Immunogenic compositions against influenza

Abstract

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

Description

[0001] PC073225A

[0002] IMMUNOGENIC COMPOSITIONS AGAINST INFLUENZA

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit under 35 U. S. C. § 119(e) of U. S. Provisional Patent Application Serial No. 63 / 734,180, filed December 15, 2024, U. S. Provisional Patent Application Serial No.

[0005] 63 / 735,876, filed December 18, 2024, U. S. Provisional Patent Application Serial No. 63 / 738,522, filed December 24, 2024, U. S. Provisional Patent Application Serial No. 63 / 742,423, filed January 6, 2025, and U. S. Provisional Patent Application Serial No. 63 / 912,061, filed November s, 2025, the disclosures of which are hereby incorporated by reference in their entirety.

[0006] SEQUENCE LISTING

[0007] This application is being filed electronically via Patent Center and includes an electronically submitted sequence listing in.xml format. The.xml file contains a sequence listing entitled “PC073225A_SEQListing_ST26.xml” created on December 14, 2025, and having a size of 385 KB. The sequence listing contained in this.xml file is part of the specification and is hereby incorporated by reference herein in its entirety.

[0008] FIELD

[0009] The 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.

[0010] BACKGROUND

[0011] Influenza viruses are members of the Orthomyxoviridae family, and are classified into three types (A, B, and C), based on antigenic differences between their nucleoprotein (NP) and matrix (M) protein.

[0012] The genome of influenza A virus includes eight molecules (seven for influenza C virus) of linear, negative polarity, single-stranded RNAs, which encode several polypeptides including: the RNA-directed RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP), which form the nucleocapsid; the matrix proteins (M1, M2, which is also a surface-exposed protein embedded in the virus membrane); two surface glycoproteins, which project from the lipoprotein envelope: hemagglutinin (HA) and neuraminidase (NA); and nonstructural proteins (NS1 and NS2).

[0013] Hemagglutinin is the major envelope glycoprotein of influenza A and B viruses, and hemagglutinin-esterase (HE) of influenza C viruses is a protein homologous to HA.

[0014] A challenge for therapy and prophylaxis against influenza and other infections using traditional vaccines is the limitation of vaccines in breadth, providing protection only against closely related subtypes. In addition, the length of time required to complete current standard influenza virus vaccine production processes inhibits the rapid development and production of an adapted vaccine in a pandemic situation.

[0015] There is a need for improved immunogenic compositions against influenza. SUMMARY

[0016] The unmet needs for improved immunogenic compositions against influenza, among other things, are provided herein. In one aspect, the disclosure relates to an improved polypeptide derived from influenza virus, wherein the polypeptide has mutations in a fusion peptide and fusion peptide proximal regions (FPPR), relative to the corresponding wild-type influenza polypeptide. In preferred embodiments, the polypeptide is derived from an influenza hemagglutinin polypeptide. In some embodiments, the polypeptide is derived from a hemagglutinin of an influenza B virus.

[0017] In some embodiments, the influenza hemagglutinin polypeptide may be derived from hemagglutinin of an influenza virus from the B / Yamagata lineage (as represented by B / Yamagata / 16 / 88) or from the B / Victoria lineage (as represented by B / Victoria / 2 / 87). In some embodiments, the polypeptide is derived from B / Brisbane / 60 / 08, B / lowa / 06 / 2017, or B / Lee / 40.

[0018] In some embodiments, the polypeptide has at least 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identify to any one of amino acid sequences SEQ ID NO: 10-SEQ ID NO: 68. In some embodiments, the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO: 10-SEQ ID NO: 68.

[0019] As used herein, the terms "non-natural," "non-naturally occurring,” and “mutant” are used interchangeably in the context of an organism, polypeptide, or nucleic acid. The terms "non- natural" and "non-naturally occurring" and “mutant” in this context refer to a polypeptide or nucleic acid having at least one variation or mutation at an amino acid position or nucleic acid position as compared to the respective wild-type polypeptide or nucleic acid. Non-limiting examples of the at least one variation are an insertion of one or more amino acids or nucleotides, a deletion of one or more amino acids or nucleotides, or a substitution of one or more amino acids or nucleotides. In some embodiments, the polypeptides and / or nucleic acids of the present disclosure, e.g., polypeptides comprising an amino acid sequence of an influenza B virus hemagglutinin protein or nucleic acids encoding such polypeptides, are non-naturally occurring and include a deletion relative to the respective wild-type sequence at specified positions of the respective wild-type sequence. Further, when referring to "residues X to Y" of a specified sequence herein, one of ordinary skill in the art understands this to mean a contiguous sequence of the indicated amino acid residues in the respective specified sequence. In some embodiments, similar polypeptides of the present disclosure have about 40%, at least about 40%, about 45%, at least about 45%, about 50%, at least about 50%, about 55%, at least about 55%, about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% identical amino acids. In some embodiments, similar polypeptides of the present disclosure have about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% functionally identical amino acids. The "percent identity" (% identity) between two sequences is determined when sequences are aligned for maximum homology, and not including gaps or truncations as set forth in the alignment parameters. Exemplary parameters for determining relatedness of two or more amino acid sequences using the BLAST algorithm, for example, can be as provided in BLASTP. Nucleic acid sequence alignments can be performed using BLASTN. Modifications can be made to the alignment parameters to either increase or decrease the stringency of the comparison, for example, for determining the relatedness of two or more sequences. Additional sequences added to a polypeptide sequence, including but not limited to immunodetection tags, purification tags, localization sequences (presence or absence), etc., do not affect the % identity. Algorithms such as Align, BLAST, ClustalW and others can be used to compare and determine a raw sequence's similarity or identity to another sequence, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms are similarly applicable for determining nucleotide or amino acid sequence similarity or identity, and can be useful in identifying orthologs of genes of interest. Parameters for sufficient similarity to determine relatedness are computed based on well-known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 45% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance if a database of sufficient size is scanned (about 5%). For example, alignment can be performed using the Needleman-Wunsch algorithm implemented through the BALIGN tool. Default parameters may be used for the alignment and BLOSUM62 may be used as the scoring matrix. In some cases, it can be useful to use the BLAST algorithm to understand the sequence identity between an amino acid motif in a template sequence and a target sequence. Therefore, in some embodiments, BLAST is used to identify or understand the identity of a shorter stretch of amino acids (e.g., a sequence motif) between a template and a target protein. BLAST finds similar sequences using a heuristic method that approximates the Smith- Waterman algorithm by locating short matches between the two sequences. The BLAST algorithm can identify library sequences that resemble the query sequence above a certain threshold. As used herein, an amino acid position (or simply, amino acid) "corresponding to" an amino acid position in another polypeptide sequence is the position that is aligned with the referenced amino acid position when the polypeptides are aligned. The polypeptides may be aligned with maximum homology, for example, as determined by BLAST, which allows for gaps in sequence homology within protein sequences to align related sequences and domains. Alternatively, in some instances, when polypeptide sequences are aligned for maximum homology, a corresponding amino acid may be the nearest amino acid to the identified amino acid that is within the same amino acid biochemical grouping- i.e., the nearest acidic amino acid, the nearest basic amino acid, the nearest aromatic amino acid, etc., to the identified amino acid. By "substantially identical," with reference to a nucleic acid sequence (e.g., a gene, RNA, or cDNA) or amino acid sequence (e.g., a protein or polypeptide) is meant one that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% at least 98%, or at least 99% nucleotide or amino acid identity, respectively, to a reference sequence.

[0020] In one aspect, the disclosure relates to a nucleic acid encoding a polypeptide derived from an influenza polypeptide, preferably a hemagglutinin polypeptide, that comprises a fusion peptide and proximal regions (FPPR), wherein the FPPR comprises a deletion of at least three to seven amino acid residues between amino acid positions 369 and 382, more preferably 352 and 382, corresponding to the amino acid positions of SEQ ID NO: 9. In some preferred embodiments, the disclosure relates to a nucleic acid encoding a polypeptide derived from an influenza polypeptide, preferably a hemagglutinin polypeptide, that comprises a fusion peptide and proximal regions (FPPR), wherein the FPPR comprises a deletion of at least three to seven amino acid residues between amino acid positions 352 and 382, corresponding to the amino acid positions of SEQ ID NO: 9.

[0021] In one aspect, the disclosure relates to an immunogenic composition including: (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen, said antigen including 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, said second antigen including at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the first and second RNA polynucleotides are formulated in a lipid nanoparticle (LNP). In some embodiments, the first and second antigens include hemagglutinin (HA), or an immunogenic fragment or variant thereof. In some embodiments, the first antigen includes an HA from a different subtype of influenza virus to the influenza virus antigenic polypeptide or an immunogenic fragment thereof of the second antigen. In some embodiments, the composition further includes (iii) a third antigen including at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the third antigen is from influenza virus but is from a different strain of influenza virus to both the first and second antigens. In some embodiments, the first, second and third RNA polynucleotides are formulated in a lipid nanoparticle.

[0022] In some embodiments, the composition further includes (iv) a fourth RNA polynucleotide having an open reading frame encoding a fourth antigen, said antigen including at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the fourth antigen is from influenza virus but is from a different strain of influenza virus to the first, second and third antigens. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in a lipid nanoparticle.

[0023] In some embodiments, each RNA polynucleotide includes a modified nucleotide. In some embodiments, the modified nucleotide is selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1 -methyl- 1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyl uridine.

[0024] In some embodiments, each RNA polynucleotide includes a 5' terminal cap, a 5’ UTR, a 3’IITR, and a 3' polyadenylation tail. In some embodiments, the 5' terminal cap includes:

[0025]

[0026] . In some embodiments, the 5’ UTR includes SEQ ID NO: 1. In some embodiments, the 3’ UTR includes SEQ ID NO: 2. In some embodiments, the 3' polyadenylation tail includes SEQ ID NO: 3.

[0027] In some embodiments, the RNA polynucleotide has an integrity greater than 85%. In some embodiments, the RNA polynucleotide has a purity of greater than 85%.

[0028] In some embodiments, the lipid nanoparticle includes 20-60 mol % ionizable cationic lipid, 5-25 mol % neutral lipid, 25-55 mol % cholesterol, and 0.5-5 mol % PEG-modified lipid.

[0029] In some embodiments, the cationic lipid includes:

[0030]

[0031] In some embodiments, the PEG-modified lipid includes:

[0032]

[0033] In some embodiments, the first antigen is HA from influenza A subtype H1 or an immunogenic fragment or variant thereof and the second antigen is HA from a different H1 strain to the first antigen or an immunogenic fragment or variant thereof. In some embodiments, the first and second antigens are HA from influenza A subtype H3 or an immunogenic fragment or variant thereof and wherein both antigens are derived from different strains of H3 influenza virus.

[0034] In some embodiments, the first and second antigens are HA from influenza A subtype H1 or an immunogenic fragment or variant thereof and the third and fourth antigens are from influenza A subtype H3 or an immunogenic fragment or variant thereof and wherein the first and second antigens are derived from different strains of H1 virus and the third and fourth antigens are from different strains of H3 influenza virus.

