA, a 4-valent modRNA-LNP vaccine encoding HA, or the approved comparator FluAd. Serum was collected 2 weeks after the second vaccination (day 42, 2wks PD2). The virus neutralizing antibody titers against A / Wisconsin / 588 / 2019 (upper left), A / Cambodia / e0926360 / 2020 (upper right), B / Phuket / 3073 / 2013 (lower left), or B / Washington / 02 / 2019 (lower right) in sera collected from mice (n = 5) were measured by a 1-Day micro-neutralization test (MNT). The 50% neutralization titers are plotted, and the geometric mean titers (GMT) at each time point are shown at the top of each graph and indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by a dotted line.

[0386] Exemplary embodiments 1. A composition comprising a self-amplifying RNA molecule comprising a 5' cap; a 5' untranslated region; a coding region for a non-structural protein derived from an alphavirus; a subgenomic promoter derived from an alphavirus; an open reading frame encoding a gene of interest; a 3' untranslated region; and a 3' polyA sequence, wherein at least 5% of the entire population of a specific nucleotide in the molecule is replaced by one or more modified or unnatural nucleotides.

[0387] 2. The 5' cap is of formula I:

[0388]

Chemical formula

[0389] 3.B 1 and B 2 is a naturally occurring base, the composition of clause 1 or 2.

[0390] 4.R 1 is methyl and R 2 is hydrogen, the composition of any one of clauses 1 to 3.

[0391] 5.B 1 is guanine, the composition of any one of clauses 1 to 4.

[0392] 6.B 1 is adenine, the composition of any one of clauses 1 to 5.

[0393] 7.B 2 is adenine, the composition of any one of clauses 1 to 5.

[0394] 8.B 2 is uracil, the composition of any one of clauses 1 to 7.

[0395] 9. The nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, the composition of any one of clauses 1 to 8.

[0396] 10.B 1 is adenine and B 2 is uracil, the composition of any one of clauses 1 to 9.

[0397] 11.B 1 is adenine and B 2 is uracil and R 1 is methyl and R 2 is hydrogen, the composition of any one of clauses 1 to 10.

[0398] 12. The nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine and B 1 is adenine and B 2is uracil, and R 1 is methyl, and R 2 is hydrogen, a composition according to any one of clauses 1 to 11.

[0399] 13. A composition according to any one of clauses 1 to 12, wherein at least 10% of the entire population of specific nucleotides in the molecule is replaced by one or more modified or unnatural nucleotides.

[0400] 14. A composition according to any one of clauses 1 to 13, wherein at least 25% of the entire population of specific nucleotides in the molecule is replaced by one or more modified or unnatural nucleotides.

[0401] 15. A composition according to any one of clauses 1 to 14, wherein at least 50% of the entire population of specific nucleotides in the molecule is replaced by one or more modified or unnatural nucleotides.

[0402] 16. A composition according to any one of clauses 1 to 15, wherein at le...

Claims

**Claim 1** A composition comprising a self - amplifying RNA (saRNA) comprising a 5′ cap; a 5′ untranslated region (5′ UTR); a coding region for a non - structural protein derived from an alphavirus; a first sub - genomic promoter derived from an alphavirus; a first open reading frame encoding a first gene of interest derived from influenza virus hemagglutinin (HA); a second sub - genomic promoter derived from an alphavirus; a second open reading frame encoding a second gene of interest derived from influenza virus; a 3′ untranslated region (3′ UTR); and a 3′ poly - A sequence. **Claim 2** The composition according to claim 1, wherein the 5′ UTR sequence has at least 70% sequence identity to SEQ ID NO: 12, the coding region of the non - structural protein comprises polynucleotide sequences having at least 70% sequence identity to SEQ ID NO: 13, polynucleotide sequences having at least 70% sequence identity to SEQ ID NO: 14, polynucleotide sequences having at least 70% sequence identity to SEQ ID NO: 15, and polynucleotide sequences having at least 70% sequence identity to SEQ ID NO: 16, the first sub - genomic promoter has a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 17, the polynucleotide sequence encoding HA has at least 70% sequence identity to SEQ ID NO: 18, the second sub - genomic promoter comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 19, the 3′ UTR comprises a polynucleotide sequence having at least 70% sequence identity to SEQ ID NO: 21, and the poly - A tail comprises at least 20 consecutive adenines. **Claim 3** The composition according to any one of claims 1 to 2, wherein the second gene of interest derived from influenza virus encodes any one polypeptide selected from HA, NA, NP, M1, M2, NS1 and NS2. **Claim 4** The composition according to any one of claims 1 to 3, wherein the 5′ cap is represented by formula II: 【Chemical 1】 **Claim 5** The composition according to any one of claims 1 to 4, wherein at least 10% of the total nucleotides within the saRNA are replaced by a modified or unnatural nucleotide selected from the group consisting of 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-methyluridine.