[0035] In some embodiments, at least the first and second RNA polynucleotides are formulated in a single lipid nanoparticle. In some embodiments, the first and second RNA polynucleotides are formulated in a single lipid nanoparticle. In some embodiments, the first, second, and third RNA polynucleotides are formulated in a single lipid nanoparticle. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in a single LNP.

[0036] In some embodiments, each of the RNA polynucleotides is formulated in a single LNP, wherein each single LNP encapsulates the RNA polynucleotide encoding one antigen. In some embodiments, the first RNA polynucleotide is formulated in a first LNP; and the second RNA polynucleotide is formulated in a second LNP. In some embodiments, the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; and the third RNA polynucleotide is formulated in a third LNP. In some embodiments, the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; the third RNA polynucleotide is formulated in a third LNP; and the fourth RNA polynucleotide is formulated in a fourth LNP.

[0037] In another aspect, the disclosure relates to any of the immunogenic compositions described herein, for use in the eliciting an immune response against influenza.

[0038] In another aspect, the disclosure relates to a method of eliciting an immune response against influenza disease, including administering an effective amount of any of the immunogenic compositions described herein.

[0039] In another aspect, the disclosure relates to a method of purifying an RNA polynucleotide synthesized by in vitro transcription. The method includes ultrafiltration and diafiltration. In some embodiments, the method does not comprise a chromatography step. In some embodiments, the purified RNA polynucleotide is substantially free of contaminants comprising short abortive RNA species, long abortive RNA species, double- stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcription enzymes, residual solvent and / or residual salt. In some embodiments, the residual plasmid DNA is < 500 ng DNA / mg RNA. In some embodiments, the yield of the purified mRNA is about 70% to about 99%. In some embodiments, purity of the purified mRNA is between about 60% and about 100%. In some embodiments, purity of the purified mRNA is between about 85%-95%.

[0040] In some embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein. In some embodiments, the disclosure provides an expression construct comprising a nucleic acid described herein. In some embodiments, the disclosure provides a method of inducing an immunological response against an influenza B virus in a subject in need thereof, comprising administering to the subject an immunologically effective amount of a polypeptide or protein trimer described herein, the immunogenic composition described herein, or combination thereof.

[0041] DESCRIPTION OF THE DRAWINGS

[0042] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0043] FIG. 1A-H Functional Anti-HA Antibodies Elicited by Immunization of Mice with Monovalent or Quadrivalent LNP-Formulated modRNA Encoding Influenza HA from one of four vaccine strains (H1N1 A / Wisconsin / 588 / 2019 [FIG. 1A-B], H3N2 A / Cambodia / e0826360 / 2020 [FIG. 1C-D], By / Phuket / 3073 / 2013 [FIG. 1E-F], Bv / Washington / 02 / 2019 [FIG. 1 G-H]) as measured by mean neutralization titer (MNT).

[0044] FIG. 2 Example of a mutant (#24)(also referred to herein as any one of A355-363 and pBV-024 (SEQ ID NO: 33) shows increased potency when compared to wild type FluB.

[0045] FIG. 3 Correlation of In Vitro Expression in Human HeLa Cells determined using monoclonal antibody CR8071 vs polyclonal antibody B295 - HA variants of Influenza virus. Selected variants are labeled with their SEQ NO IDs.

[0046] FIG. 4 Correlation of In Vitro Expression in Human HeLa Cells measured using monoclonal antibodies CR8071 and CR9114 of HA variants of Influenza virus. Selected variants are labeled with their SEQ ID NOs. CR8071 and CR9114 recognize different epitopes of the HA.

[0047] FIG. 5 Functional Anti-HA Antibodies Elicited by Immunization of Mice with Monovalent or Quadrivalent LNP-Formulated modRNA Encoding Influenza HA as Measured By MNT. In vivo immunogenicity elicited by HA variants of influenza B virus determined by neutralization antibody titer, 3 weeks post dose 1. FIG. 6 In vivo immunogenicity elicited by HA variants of influenza B virus measured by neutralization antibody titer, 2 weeks post dose 2.

[0048] FIG. 7 Correlation of In vivo immunogenicity elicited by HA variants of influenza B virus measured by neutralization antibody titer, 2 weeks post dose 2, and their in vitro expression in human cells. DS, drug substances; DR, Drug product.

[0049] FIG. 8A-C Same design principle can be applied to HA of other influenza B virus strains to improve the immunogenicity against those virus strains. In this example, the HA variant with the mutation equivalent to SEQ ID NO: 47 was engineered in Washington and Colorado sublineage of Victory strain of influenza B virus, and Phuket sublineage of Yamagata strain. Improvements on immunogenicity measured by neutralization antibody titers were observed.

[0050] FIG. 9A-C CleanCap AG saRNA performed better than enzymatically capped saRNA in THP-1 cells. In the number of cells expressing antigen encoded by saRNA & geometric mean fluorescence intensity (GMFI) of the antigen in saRNA-transfected cells (the antigen copy number). FIG. 9C depicts A / Wisc / 588 / 19 HA expression in THP-1. THP-1 cells were transfected with either saRNA-TC83-A / Wisc / 588 / 19 HA-40A or bicistronic saRNA-TC83-A / Wisc / 588 / 19 HA-NA-80A either with no nucleoside modifications, m5C, Hm5C, or2'Ome-G incorporation (11 -point, 2-fold dilution series starting from 1000ng). Number of HA expressing cells (% HA+ cells) (FIG. 9A), total HA expression per HA positive cell (Geometric mean fluorescence intensity (GMFI)) (FIG. 9B), and number of live cells (% live cells) (FIG. 9C), were determined by flow cytometry at 22 hrs post transfection. Results are presented as mean ± standard deviation for each group from a representative experiment.

[0051] FIG. 10A-B. FIG. 10A depicts % of encapsulation and LNP size (diameter in nanometers [d.nm]) of LNPs before dialysis and after filtration. FIG. 10B depicts % of positive express in HEK293T cells when comparing LNP formulations in the presence of egg sphingomyelin (ESM) and cholesterol against a benchmark LNP formulation in the absence of ESM and cholesterol.

[0052] FIG. 11A-C. FIG. 11A depicts testing various LNP formulations and measuring LNP size (d.nm), wherein the samples tested are described in Table 54. Successful combination of sitosterol and sphingomyelin (SM) can be achieved using PBS as buffer. FIG. 11B depicts fraction of positive expression in HEK293T, testing samples as described respectively in Table 52. FIG. 11C depicts mean fluorescence intensity (MFI) of samples as described respectively in Table 52.

[0053] FIG. 12A-B. FIG. 12A depicts LNP size change of samples with various cationic lipid and ESM ratios. Samples tested are respectively described in Table 54. FIG. 12B depicts % encapsulation efficiency (EE) change of samples with various cationic lipid and ESM ratios. Samples tested are respectively described in Table 54.

[0054] FIG. 13A-B. FIG. 13A depicts % of positive expression in HEK293T cells of samples with various cationic lipid and ESM ratios. Samples tested are respectively described in Table 55. FIG. 13B depicts MFI of positive expression cells from samples respectively described in Table 55.

[0055] FIG. 14A-G. HA-specific antibody titers were measured by a hemagglutination inhibition assay (HAI) (Fig. 14A) and a microneutralization assay test (MNT) (Fig. 14B). Splenocytes were harvested two weeks after the second immunization, stimulated with peptides spanning the H1N1 HA protein from the vaccine strain (A / Wisconsin / 588 / 2019), and assessed by intracellular cytokine staining for CD4+T cells expressing IFN-y, IL-4, IL-2, TNF-a and / or CD154, and CD8+T cells expressing IFN-y, TNF-a and / or CD107a. Immunization with two doses of mIRV induced a higher percentage of IFN-g-producing CD4+T cells than IL-4-producing CD4+ T cells (FIG. 14C), indicative of a Th1-biased response, whereas two doses of QIV induced a higher percentage of IL-4+CD4+T cells than IFN-y+CD4+ T cells (FIG. 14D), indicative of a Th2-biased response. A strong polyfunctional (IFN-g+, I L-2+, TNF-a+, CD154+) CD4+T cell response was also observed with the mIRV vaccine, but not with QIV (FIG. 14E). In addition, immunization with mIRV induced higher levels of IFN-g+CD8+T cells (FIG. 14F) and polyfunctional (IFN-g+, TNF-a+, CD107a+) CD8+ T cells (FIG. 14G) compared to QIV.

[0056] FIG. 15A-F. HA-specific antibodies were measured by HAI (FIG. 15A-B) and MNT (FIG. 15C-D). T cell immunity was quantified by measuring cytokine-expressing peripheral CD4+and CD8+T cells after ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with HA peptide pools derived from the H1N1 vaccine strain (FIG 15E-F).

[0057] FIG. 16A-B. Mice were immunized with two doses of qlRV or licensed adjuvanted QIV 28 days apart. Two weeks after the second immunization, functional antibodies against each of the four strains encoded by the vaccines were measured by HAI (FIG. 16A) and MNT (FIG. 16B).

[0058] FIG. 17A-D. Two weeks after the second immunization, virus neutralization titers against the vaccine-matched strains were measured by MNT. Neutralization titers elicited by mIRV, tIRV, or qlRV against the shared vaccine strains (H1N1, H3N2, and B / Vic) were not statistically different (FIG. 17A-C) indicating an absence of interference. Neutralization titers against B / Yamagata elicited by mIRV and qlRV were also not statistically different (FIG. 17D).

[0059] FIG. 18A-D. Hematology findings were consistent with an inflammatory leukogram and included higher neutrophil counts on Days 3 and 17; a higher incidence of hyper-segmented neutrophils on Day 17; and higher monocytes, eosinophils, and / or large unstained cells on Days 3 and 17 (FIG. 18A). Findings consistent with an acute phase response were also noted for both vaccines and included higher fibrinogen on Day 17; higher globulin and / or lower albumin on Days 3 and 17; and higher alpha-2 macroglobulin (A2M) and alpha-1 -acid glycoprotein (A1AGP) on Days 3 and 17 (FIG. 18B-D).

[0060] FIG. 19 depicts Mean Influenza Challenge Virus Viral Load by qRT-PCR by Day, Per Protocol Population, wherein treatment groups tested are monovalent modRNA HA (N=55), QIV comparator (N=48), and placebo (N=52). FIG. 20 depicts a Box Plot of Area Under the Curve of Influenza Challenge Virus Viral Load by qRT-PCR by Day, Per Protocol Population. Treatment groups tested are monovalent modRNA HA (N=55), QIV comparator (N=48), and placebo (N=52). Whiskers represent the minimum and maximum, the box represents the interquartile range with the line representing the median, the diamond representing the mean, and the circles showing the values for each individual. FIG. 21 depicts a Forest Plot of Vaccine Efficacy for qRT-PCR Confirmed Moderately Severe Influenza Infection, Per Protocol Population, i.e., QIV comparator and monovalent modRNA HA.

[0061] FIG. 22 depicts 50% neutralization titer results 3 weeks post dose 1 against H1 N1 A / California strain from drug product formulations respectively described in Table 57 and Table 58.

[0062] FIG. 23 depicts 50% neutralization titer results 3 weeks post dose 1 against RSV M37 strain from drug product formulations respectively described in Table 57 and Table 58.

[0063] FIG. 24 depicts 50% neutralization titer results 3 weeks post dose 1 against RSV B18537 strain from drug product formulations respectively described in Table 57 and Table 58.

[0064] FIG. 25A-C. FIG. 25A depicts 50% neutralization titer results 3 weeks post dose 1 against Influenza B / Colorado strain from Bv / Colorado / 06 / 2017-HA-A370-374 samples respectively described in Table 61. FIG. 25B depicts 50% neutralization titer results 3 weeks post dose 1 against Influenza B / Washington strain from Bv / Washington / 02 / 2019-HA-A369-373 samples respectively described in Table 61. FIG. 25C depicts 50% neutralization titer results 3 weeks post dose 1 against Influenza B / Phuket strain from By / Phuket / 3073 / 2013-HA-A371-375 samples respectively described in Table 61. FIG. 25D depicts 50% neutralization titer results 3 weeks post dose 1 against Influenza B / Austria strain from mutant B / Austria samples respectively described in Table 62.

[0065] FIG. 26. FIG. 26A depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza B / Colorado strain from Bv / Colorado / 06 / 2017-HA-A370-374 samples respectively described in Table 61. FIG. 26B depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza B / Washington strain from Bv / Washington / 02 / 2019-HA-A369-373 samples respectively described in Table 61. FIG. 26C depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza B / Phuket strain from By / Phuket / 3073 / 2013-HA-A371-375 samples respectively described in Table 61. FIG. 26D depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza B / Austria strain from mutant B / Austria samples respectively described in Table 62.

[0066] FIG. 27 depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza B / Austria strain from mutant B / Austria samples. Flu B modRNA constructs bearing a deletion within the HA fusion peptide, A(369-373), led to higher neutralizing antibody titers in-vivo compared to WT benchmark control.

[0067] FIG. 28 depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza B / Austria strain from samples respectively described in Table 60. FIG. 29 depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza H1N1 A / Wisconsin / 67 / 2022 strain from samples respectively described in Table 63.

[0068] FIG. 30A-B. FIG. 30A depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza H1N1 A / Wisconsin / 67 / 2022 strain from samples respectively described in Table 63.

[0069] FIG. 30B depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza H3N2 A / Darwin / 06 / 2021 strain from samples respectively described in Table 63.

[0070] FIG. 31 depicts 50% neutralization titer results 3 weeks post dose 1 against Influenza Bv / Austria strain from samples respectively described in Table 63. The “?” in the FIG. represents “A.”

[0071] FIG. 32A-B. FIG. 32A depicts 50% neutralization titer results 3 weeks post dose 1 against Influenza H1N1 A / Wisconsin / 67 / 2022 strain from samples respectively described in Table 63, wherein the “?” represents “A.” FIG. 32B depicts 50% neutralization titer results 3 weeks post dose 1 against Influenza H3N2 A / Darwin / 06 / 2021 strain from samples respectively described in Table 63. The “?” in the FIG. represents “A.”

[0072] FIG. 33 depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza Bv / Austria strain from samples respectively described in Table 63. The “?” in the FIG. represents “A.”

[0073] FIG. 34A-B. FIG. 34A depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza H1N1 A / Wisconsin / 67 / 2022 strain from samples respectively described in Table 63. The “?” in the FIG. represents “A.” FIG. 34B depicts 50% neutralization titer results 2 weeks post dose 2 against Influenza H3N2 A / Darwin / 06 / 2021 strain from samples respectively described in Table 63. The “?” in the FIG. represents “A.”

[0074] FIG. 35 depicts EC50 values obtained in a comparison of wild-type (WT) and mutant B / Catalonia / 3514402NS / 2023, B / Netherlands / 10335 / 2023, B / Brisbane / 60 / 2008, and B / Catalonia / 2279261 NS / 2023 polypeptides.

[0075] FIG. 36 depicts EC50 values obtained in a comparison of wild-type (WT) and mutant B / Brisbane / 60 / 2008, B / Catalonia / 2279261 NS / 2023, B / Catalonia / 3514NS / 2023, and B / Netherlands / 10335 / 2023 polypeptides

[0076] FIG. 37 depicts EC50 values obtained in a comparison of wild-type (WT) and mutant B / Netherlands / 10335 / 2023, B / Catalonia / 351440NS / 2023, B / Brisbane / 60 / 2008, and B / Catalonia / 2279261 NS / 2023 polypeptides.

[0077] FIG. 38 depicts EC50 values obtained in a comparison of wild-type (WT) and mutant B / Colorado / 02 / 2017 and B / Washington polypeptides.

[0078] FIG. 39A-B depicts EC50 values obtained in a comparison of wild-type (WT) and mutant influenza B (FIG. 39A) and influenza A (FIG. 39B) polypeptides

[0079] FIG. 40 depicts qRT-PCR confirmed viral load. Shown in FIG.40A (panel A) is the mean viral load overtime after challenge and a box plot of viral load AUC from challenge to Day 8. Shown in FIG.40B (panel B) is a box plot of peak viral load after challenge. Whiskers represent the minimum and maximum, boxes represent the interquartile range with the line representing the median. The diamonds represent the mean and the circles represent individual values. Data are in the per-protocol population. Estimate of the difference between the groups and the associated 95% Cis were based on the Hodges-Lehman method. P values were determined with the Wilcoxon rank sum test. AUC, area under the concentration-time curve; A, difference; modRNA, nucleoside-modified mRNA vaccine; QIV, quadrivalent influenza vaccine; qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction.

[0080] FIG. 41A-C depicts HAI GMTs and GMFRs (FIG. 41A), and percentages of participants with seroconversion (FIG. 41 B), and HAI titers >1:40 (FIG. 41 C). Data are for the immunogenicity population. Error bars show 95% Cis. GMT, geometric mean titer; GMFR, geometric mean fold rise; HAI, hemagglutination inhibition; modRNA, nucleoside-modified mRNA vaccine; QIV, quadrivalent influenza vaccine.

[0081] FIG. 42A-D depicts GMFRs (95% Cis) of CD4+ T-cells producing IFNy (FIG. 42A); CD4 T-cells producing IFNy, IL-2, and TNFa (FIG. 42B); CD8+ T-cells producing IFNy (FIG. 42C), and CD4 T-cells producing IL-4 (FIG. 42D), in response to HA from H1N1. Data are for the immunogenicity population. T-cell responses were analyzed by ICS. Peripheral blood mononuclear cells collected before vaccination, 8 days after vaccination, and 28 days after vaccination were stimulated with H1 -specific peptide pools and ICS was used to measure the frequency of specific T-cells shown. GMFRs are compared to pre-vaccination levels. GMFR, geometric mean fold rise; ICS, intracellular cytokine staining; IFN, interferon; IL, interleukin; modRNA, nucleoside-modified mRNA vaccine; QIV, quadrivalent influenza vaccine; TNF, tumor necrosis factor.

[0082] FIG. 43A-B: In vitro expression of influenza HA from RNA encoding wild-type or alternate B / Victoria HA (“altB,” comprising a mutated HA cleavage site). LNP-formulated monovalent RNA (mIRV) encoding wild-type (wt) or altB HA from B / Austria / 1359417 / 2021 (B / Victoria) was transfected into (FIG. 43A) HEK293T cells and (FIG. 43B) primary human skeletal muscle cells.

[0083] (FIG. 43A) Protein expression in HEK293T cells was measured using a broadly reactive, prefusion conformation-specific anti-HA monoclonal antibody. Cells were labeled and the percentage of live cells expressing HA protein was enumerated using an image reader (Cytation 5 Cell Imaging Multimode Reader, BioTek). Expression was measured by quantifying the number of cells that had a positive signal for bound anti-HA antibody. Data shown is mean with standard deviation of duplicate measurements from one experiment. (FIG. 43B) Primary human skeletal muscle cells were lysed with a detergent solution and liquid chromatography-mass spectrometry proteomics analysis was performed. HA protein abundance relative to -8,000 non-specific cellular proteins was measured. Data shown is mean with standard deviation of triplicate measurements from one experiment. “DP” refers to drug product.

[0084] FIG. 44: In vitro expression of influenza HA from RNA encoding wild-type or altB HA in a panel of B / Victoria lineage viruses. LNP-formulated monovalent RNA (mIRV) encoding wild-type (wt) or altB HA (comprising a mutated HA cleavage site) from B / Victoria lineage strains B / Brisbane / 60 / 2008, B / Colorado / 06 / 2017, B / Austria / 1359417 / 2021, B / Catalonia / 3514402NS / 2023, and B / Netherlands / 10335 / 2023 was transfected into HEK293T cells. Protein expression was measured using a broadly reactive, prefusion conformation-specific anti-HA monoclonal antibody. Cells were labeled and the percentage of live cells expressing HA protein was enumerated using an image reader (Cytation 5 Cell Imaging Multimode Reader, BioTek). Expression was measured by quantifying the mean fluorescence intensity (MFI) of cells that had a positive signal for bound anti-HA antibody. Data shown is the average MFI of duplicate measurements over a range of doses (0.19-200 ng) from one experiment.

[0085] FIG. 45A-B: Functional antibody and virus neutralization titers elicited by immunization of mice with monovalent influenza RNA vaccines encoding wild-type or alternate B / Victoria HA. Female BALB / c mice were immunized IM ~4 weeks apart (Days 0 and 27) with 0.2 pg of LNP-formulated monovalent RNA vaccine encoding wild-type (wt) or altB HA from

[0086] B / Austria / 1359417 / 2021 (B / Victoria). Functional antibody and virus neutralization titers against B / Austria / 1359417 / 2021 (B / Victoria) were measured by (FIG. 45A) HAI and (FIG. 45B) MNT assays, respectively, on Day 41 (2 weeks post-dose 2). All titers are reported as geometric mean titer (GMT) with 95% confidence interval. Each data point represents one animal.

[0087] Statistical comparisons were performed using a two-sample t-test, * indicates p<0.01. LCD refers to limit of detection.

[0088] FIG. 46: Functional antibody and virus neutralization titers elicited by immunization of mice with trivalent influenza RNA vaccines encoding wild-type or alternate B / Victoria HA. Female BALB / c mice were immunized IM 4 weeks apart (Days 0 and 28) with 1.2 pg of LNP-formulated trivalent RNA vaccine (comprising 0.2 pg H1N1, 0.2 pg H3N2, and 0.8 pg B / Victoria) encoding wild-type HA from A / Wisconsin / 67 / 2022 (H1N1) and A / Darwin / 6 / 2021 (H3N2), and either wild-type (wt) or altB HA from B / Austria / 1359417 / 2021 (B / Victoria). Functional antibody and virus neutralization titers against the vaccine strains were measured by (A) HAI and (B) MNT assays, respectively, on Day 42 (2 weeks post-dose two). All titers are reported as geometric mean titer (GMT) with 95% confidence interval. Each data point represents one animal. Statistical comparisons were performed using two-way analysis of variance (ANOVA), * indicates p<0.01; ns refers to “not significant;” LOD refers to “limit of detection.”