6. The composition according to any one of claims 1 to 5, wherein the nucleotide immediately downstream (from 5' to 3') of the 5' cap contains guanine.

7. The composition according to any one of claims 1 to 6, wherein at least 50% of the total saRNA molecules are full-length.

8. The composition according to any one of claims 1 to 7, wherein at least 80% of the total saRNA molecules are full-length.

9. The composition according to any one of claims 1 to 8, wherein the saRNA forms a complex with or associates with lipid nanoparticles (LNP) comprising ionizable lipids, neutral lipids, steroids, and lipids conjugated with polymers.

10. The saRNA is (i) 【Chemical 2】 at least one cationic lipid according to; (ii) at least one neutral lipid comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid comprising cholesterol; and (iv) 【Chemical 3】 wherein n has an average value ranging from 30 to 60 The composition according to any one of claims 1 to 9, which forms a complex with or associates with lipid nanoparticles (LNP) comprising at least one PEG-lipid according to.

11. The composition according to any one of claims 1 to 10, wherein (i) to (iv) are present in a molar ratio of about 20 to 60% cationic lipid, 5 to 25% neutral lipid, 25 to 55% sterol, and 0.5 to 15% PEG-lipid.

12. The composition according to any one of claims 1 to 11, wherein the saRNA comprises at least one poly(A) sequence comprising 30 to 200 adenosine nucleotides.

13. A composition comprising a self-amplifying RNA (saRNA) comprising a 5' cap; a 5' untranslated region (5'UTR); a coding region for a non-structural protein derived from an alphavirus; a subgenomic promoter derived from an alphavirus; an open reading frame encoding a gene of interest derived from an influenza virus; a 3' untranslated region (3'UTR); and a 3' polyA sequence, wherein at least 25% of the entire population of specific nucleotides within the saRNA is replaced by one or more modified or unnatural nucleotides selected from the group consisting of 5-methyluridine, N1-methylpseudouridine, 5-methoxyuridine, and 5-methylcytosine.

14. The composition according to claim 13, wherein the 5' cap is of formula II: [Chemical Formula 4] as represented by.

15. The composition according to any one of claims 13 to 14, wherein the nucleotide immediately downstream (5' to 3') of the 5' cap contains guanine.

16. The composition according to any one of claims 13 to 15, wherein the saRNA molecule is encapsulated in, bound to, or adsorbed to liposomes, lipid nanoparticles, polyplexes, cochleates, virosomes, immunostimulatory complexes, microparticles, microspheres, nanospheres, unilamellar vesicles, multilamellar vesicles, water-in-oil emulsions, oil-in-water emulsions, emulsomes, polycationic peptides, cationic nanoemulsions, or combinations thereof.

17. The composition according to any one of claims 13 to 16, wherein at least 50% of the entire saRNA molecule is full-length.

18. The composition according to any one of claims 13 to 17, wherein at least 80% of the entire saRNA molecule is full-length.

19. Use of the composition according to any one of claims 1 to 18 in the manufacture of a medicament for inducing an immune response in a subject.

20. Use according to claim 19, wherein the composition elicits an immune response including a T cell response.

21. A composition according to any one of claims 1 to 18, comprising a plurality of said saRNAs encapsulated in lipid nanoparticles.

22. A composition according to any one of claims 1 to 18, comprising four of said saRNAs encapsulated in lipid nanoparticles.

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