[0089] FIG. 47 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for SARS-CoV-2 Omicron KP.2 neutralizing titers - Cohort 1 - evaluable SARS-CoV-2 immunogenicity population. Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0090] FIG. 48 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for SARS-CoV-2 Omicron KP.2 neutralizing titers - Cohort 2 - evaluable SARS-CoV-2 immunogenicity population. Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0091] FIG. 49 depicts a forest plot of geometric mean ratios (GM Rs) and difference of seroresponse rate (SRR) at 1 month after vaccination - Cohort 1 - evaluable SARS-CoV-2 immunogenicity population - Assay Stain: SARS-CoV-2 Omicron KP.2 (NT50) - Comparison to TIV + BNT162b2 (30 ug). Abbreviations include: GMR = geometric mean ratio; LS = least squares; NT50 = 50% neutralizing titer; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SRR = seroresponse rate. Notes include: Model-based GMRs and 2-sided 95% Cis were calculated by exponentiating the difference in LS means and the corresponding Cis between the two comparative vaccine groups based on analysis of logarithmically transformed assay results using a linear regression model with terms of the baseline assay results (log scale), age, and vaccine group. A separate model was fit for each comparison. Difference and the associated 2-sided 95% Cl based on the Miettinen and Nurminen method for the difference in proportions, expressed as a percentage (the corresponding vaccine group - BNT162b2 (30 ug) group or TIV + BNT162b2 (30 ug) group).

[0092] FIG. 50 depicts a forest plot of geometric mean ratios (GMRs) and difference of seroresponse rate (SRR) at 1 month after vaccination - Cohort 2 - evaluable SARS-CoV-2 immunogenicity population - Assay Stain: SARS-CoV-2 Omicron KP.2 (NT50) - Comparison to EIV + BNT162b2 (30 ug). Abbreviations include: GMR = geometric mean ratio; LS = least squares; NT50 = 50% neutralizing titer; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SRR = seroresponse rate. Notes include: Model-based GMRs and 2-sided 95% Cis were calculated by exponentiating the difference in LS means and the corresponding Cis between the two comparative vaccine groups based on analysis of logarithmically transformed assay results using a linear regression model with terms of the baseline assay results (log scale), age, and vaccine group. A separate model was fit for each comparison. Difference and the associated 2-sided 95% Cl based on the Miettinen and Nurminen method for the difference in proportions, expressed as a percentage (the corresponding vaccine group - BNT162b2 (30 ug) group or EIV + BNT162b2 (30 ug) group). FIG. 51 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for strain-specific HAI titers - Cohort 1 - evaluable HAI immunogenicity population - strain:

[0093] A / Wisconsin / 67 / 2022 (H1N1). Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; HAI = hemagglutination inhibition assay. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0094] FIG. 52 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for strain-specific HAI titers - Cohort 1 - evaluable HAI immunogenicity population - strain:

[0095] A / Massachusetts / 18 / 2022 (H3N2). Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; HAI = hemagglutination inhibition assay. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0096] FIG. 53 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for strain-specific HAI titers - Cohort 1 - evaluable HAI immunogenicity population - strain:

[0097] B / Austria / 1359417 / 2021 (Victoria). Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; HAI = hemagglutination inhibition assay. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0098] FIG. 54 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for strain-specific HAI titers - Cohort 2 - evaluable HAI (cell-based) immunogenicity population - strain: A / Wisconsin / 67 / 2022 (H1N1). Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; HAI = hemagglutination inhibition assay. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0099] FIG. 55 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for strain-specific HAI titers - Cohort 2 - evaluable HAI (cell-based) immunogenicity population - strain: A / Massachusetts / 18 / 2022 (H3N2). Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; HAI = hemagglutination inhibition assay. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0100] FIG. 56 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for strain-specific HAI titers - Cohort 2 - evaluable HAI (cell-based) immunogenicity population - strain: B / Austria / 1359417 / 2021 (Victoria). Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; HAI = hemagglutination inhibition assay. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0101] FIG. 57 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for strain-specific HAI titers - Cohort 2 - evaluable HAI (egg-based) immunogenicity population - strain: A / Victoria / 4897 / 2022 (H1N1). Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; HAI = hemagglutination inhibition assay. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0102] FIG. 58 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for strain-specific HAI titers - Cohort 2 - evaluable HAI (egg-based) immunogenicity population - strain: A / Thailand / 8 / 2022 (H3N2). Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; HAI = hemagglutination inhibition assay. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0103] FIG. 59 depicts geometric mean titers (GMTs) and 95% confidence interval (Cl) for strain-specific HAI titers - Cohort 2 - evaluable HAI (egg-based) immunogenicity population - strain: B / Austria / 1359417 / 2021 (Victoria). Abbreviations include: GMT = geometric mean titer; LLOQ = lower limit of quantitation; HAI = hemagglutination inhibition assay. Notes include: D1 = before vaccination; M1 = 1 month after vaccination. Numbers / GMTs within each bar denote the number of participants with valid and determinate assay results for the specified assay at the given sampling time point, and corresponding geometric mean titers. GMTs and 2-sided 95% Cis were calculated by exponentiating the mean logarithm of the titers and the corresponding Cis (based on the Student t distribution). Assay results below the LLOQ were set to 0.5 x LLOQ.

[0104] FIG. 60 depicts a graph showing the effect of modRNA-encoded cytokines co-administered with modRNA-HA on neutralizing antibody responses in mice at 2 weeks post-dose 2 (PD2) for H1N1 A / Wisconsin / 67 / 2022. Analysis was a one-way ANOVA on log transformed data with Sidak’s multiple comparisons test of pre-selected pairs: HA + Luciferase (1:1) or HA + CXCL-13 (4.7:1) as comparators.

[0105] FIG. 61 depicts a graph showing the effect of modRNA-encoded cytokines co-administered with modRNA-HA on neutralizing antibody responses in mice at 3 weeks post-dose 1 (PD1) for H1N1 A / Wisconsin / 67 / 2022. Analysis was a one-way ANOVA on log transformed data with Dunnett’s multiple comparisons test using modRNA-HA + modRNA-Luc as a control.

[0106] FIG. 62 depicts a graph showing the effect of modRNA-encoded cytokines co-administered with modRNA-HA on neutralizing antibody responses in mice at 2 weeks post-dose 2 (PD2) for H1N1 A / Wisconsin / 67 / 2022. Analysis was a one-way ANOVA on log transformed data with Dunnett’s multiple comparisons test using modRNA-HA + modRNA-Luc as a control.

[0107] FIG. 63 depicts a graph showing the effect of modRNA-encoded cytokines co-administered with modRNA-HA on neutralizing antibody responses in mice at 7 weeks post-dose 2 (PD2) for H1N1 A / Wisconsin / 67 / 2022. Analysis was a one-way ANOVA on log transformed data with Dunnett’s multiple comparisons test using modRNA-HA + modRNA-Luc as a control.

[0108] FIG. 64 depicts a line chart showing the effect of modRNA-encoded cytokines co-administered with modRNA-HA on neutralizing antibody responses over 80 days for H1N1 A / Wisconsin / 67 / 2022.

[0109] FIG. 65 depicts bar graphs for various Th1 related cytokines induced at 6 hours post-dose 2 (6hr PD2) and 24 hours post-dose 2 (24hr PD2) by (in pairs from I eft- to- right): Saline; Fluad (Quadrivalent); modRNA-HA (A / Wisconsin / 67 / 2022); modRNA-(HA + mlL-12p70); modRNA-(HA + mlL-21); modRNA-(HA + hlL-15); and modRNA-(HA + Luciferase). FIG. 66 depicts bar graphs for various macrophage and type-1 interferon (IFN 1) related cytokines induced at 6 hours post-dose 2 (6hr PD2) and 24 hours post-dose 2 (24hr PD2) by (in pairs from left-to-right): Saline; Fluad (Quadrivalent); modRNA-HA (A / Wisconsin / 67 / 2022); modRNA-(HA + mlL-12p70); modRNA-(HA + mlL-21); modRNA-(HA + hlL-15); and modRNA-(HA + Luciferase).

[0110] DETAILED DESCRIPTION

[0111] Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines that include polynucleotide encoding an influenza virus antigen. Influenza virus RNA vaccines, as provided herein may be used to induce a balanced immune response, comprising both cellular and humoral immunity, without many of the risks associated with DNA vaccination.

[0112] In some embodiments, the virus is a strain of Influenza A or Influenza B or combinations thereof.

[0113] In one aspect, the disclosure relates to an immunogenic composition including: (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen, said antigen including 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, said second antigen including at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the first and second RNA polynucleotides are formulated in a lipid nanoparticle (LNP). In some embodiments, the first and second antigens include hemagglutinin (HA), or an immunogenic fragment or variant thereof. In some embodiments, the first antigen includes an HA from a different subtype of influenza virus to the influenza virus antigenic polypeptide or an immunogenic fragment thereof of the second antigen. In some embodiments, the composition further includes (iii) a third antigen including at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the third antigen is from influenza virus but is from a different strain of influenza virus to both the first and second antigens. In some embodiments, the first, second and third RNA polynucleotides are formulated in a lipid nanoparticle.

[0114] In some embodiments, the composition further includes (iv) a fourth RNA polynucleotide having an open reading frame encoding a fourth antigen, said antigen including at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the fourth antigen is from influenza virus but is from a different strain of influenza virus to the first, second and third antigens. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in a lipid nanoparticle.

[0115] In some embodiments, the RNA polynucleotides are mixed in desired ratios in a single vessel and are subsequently formulated into lipid nanoparticles. The inventors surprisingly discovered that the initial input of different RNA polynucleotides at a known ratio to be formulated in a single LNP process surprisingly resulted in LNPs encapsulating the different RNA polynucleotides in about the same ratio as the input ratio. The results were surprising in view of the potential for the manufacturing process to favor one RNA polynucleotide to another when encapsulating the RNA polynucleotides into an LNP. Such embodiments may be referred herein as "pre-mix". Accordingly, in some embodiments, first and second RNA polynucleotides are formulated in a single lipid nanoparticle. In some embodiments, the first, second, third, and fourth RNA polynucleotides are formulated in a single LNP. In some embodiments, the first, second, third, fourth, and fifth RNA polynucleotides are formulated in a single LNP. In some embodiments, the first, second, third, fourth, fifth, and sixth RNA polynucleotides are formulated in a single LNP. In some embodiments, the first, second, third, fourth, fifth, sixth, and seventh RNA polynucleotides are formulated in a single LNP. In some embodiments, the first, second, third, fourth, fifth, sixth, seventh, and eighth RNA polynucleotides are formulated in a single LNP.

[0116] In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide in the mix of RNA polynucleotides prior to formulation into LNPs 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.

[0117] In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide in the mix of RNA polynucleotides prior to formulation into LNPs 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.

[0118] In some embodiments, the molar ratio of the first RNA polynucleotide to the fourth RNA polynucleotide in the mix of RNA polynucleotides prior to formulation into LNPs 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 mix of RNA polynucleotides prior to formulation into LNPs 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 mix of RNA polynucleotides prior to formulation into LNPs 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 mix of RNA polynucleotides prior to formulation into LNPs 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 mix of RNA polynucleotides prior to formulation into LNPs 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.

[0119] Self-amplifying RNA (saRNA)

[0120] In some embodiments, the RNA molecule, such as the first RNA molecule, is an saRNA. “saRNA,” “self-amplifying RNA,” and “replicon” refer to RNA with the ability to replicate itself. Selfamplifying RNA molecules may be produced by using replication elements derived from a virus or viruses, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the protein of interest, e.g., an antigen. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNAs and so the encoded gene of interest, e.g., a viral antigen, can become a major polypeptide product of the cells.

[0121] In some embodiments, the self-amplifying RNA includes at least one or more genes selected from any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins. In some embodiments, the self-amplifying RNA may also include 5'- and 3 '-end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0122] In some embodiments, the self-amplifying RNA molecule is not encapsulated in a viruslike particle. Self-amplifying RNA molecules described herein may be designed so that the self- amplifying RNA molecule cannot induce production of infectious viral particles. This may be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary to produce viral particles in the self-amplifying RNA. For example, when the selfamplifying RNA molecule is based on an alphavirus, such as Sinbis 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, may be omitted.

[0123] In some embodiments, a self-amplifying RNA molecule described herein encodes (i) an RNA- dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a viral antigen. In some embodiments, the polymerase may be an alphavirus replicase, e.g., including any one of alphavirus protein 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 selfamplifying RNA molecules does not include a modified nucleotide (e.g., pseudouridine, N6-methyladenosine, 5- methylcytidine, 5-methyluridine).

[0124] The saRNA construct may encode at least one non-structural protein (NSP), disposed 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 disposed 5’ of the sequences encoding the peptide or polypeptide of interest. Thus, the sequence encoding at least one NSP may be disposed 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 RNA 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 membrane anchor of the replication complex (RC). nsP2 is an RNA helicase and the protease responsible for the ns polyprotein processing. nsP3 interacts with several host proteins and may modulate protein poly- and mono-ADP-ribosylation. nsP4 is the core viral RNA-dependent RNA polymerase. In some embodiments, the polymerase may be an alphavirus replicase, e.g., comprising one or more of alphavirus proteins nsP1, nsP2, nsP3, and nsP4.

[0125] Whereas natural alphavirus genomes encode structural virion proteins in addition to the non- structural replicase polypeptide, in some embodiments, the self-amplifying RNA molecules do not encode alphavirus structural proteins. In some embodiments, the self-amplifying RNA may lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA that includes virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike a wild-type alphavirus, the self-amplifying RNA molecule cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wild-type viruses can be absent from self-amplifying RNAs of the present disclosure and their place can be taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins.

[0126] In some embodiments, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame can encode a replicase; the second (3') open reading frame can encode a polypeptide comprising an antigen of interest. In some embodiments the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode further antigens or to encode accessory polypeptides.

[0127] In some embodiments, the second RNA or the saRNA molecule further includes (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.

[0128] Optionally, self-amplifying RNA molecules described herein may also be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection.

[0129] In some embodiments, the saRNA molecule is alphavirus-based. Alphaviruses include a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Exemplary viruses and virus subtypes within the alphavirus genus include Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. As such, the self-amplifying RNA described herein may incorporate 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 alphavirus family. In some embodiments, the self-amplifying RNA described herein may incorporate sequences derived from a mutant or wild-type virus sequence, e.g., the attenuated TC83 mutant of VEEV has been used in saRNAs.

[0130] Alphavirus-based saRNAs are (+)-stranded saRNAs that may be translated after delivery to a cell, which leads to translation of a replicase (or replicase- transcriptase). The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic (-)-strand copies of the (+)-strand delivered RNA. These (-)-strand transcripts may themselves be transcribed to give further copies of the (+)-stranded parent RNA and also to give a subgenomic transcript which encodes the desired gene product. Translation of the subgenomic transcript thus leads to in situ expression of the desired gene product by the infected cell. Suitable alphavirus saRNAs may use a replicase from a sindbis virus, a semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, or mutant variants thereof.

[0131] In some embodiments, the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, such as a positive-stranded RNA virus, and in particular a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus. Suitable wildtype 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 encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR- 66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo 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), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250 ATCC VR- 1249, ATCC VR-532), Western equine encephalomyelitis (ATCC VR-70, ATCC VR- 1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375). In some aspects, one or more of the alphaviruses in the list may be excluded.

[0132] In some embodiments, the self-amplifying RNA molecules described herein are larger than other types of RNA (e.g., saRNA). Typically, the self-amplifying RNA molecules described herein include at least about 4 kb. For example, the self-amplifying RNA may be equal to any one of, at least any one of, at most any one of, or between any two 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. In some instances the self-amplifying RNA may include 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 about 4 kb to about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb.

[0133] In some embodiments, the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more of polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The polypeptides generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences. In some embodiments, the saRNA molecule may encode one polypeptide of interest or more, such as an antigen or more than one antigen, e.g., two, three, four, five, six, seven, eight, nine, ten, or more polypeptides. Alternatively, or in addition, one saRNA molecule may also encode more than one polypeptide of interest or more, such as an antigen, e.g., a bicistronic, or tricistronic RNA molecule that encodes different or identical antigens. An exemplary bicistronic saRNA encoding HA and NA include the sequence set forth in SEQ ID NO: 70 (published as SEQ ID NO: 9 of PCT / IB2023 / 057034, published as WQ2024 / 013625). Another exemplary bicistronic saRNA includes the the sequence set forth in SEQ ID NO: 71 (published as SEQ ID NO: 10 of PCT / IB2023 / 057034, published as WQ2024 / 013625). See, for example, International patent application PCT / IB2023 / 057034, published as WQ2024 / 013625, entitled, “Self-amplifying rna encoding an influenza virus antigen,” (Pfizer Inc.) filed on July 7, 2023, which is incorporated by reference in its entirety and describes saRNA molecules and bicistronic saRNA.

[0134] The term "linked" as used herein refers to a first amino acid sequence or polynucleotide sequence covalently or non-covalently joined to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence can be directly joined or juxtaposed to the second amino acid or polynucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. The term "linked" means not only a fusion of a first RNA molecule to a RNA molecule at the 5’-end or the 3’-end, but also includes insertion of the whole first RNA molecule into any two nucleotides in the second RNA molecule. The first second RNA molecule can be linked to a second RNA molecule by a phosphodiester bond or a linker. The linker can be, e.g., a polynucleotide.

[0135] In some embodiments, the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. In some embodiments, the self-amplifying RNA described herein may encode epitopes capable of eliciting either a helper T-cell response or a cytotoxic T-cell response or both.

[0136] In some embodiments, the saRNA molecule is purified, e.g., such as by filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration.

[0137] Some embodiments of the disclosure are directed to a composition comprising a selfamplifying RNA molecule comprising a 5’ Cap, a 5’ untranslated region, a coding region comprising a sequence encoding an RNA-dependent RNA polymerase (also referred to as a “replicase”), a subgenomic promoter, such as one derived from an alphavirus, an open reading frame encoding a gene of interest (e.g., an antigen derived from influenza virus), a 3’ untranslated region, and a 3’ poly A sequence. In some embodiments, at least 5% of a total population of a particular nucleotide in the saRNA molecule has been replaced with one or more modified or unnatural nucleotides.

[0138] In some embodiments, the saRNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below.

[0139] In some embodiments, the saRNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below. In some embodiments, the RNA may include a 5' cap comprising a 7'-methylguanosine, and the first 1, 2 or 35' ribonucleotides may be methylated at the 2' position of the ribose. In alternative embodiments, each RNA polynucleotide encoding a particular antigen is formulated in an individual LNP, such that each LNP encapsulates an RNA polynucleotide encoding identical antigens. Such embodiments may be referred herein as "post-mix". Accordingly, in some embodiments, the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; the third RNA polynucleotide is formulated in a third LNP; the fourth RNA polynucleotide is formulated in a fourth LNP; the fifth RNA polynucleotide is formulated in a fifth LNP; the sixth RNA polynucleotide is formulated in a sixth LNP; the seventh RNA polynucleotide is formulated in a seventh LNP; and the eighth RNA polynucleotide is formulated in an eighth LNP.

[0140] In some embodiments, the molar ratio of the first LNP to the second LNP in the mix of LNPs prior to formulation into LNPs 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.

[0141] In some embodiments, the molar ratio of the first LNP to the third LNP in the mix of LNPs prior to formulation into LNPs 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.

[0142] In some embodiments, the molar ratio of the first LNP to the fourth LNP in the mix of LNPs prior to formulation into LNPs 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 mix of LNPs prior to formulation into LNPs 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 mix of LNPs prior to formulation into LNPs 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 mix of LNPs prior to formulation into LNPs 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 mix of LNPs prior to formulation into LNPs 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.

[0143] Surprisingly, the inventors discovered that regardless of the process, the resulting ratio of RNA polynucleotide was comparable whether the plurality of RNA polynucleotides are mixed prior to formulation in an LNP (pre-mixed) or whether the RNA polynucleotides encoding a particular antigen is formulated in an individual LNP and the plurality of LNPs for different antigens are mixed (post-mixed). As a result of the discovery, there may be an option for medical professionals to mix and administer different ratios of antigens depending on the influenza season, particularly when the individual LNPs encapsulate RNA for a single antigen.

[0144] In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or 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 comprise a head domain. In some embodiments, the hemagglutinin protein comprises a portion of the head domain. In some embodiments, the hemagglutinin protein does not comprise a cytoplasmic domain. In some embodiments, the hemagglutinin protein comprises a portion of the cytoplasmic domain. In some embodiments, the truncated hemagglutinin protein comprises a portion of the transmembrane domain.

[0145] Some embodiments provide influenza vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a hemagglutinin protein and a pharmaceutically acceptable carrier or excipient, formulated within a cationic lipid nanoparticle. In some embodiments, the hemagglutinin protein is selected from H1, H7 and H10. In some embodiments, the RNA polynucleotide further encodes neuraminidase (NA) protein. In some embodiments, the hemagglutinin protein is derived from a strain of Influenza A virus or Influenza B virus or combinations thereof. In some embodiments, the Influenza virus is selected from H1N1, H3N2, H7N9, and H10N8.

[0146] In some embodiments, the virus is a strain of Influenza A or Influenza B or combinations thereof. In some embodiments, the strain of Influenza A or Influenza B is associated with birds, pigs, horses, dogs, humans, or non-human primates. In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or fragment thereof. In some embodiments, the hemagglutinin protein is H7 or H10 or a fragment thereof. In some embodiments, the hemagglutinin protein comprises a portion of the head domain (HA1). In some embodiments, the hemagglutinin protein comprises a portion of the cytoplasmic domain. In some embodiments, the truncated hemagglutinin protein. In some embodiments, the protein is a truncated hemagglutinin protein comprises a portion 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 polypeptides of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference protein 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length.

[0147] In some embodiments, an Influenza RNA composition includes an RNA encoding an antigenic fusion protein. Thus, the encoded antigen or antigens may include two or more proteins (e.g., protein and / or protein fragment) joined together. Alternatively, the protein to which a protein antigen is fused does not promote a strong immune response to itself, but rather to the influenza antigen. Antigenic fusion proteins, in some embodiments, retain the functional property from each original protein.

[0148] Some embodiments provide methods of preventing or treating influenza viral infection comprising administering to a subject any of the vaccines described herein. In some embodiments, the antigen specific immune response comprises a T cell response. In some embodiments, the antigen specific immune response comprises a B cell response. In some embodiments, the antigen specific immune response comprises both a T cell response and a B cell response. In some embodiments, the method of producing an antigen specific immune response involves a single administration of the vaccine. In some embodiments, the vaccine is administered to the subject by intradermal, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration.

[0149] In some embodiments, the RNA (e.g., mRNA) polynucleotides or portions thereof may encode one or more polypeptides or fragments thereof of an influenza strain as an antigen. mRNA vaccines of the disclosure

[0150] The present disclosure relates to mRNA vaccines in general. Several mRNA vaccine platforms are available in the prior art. The basic structure of in vitro transcribed (IVT) mRNA closely resembles “mature” eukaryotic mRNA and includes (i) a protein-encoding open reading frame (ORF), flanked by (ii) 5' and 3' untranslated regions (UTRs), and at the end sides (iii) a 7-methyl guanosine 5' cap structure and (iv) a 3' poly(A) tail. The non-coding structural features play important roles in the pharmacology of mRNA and can be individually optimized to modulate the mRNA stability, translation efficiency, and immunogenicity. By incorporating modified nucleosides, mRNA transcripts referred to as “nucleoside-modified mRNA” can be produced with reduced immunostimulatory activity, and therefore an improved safety profile can be obtained. In addition, modified nucleosides allow the design of mRNA vaccines with strongly enhanced stability and translation capacity, as they can avoid the direct antiviral pathways that are induced by type IFNs and are programmed to degrade and inhibit invading mRNA. For instance, the replacement of uridine with pseudouridine in IVT mRNA reduces the activity of 2'-5'-oligoadenylate synthetase, which regulates the mRNA cleavage by RNase L. In addition, lower activities are measured for protein kinase R, an enzyme that is associated with the inhibition of the mRNA translation process.

[0151] Besides the incorporation of modified nucleotides, other approaches have been validated to increase the translation capacity and stability of mRNA. One example is the development of “sequence-engineered mRNA”. Here, mRNA expression can be strongly increased by sequence optimizations in the ORF and UTRs of mRNA, for instance by enriching the GO content, or by selecting the UTRs of natural long-lived mRNA molecules. Another approach is the design of “self-amplifying mRNA” constructs. These are mostly derived from alphaviruses and contain an ORF that is replaced by the antigen of interest together with an additional ORF encoding viral replicase. The latter drives the intracellular amplification of mRNA and can therefore significantly increase the antigen expression capacity.

[0152] Also, several modifications have been implemented at the end structures of mRNA. Antireverse cap (ARCA) modifications can ensure the correct cap orientation at the 5' end, which yields almost complete fractions of mRNA that can efficiently bind the ribosomes. Other cap modifications, such as phosphorothioate cap analogs, can further improve the affinity towards the eukaryotic translation initiation factor 4E, and increase the resistance against the RNA decapping complex.

[0153] Conversely, by modifying its structure, the potency of mRNA to trigger innate immune responses can be further improved, but to the detriment of translation capacity. By stabilizing the mRNA with either a phosphorothioate backbone, or by its precipitation with the cationic protein protamine, antigen expression can be diminished, but stronger immune-stimulating capacities can be obtained.

[0154] In one aspect the invention relates to an immunogenic composition comprising an mRNA molecule that encodes one or more polypeptides or fragments thereof of an influenza strain as an antigen.

[0155] In some embodiments, the mRNA molecule comprises a nucleoside-modified mRNA. mRNA useful in the disclosure typically include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5 '-terminus of the first region (e.g., a 5 -UTR), a second flanking region located at the 3 '-terminus of the first region (e.g., a 3 -UTR), at least one 5 '-cap region, and a 3 '-stabilizing region. In some embodiments, the mRNA of the disclosure further includes a poly-A region or a Kozak sequence (e.g., in the 5 '-UTR). In some cases, mRNA of the disclosure may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, mRNA of the disclosure may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a poly A sequence, and / or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside). For example, the 3 '-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2'-0-methyl nucleoside and / or the coding region, 5 '-UTR, 3 '-UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5- methoxyuridine), a 1 -substituted pseudouridine (e.g., 1-methyl-pseudouridine), and / or a 5- substituted cytidine (e.g., 5-methyl-cytidine).

[0156] The compositions described herein comprise at least one RNA polynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, for example, is transcribed in vitro from template DNA, referred to as an “in vitro transcription template.” In some embodiments, an in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a polyA tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

[0157] A “5' untranslated region” (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.

[0158] In preferred embodiments, the 5’ UTR comprises SEQ ID NO: 1.

[0159] A “3' untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.

[0160] In preferred embodiments, the 3’ UTR comprises SEQ ID NO: 2.

[0161] An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG orTGA) and encodes a polypeptide.

[0162] A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation.

[0163] In preferred embodiments, the 3' polyadenylation tail comprises SEQ ID NO: 3.

[0164] In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides. For example, a polynucleotide may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides).

[0165] In some embodiments, a LNP includes one or more RNAs, and the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N: P ratio. The N: P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA. In general, a lower N: P ratio is preferred. The one or more RNA, lipids, and amounts thereof may be selected to provide an N: P ratio from about 2: 1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22: 1, 24: 1, 26: 1, 28: 1, or 30: 1. In certain embodiments, the N: P ratio may be from about 2: 1 to about 8: 1. In other embodiments, the N: P ratio is from about 5: 1 to about 8: 1. For example, the N: P ratio may be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N: P ratio may be about 5.67: 1.

[0166] mRNA of the disclosure may include one or more naturally occurring components, including any of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3 '-UTR, (d) the poly A tail, and any combination of (a, b, c, or d above) comprise naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).

[0167] mRNA of the disclosure may include one or more alternative components, as described herein, which impart useful properties including increased stability and / or the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced. For example, a modRNA may exhibit reduced degradation in a cell into which the modRNA is introduced, relative to a corresponding unaltered mRNA. These alternative species may enhance the efficiency of protein production, intracellular retention of the polynucleotides, and / or viability of contacted cells, as well as possess reduced immunogenicity.

[0168] mRNA of the disclosure may include one or more modified (e.g., altered or alternative) nucleobases, nucleosides, nucleotides, or combinations thereof. The mRNA useful in a LNP can include any useful modification or alteration, such as to the nucleobase, the sugar, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage I to the phosphodiester backbone). In certain embodiments, alterations (e.g., one or more alterations) are present in each of the nucleobase, the sugar, and the internucleoside linkage. Alterations according to the present disclosure may be alterations of ribonucleic acids (RNAs), e.g., the substitution of the 2'-OH of the ribofuranosyl ring to 2'-H, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or hybrids thereof. Additional alterations are described herein.

[0169] mRNA of the disclosure may or may not be uniformly altered along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may or may not be uniformly altered in a mRNA, or in a given predetermined sequence region thereof. In some instances, all nucleotides X in a mRNA (or in a given sequence region thereof) are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0170] Different sugar alterations and / or internucleoside linkages (e.g., backbone structures) may exist at various positions in a polynucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. An alteration may also be a 5'- or 3 '-terminal alteration. In some embodiments, the polynucleotide includes an alteration at the 3 '-terminus. The polynucleotide may contain from about 1% to about 100% alternative nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of a canonical nucleotide (e.g., A, G, U, or C).

[0171] Polynucleotides may contain at a minimum zero and at maximum 100% alternative nucleotides, or any intervening percentage, such as at least 5% alternative nucleotides, at least 10% alternative nucleotides, at least 25% alternative nucleotides, at least 50% alternative nucleotides, at least 80% alternative nucleotides, or at least 90% alternative nucleotides. For example, polynucleotides may contain an alternative pyrimidine such as an alternative uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in a polynucleotide is replaced with an alternative uracil (e.g., a 5-substituted uracil). The alternative uracil can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some instances, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the polynucleotide is replaced with an alternative cytosine (e.g., a 5-substituted cytosine). The alternative cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

[0172] In some instances, nucleic acids do not substantially induce an innate immune response of a cell into which the polynucleotide (e.g., mRNA) is introduced. Features of an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc., and / or 3) termination or reduction in protein translation.

[0173] In some embodiments, the mRNA comprises one or more alternative nucleoside or nucleotides. The alternative nucleosides and nucleotides can include an alternative nucleobase. A nucleobase of a nucleic acid is an organic base such as a purine or pyrimidine or a derivative thereof. A nucleobase may be a canonical base (e.g., adenine, guanine, uracil, thymine, and cytosine). These nucleobases can be altered or wholly replaced to provide polynucleotide molecules having enhanced properties, e.g., increased stability such as resistance to nucleases. Non-canonical or modified bases may include, for example, one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction.

[0174] In some embodiments, the nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having an alternative uracil include pseudouridine (ip), pyridin-4- one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio- uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy -uracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m3U), 5-methoxy-uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1 -carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm5s2U), 5-aminomethyl-2-thio-uracil (nm5s2u), 5-methylaminomethyl-uracil (mnm5U), 5-methylaminomethyl-2-thio-uracil (mnm5s2U), 5-methylaminomethyl-2-seleno-uracil (mnm5se2U), 5-carbamoylmethyl-uracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uracil (cmnm5s2U), 5-propynyl-uracil, 1- propynyl-pseudouracil, 5-taurinomethyl-uracil (xm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uracil(xm5s2U), 1 -taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1Ψ), 5-methyl-2- thio-uracil (m5s2U), 1-methyl-4-thio-pseudouridine (m1xΨ), 4-thio- 1-methyl-pseudouridine, 3- methyl-pseudouridine (m \| / ), 2 -thio- 1-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-thio-l -methyl- 1-deaza-pseudouri dine, dihydrouracil (D), dihydropseudouridine, 5,6- di hydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-th io- uracil, 4-methoxy- pseudouridine, 4-methoxy -2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ip), 5- (isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm5s2U), 5,2'-0-dimethyl-uridine (m5Um), 2-thio-2'-O_methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-0-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-0-methyl- uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm5Um), 3,2'-0- dimethyl-uridine (m Um), and 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm5Um), 1- thio-uracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil, and 5-[3-(l-E-propenylamino)]uracil. In some embodiments, the nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides having an alternative cytosine include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio-pseudoisocy tidine, 4-thio- 1 -methy 1-pseudoisocy tidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5 -methy 1- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5- methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1 -methyl-pseudoisocytidine, lysidine (k2C), 5,2'-0-dimethyl-cytidine (m5Cm), N4-acetyl-2'-0-methyl-cytidine (ac4Cm), N4,2'-0-dimethyl-cytidine (m4Cm), 5-formyl-2'-0-methyl-cytidine (f5Cm), N4, N4,2'-0- trimethyl-cytidine (m42Cm), 1 -thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine.

[0175] In some embodiments, the nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methy 1-adenine (ml A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl- adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6, N6-dimethyl- adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy -adenine, N6,2'-0-dimethyl-adenosine (m6Am), N6, N6,2'-0-trimethyl-adenosine (m62Am), l,2'-0-dimethyl-adenosine (ml Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0176] In some embodiments, the nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQi), archaeosine (G+), 7-deaza-8-aza-guanine, 6- thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6- thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1 -methyl-guanine (mIG), N2- methyl-guanine (m2G), N2, N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2, N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1 -methyl-6-thio- guanine, N2-methyl-6-thio-guanine, N2, N2-dimethyl-6-thio-guanine, N2-methyl-2'-0-methyl- guanosine (m2Gm), N2, N2-dimethyl-2'-0-methyl-guanosine (m22Gm), 1 -methyl-2'-0-methyl- guanosine (mIGm), N2,7-dimethyl-2'-0-methyl-guanosine (m2,7Gm), 2'-0-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 1 -thio-guanine, and O-6-methyl-guanine.

[0177] The alternative nucleobase of a nucleotide can be independently a purine, a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can be an alternative to adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3 -deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a] 1, 3, 5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; or 1,3,5 triazine. When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and / or derivatives thereof, e.g., A includes adenine or adenine analogs, e.g., 7-deaza adenine).

[0178] The mRNA may include a 5 '-cap structure. The 5 '-cap structure of a polynucleotide is involved in nuclear export and increasing polynucleotide stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for polynucleotide stability in the cell and translation competency through the association of CBP with poly -A binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5 '-proximal introns removal during mRNA splicing.

[0179] Endogenous polynucleotide molecules may be 5 '-end capped generating a 5 '-ppp-5' -triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the polynucleotide. This 5 '-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5 ' end of the polynucleotide may optionally also be 2'-0-methylated. 5 '-decapping through hydrolysis and cleavage of the guanylate cap structure may target a polynucleotide molecule, such as an mRNA molecule, for degradation. Alterations to polynucleotides may generate a non-hydrolyzable cap structure preventing decapping and thus increasing polynucleotide half-life. Because cap structure hydrolysis requires cleavage of 5 '-ppp-5' phosphorodiester linkages, alternative nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5 '-ppp-5 ' cap.

[0180] Additional alternative guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides. Additional alterations include, but are not limited to, 2'-0-methylation of the ribose sugars of 5'-terminal and / or 5 '-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxy group of the sugar. Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of an mRNA molecule.

[0181] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type, or physiological) 5 '-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / linked to a polynucleotide. For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanosines linked by a 5 '-5 '-triphosphate group, wherein one guanosine contains an N7-methyl group as well as a 3'-0-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7G-3'mppp-G, which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unaltered, guanosine becomes linked to the 5 '-terminal nucleotide of the capped polynucleotide (e.g., an mRNA). The N7- and 3'-0-methylated guanosine provides the terminal moiety of the capped polynucleotide (e.g., mRNA). Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5 '-triphosphate-5 '-guanosine, m7Gm- ppp-G).

[0182] A cap may be a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group such as the dinucleotide cap analogs described in US Patent No.

[0183] 8,519,110, the cap structures of which are herein incorporated by reference.

[0184] Alternatively, a cap analog may be a N7-(4-chlorophenoxy ethyl) substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxy ethyl) substituted dinucleotide cap analogs include a N7-(4-chlorophenoxyethyl)-G(5 )ppp(5 ')G and a N7-(4-chlorophenoxyethyl)-m3 '-OG(5 )ppp(5 ')G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the cap structures of which are herein incorporated by reference). In other instances, a cap analog useful in the polynucleotides of the present disclosure is a 4-chloro / bromophenoxy ethyl analog.

[0185] While cap analogs allow for the concomitant capping of a polynucleotide in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from endogenous 5 '-cap structures of polynucleotides produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.

[0186] Alternative polynucleotides may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function, and / or structure as compared to synthetic features or analogs of the prior art, or which outperforms the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects. Non-limiting examples of more authentic 5 '-cap structures useful in the polynucleotides of the present disclosure are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5'-endonucleases, and / or reduced 5'-decapping, as compared to synthetic 5 '-cap structures known in the art (or to a wild-type, natural or physiological 5 '-cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5 '-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide wherein the cap guanosine contains an N7-methylation and the 5 '-terminal nucleotide of the polynucleotide contains a 2'-0-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency, cellular stability, and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 ' cap analog structures known in the art. Other exemplary cap structures include 7mG(5 ')ppp(5 ')N,pN2p (Cap 0), 7mG(5 ')ppp(5 ')NlmpNp (Cap 1), 7mG(5 ')-ppp(5')NlmpN2mp (Cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (Cap 4).

[0187] Because the alternative polynucleotides may be capped post-transcriptionally, and because this process is more efficient, nearly 100% of the mRNA may be capped. This is in contrast to -80% when a cap analog is linked to a polynucleotide in the course of an in vitro transcription reaction. 5 '-terminal caps may include endogenous caps or cap analogs. A 5 '-terminal cap may include a guanosine analog. Useful guanosine analogs include inosine, N1-methyl- guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine. In some cases, a polynucleotide contains a modified 5 '-cap. A modification on the 5 '-cap may increase the stability of polynucleotide, increase the half-life of the polynucleotide, and could increase the polynucleotide translational efficiency. The modified 5 '-cap may include, but is not limited to, one or more of the following modifications: modification at the 2'- and / or 3 '-position of a capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH2), a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety. A 5'-UTR may be provided as a flanking region to the mRNA. A 5’ -UTR may be homologous or heterologous to the coding region found in a polynucleotide. Multiple 5 '-UTRs may be included in the flanking region and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical alterations, before and / or after codon optimization.

[0188] In one embodiment, an ORF encoding an antigen of the disclosure is codon optimized. Codon optimization methods are known in the art. For example, an ORF of any one or more of the sequences provided herein may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art — non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms. To alter one or more properties of an mRNA, 5 '-UTRs which are heterologous to the coding region of an mRNA may be engineered. The mRNA may then be administered to cells, tissue or organisms and outcomes such as protein level, localization, and / or half-life may be measured to evaluate the beneficial effects the heterologous 5 ' -UTR may have on the mRNA. Variants of the 5 '-UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G. 5 '-UTRs may also be codon-optimized, or altered in any manner described herein.

[0189] mRNAs may include a stem loop such as, but not limited to, a histone stem loop. The stem loop may be a nucleotide sequence that is about 25 or about 26 nucleotides in length. The histone stem loop may be located 3 '-relative to the coding region (e.g., at the 3 '-terminus of the coding region). As a non-limiting example, the stem loop may be located at the 3 '-end of a polynucleotide described herein. In some cases, an mRNA includes more than one stem loop (e.g., two stem loops). A stem loop may be located in a second terminal region of a polynucleotide. As a non-limiting example, the stem loop may be located within an untranslated region (e.g., 3'-UTR) in a second terminal region. In some cases, a mRNA which includes the histone stem loop may be stabilized by the addition of a 3 '-stabilizing region (e.g., a 3'-stabilizing region including at least one chain terminating nucleoside). Not wishing to be bound by theory, the addition of at least one chain terminating nucleoside may slow the degradation of a polynucleotide and thus can increase the half-life of the polynucleotide. In other cases, a mRNA, which includes the histone stem loop may be stabilized by an alteration to the 3 '-region of the polynucleotide that can prevent and / or inhibit the addition of oligio(U). In yet other cases, a mRNA, which includes the histone stem loop may be stabilized by the addition of an oligonucleotide that terminates in a 3 '-deoxynucleoside, 2', 3 '-dideoxynucleoside 3 '-0-methylnucleosides, 3 -0- ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and / or described herein. In some instances, the mRNA of the present disclosure may include a histone stem loop, a poly-A region, and / or a 5 '-cap structure. The histone stem loop may be before and / or after the poly-A region. The polynucleotides including the histone stem loop and a poly-A region sequence may include a chain terminating nucleoside described herein. In other instances, the polynucleotides of the present disclosure may include a histone stem loop and a 5 '-cap structure. The 5 '-cap structure may include, but is not limited to, those described herein and / or known in the art. In some cases, the conserved stem loop region may include a miR sequence described herein. As a non-limiting example, the stem loop region may include the seed sequence of a miR sequence described herein. In another non-limiting example, the stem loop region may include a miR- 122 seed sequence. mRNA may include at least one histone stem-loop and a poly-A region or polyadenylation signal. In certain cases, the polynucleotide encoding for a histone stem loop and a poly-A region or a polyadenylation signal may code for a pathogen antigen or fragment thereof. In other cases, the polynucleotide encoding for a histone stem loop and a poly-A region or a polyadenylation signal may code for a therapeutic protein. In some cases, the polynucleotide encoding for a histone stem loop and a poly-A region or a polyadenylation signal may code for a tumor antigen or fragment thereof. In other cases, the polynucleotide encoding for a histone stem loop and a poly-A region or a polyadenylation signal may code for an allergenic antigen or an autoimmune self-antigen.

[0190] An mRNA may include a polyA sequence and / or polyadenylation signal. A polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof. A polyA sequence may be a tail located adjacent to a 3' untranslated region of a nucleic acid. During RNA processing, a long chain of adenosine nucleotides (poly-A region) is normally added to messenger RNA (mRNA) molecules to increase the stability of the molecule.

[0191] Immediately after transcription, the 3'-end of the transcript is cleaved to free a 3'-hydroxy. Then poly-A polymerase adds a chain of adenosine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A region that is between 100 and 250 residues long. Unique poly-A region lengths may provide certain advantages to the alternative polynucleotides of the present disclosure. Generally, the length of a poly-A region of the present disclosure is at least 30 nucleotides in length. In another embodiment, the poly-A region is at least 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 70 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1700 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 1900 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In some instances, the poly-A region may be 80 nucleotides, 120 nucleotides, 160 nucleotides in length on an alternative polynucleotide molecule described herein. In other instances, the poly-A region may be 20, 40, 80, 100, 120, 140 or 160 nucleotides in length on an alternative polynucleotide molecule described herein. In some cases, the poly-A region is designed relative to the length of the overall alternative polynucleotide. This design may be based on the length of the coding region of the alternative polynucleotide, the length of a particular feature or region of the alternative polynucleotide (such as mRNA) or based on the length of the ultimate product expressed from the alternative polynucleotide. When relative to any feature of the alternative polynucleotide (e.g., other than the mRNA portion which includes the poly-A region) the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than the additional feature. The poly-A region may also be designed as a fraction of the alternative polynucleotide to which it belongs. In this context, the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A region. In certain cases, engineered binding sites and / or the conjugation of mRNA for poly-A binding protein may be used to enhance expression. The engineered binding sites may be sensor sequences which can operate as binding sites for ligands of the local microenvironment of the mRNA. As a non-limiting example, the mRNA may include at least one engineered binding site to alter the binding affinity of poly-A binding protein (PABP) and analogs thereof. The incorporation of at least one engineered binding site may increase the binding affinity of the PABP and analogs thereof.

[0192] Additionally, multiple distinct mRNA may be linked together to the PABP (poly-A binding protein) through the 3'-end using alternative nucleotides at the 3'- terminus of the poly-A region. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12 hours, 24 hours, 48 hours, 72 hours, and day 7 post-transfection. As a non-limiting example, the transfection experiments may be used to evaluate the effect on PABP or analogs thereof binding affinity as a result of the addition of at least one engineered binding site. In certain cases, a poly-A region may be used to modulate translation initiation. While not wishing to be bound by theory, the poly-A region recruits PABP which in turn can interact with translation initiation complex and thus may be essential for protein synthesis. In some cases, a poly-A region may also be used in the present disclosure to protect against 3 '-5 '-exonuclease digestion. In some instances, an mRNA may include a polyA-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A region. The resultant mRNA may be assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A region of 120 nucleotides alone. In some cases, mRNA may include a poly-A region and may be stabilized by the addition of a 3 '-stabilizing region. The mRNA with a poly-A region may further include a 5 '-cap structure. In other cases, mRNA may include a poly-A-G Quartet. The mRNA with a poly-A-G Quartet may further include a 5 '-cap structure. In some cases, the 3 '-stabilizing region which may be used to stabilize mRNA includes a poly-A region or poly-A-G Quartet. In other cases, the 3 '-stabilizing region which may be used with the present disclosure include a chain termination nucleoside such as 3 '-deoxyadenosine (cordycepin), 3 '-deoxyuridine, 3 '- deoxycytosine, 3 '-deoxyguanosine, 3 '-deoxy thymine, 2', 3'-dideoxynucleosides, such as 2', 3 '- dideoxyadenosine, 2', 3 '-dideoxyuridine, 2', 3 '-dideoxycytosine, 2', 3 '- dideoxyguanosine, 2', 3 '-dideoxythymine, a 2'-deoxynucleoside, or an O-methylnucleoside. In other cases, mRNA which includes a polyA region or a poly-A-G Quartet may be stabilized by an alteration to the 3 '-region of the polynucleotide that can prevent and / or inhibit the addition of oligio(U). In yet other instances, mRNA which includes a poly-A region or a poly-A-G Quartet may be stabilized by the addition of an oligonucleotide that terminates in a 3 '-deoxynucleoside, 2', 3 '-dideoxynucleoside 3 -O- methylnucleosides, 3 '-O- ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and / or described herein.

[0193] In an embodiment, the mRNA vaccines of the disclosure comprise lipids. The lipids and modRNA can together form nanoparticles. The lipids can encapsulate the mRNA in the form of a lipid nanoparticle (LNP) to aid cell entry and stability of the RNA / lipid nanoparticles.

[0194] Lipid nanoparticles may include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic. A LNP may be designed for one or more specific applications or targets. The elements of a LNP may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a LNP may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combinations of elements. The efficacy and tolerability of a LNP formulation may be affected by the stability of the formulation.

[0195] Lipid nanoparticles may be designed for one or more specific applications or targets. For example, a LNP may be designed to deliver a therapeutic and / or prophylactic such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal's body.

[0196] Physiochemical properties of lipid nanoparticles may be altered to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs. The therapeutic and / or prophylactic included in a LNP may also be selected based on the desired delivery target or targets. For example, a therapeutic and / or prophylactic may be selected for a particular indication, condition, disease, or disorder and / or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery). In certain embodiments, a LNP may include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest. Such a composition may be designed to be specifically delivered to a particular organ. In some embodiments, a composition may be designed to be specifically delivered to a mammalian liver. In some embodiments, a composition may be designed to be specifically delivered to a lymph node. In some embodiments, a composition may be designed to be specifically delivered to a mammalian spleen.

[0197] A LNP may include one or more components described herein. In some embodiments, the LNP formulation of the disclosure includes at least one lipid nanoparticle component. Lipid nanoparticles may include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic, such as a nucleic acid. A LNP may be designed for one or more specific applications or targets. The elements of a LNP may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a LNP may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combination of elements. The efficacy and tolerability of a LNP formulation may be affected by the stability of the formulation.

[0198] In some embodiments, for example, a polymer may be included in and / or used to encapsulate or partially encapsulate a LNP. A polymer may be biodegradable and / or biocompatible. A polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, a polymer may include 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), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEG), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, 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 copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, 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), and polyglycerol.

[0199] Surface altering agents may 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), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin (34, dornase alfa, neltenexine, and erdosteine), and DNases (e.g., rhDNase). A surface altering agent may be disposed within a nanoparticle and / or on the surface of a LNP (e.g., by coating, adsorption, covalent linkage, or other process).

[0200] A LNP may also comprise one or more functionalized lipids. For example, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction. In particular, a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition, or imaging. The surface of a LNP may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0201] In addition to these components, lipid nanoparticles may include any substance useful in pharmaceutical compositions. For example, the lipid nanoparticle may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, surface active agents, buffering agents, preservatives, and other species.

[0202] Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxy ethylene sorbitan monolaurate [TWEENO20], polyoxy ethylene sorbitan [TWEEN® 60], polyoxy ethylene 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 [BRU® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0203] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, free radical scavengers, antimicrobial 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 hydroxy toluene, 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 antimicrobial 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.

[0204] Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, 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. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™, KATHON™, and / or EUXYL®. An exemplary free radical scavenger includes butylated hydroxytoluene (BHT or butylhydroxytoluene) or deferoxamine.

[0205] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, 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 mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, Tris buffer, and / or combinations thereof.

[0206] In some embodiments, the formulation including a LNP may further include a salt, such as a chloride salt. In some embodiments, the formulation including a LNP may further includes a sugar such as a disaccharide. In some embodiments, the formulation further includes a sugar but not a salt, such as a chloride salt. In some embodiments, a LNP may further include one or more small hydrophobic molecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof). The characteristics of a LNP may depend on the components thereof. For example, a LNP including cholesterol as a structural lipid may have different characteristics than a LNP that includes a different structural lipid. As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties. As defined herein, “sterols” are 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.

[0207] The lipid nanoparticle compositions may include one or more structural lipids.

[0208] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, p-sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are 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 certain embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is alpha-tocopherol. In some preferred embodiments, the

[0209] (SN792). In some preferred embodiments, the

[0210]

[0211] embodiments, the sterol comprises stigmasterol, In some preferred embodiments, the sterol comprises sitostanol.

[0212] In some embodiments, the structural lipid is a sitosterol, a stigmasterol, a campesterol, a sitostanol, a campestanol, a brassicasterol, a fucosterol, beta-sitosterol, stigmastanol, beta-sitostanol, ergosterol, lupeol, cycloartenol, A5-avenaserol, A7-avenaserol or a A7-stigmasterol, including analogs, salts or esters thereof, alone or in combination. In some embodiments, the sterol component of a LNP of the disclosure is a single phytosterol. In some embodiments, the phytosterol component of a LNP of the disclosure is a mixture of different phytosterols (e.g. 2, 3, 4, 5 or 6 different phytosterols). In some embodiments, the phytosterol component of an LNP of the disclosure is a blend of one or more phytosterols and one or more zoosterols, such as a blend of a phytosterol (e.g., a sitosterol, such as beta-sitosterol) and cholesterol. In some embodiments, the phytosterol is p-sitosterol, campesterol, sigmastanol, or any combination thereof. In some embodiments, the phytosterol is p-sitosterol. In some embodiments, the one or more structural lipids comprises a mixture of p-sitosterol, campesterol, and stigmasterol. In some embodiments, the one or more structural lipids comprises a mixture of p-sitosterol and cholesterol. In one embodiment, the structural lipid is selected from selected from p-sitosterol and cholesterol. In an embodiment, the structural lipid is p-sitosterol. In an embodiment, the structural lipid is cholesterol.

[0213] In some embodiments, the one or more structural lipids comprises about 35% to about 85% of p-sitosterol, about 5% to about 35% stigmasterol, and about 5% to about 35% of campesterol. In some embodiments, the one or more structural lipids comprises about 40% to about 80% of p-sitosterol, about 10% to about 30% stigmasterol, and about 10% to about 30% of campesterol. In some embodiments, the one or more structural lipids comprises about 40% to about 70% of p-sitosterol, about 10% to about 25% stigmasterol, and about 10% to about 25% of campesterol. In some embodiments, the one or more structural lipids comprises about 40% to about 70% of p-sitosterol, about 15% to about 25% stigmasterol, and about 15% to about 25% of campesterol. In some embodiments, the one or more structural lipids comprises about 35% to about 45% of p-sitosterol, about 20% to about 30% stigmasterol, and about 20% to about 30% of campesterol. In some embodiments, the one or more structural lipids comprises about 40% to about 50% of p-sitosterol, about 25% to about 35% stigmasterol, and about 25% to about 35% of campesterol. In some embodiments, the one or more structural lipids comprises about 65% to about 75% of p-sitosterol, about 5% to about 15% stigmasterol, and about 5% to about 15% of campesterol. In some embodiments, the one or more structural lipids comprises about 35% to about 85% of p-sitosterol, about 5% to about 35% stigmasterol, and 0% of campesterol. In some embodiments, the one or more structural lipids comprises about 40% to about 80% of p-sitosterol, about 10% to about 30% stigmasterol, and 0% of campesterol. In some embodiments, the one or more structural lipids comprises about 40% to about 70% of p-sitosterol, about 10% to about 25% stigmasterol, and 0% of campesterol. In some embodiments, the one or more structural lipids comprises about 40% to about 70% of p-sitosterol, about 15% to about 25% stigmasterol, and 0% of campesterol. In some embodiments, the one or more structural lipids comprises about 35% to about 45% of p-sitosterol, about 20% to about 30% stigmasterol, and 0% of campesterol. In some embodiments, the one or more structural lipids comprises about 40% to about 50% of p-sitosterol, about 25% to about 35% stigmasterol, and 0% of campesterol. In some embodiments, the one or more structural lipids comprises about 65% to about 75% of p-sitosterol, about 5% to about 15% stigmasterol, and 0% of campesterol. Accordingly, in some preferred embodiments, the composition does not comprise campesterol. In some embodiments, the composition comprises one or more structural lipids comprises about 10% to about 30% of cholesterol, about 10% to about 30% p-sitosterol, and about 10% to about 30% stigmasterol, and 0% campesterol. See, for example, Error! Reference source not found.. In some embodiments, the composition further comprises about 30-50% cationic lipid and about 5-25% phospholipid.

[0214] In some embodiments, the mol % of the one or more structural lipids is between about 1% and 50% of the mol % of the compound having the structure of any of the foregoing compounds present in the lipid nanoparticle. In some embodiments, the mol % of the one or more structural lipids is between about 10% and 40% of the mol % of the compound having the structure of any of the foregoing compounds present in the lipid nanoparticle. In some embodiments, the mol % of the one or more structural lipids is between about 20% and 30% of the mol % of the compound having the structure of any of the foregoing compounds present in the lipid nanoparticle. In some embodiments, the mol % of the one or more structural lipids is about 30% of the mol % of the compound having the structure of any of the foregoing compounds present in the lipid nanoparticle.

[0215] In some embodiments, the lipid nanoparticle compositions described herein can comprise about 20 mol% to about 60 mol% structural lipid. In some embodiments, the lipid nanoparticle compositions comprise about 30 mol% to about 50 mol% of structural lipid. In some embodiments, the lipid nanoparticle compositions comprise about 35 mol% to about 45 mol% of structural lipid. In some embodiments, the lipid nanoparticle compositions comprise about 37 mol% to about 42 mol% of structural lipid. In some embodiments, the lipid nanoparticle compositions comprise about 35, about 36, about 37, about 38, about 39, or about 40 mol% of structural lipid. In some embodiments, the nanoparticle comprises about 39 to about 40 mol% structural lipid.

[0216] In some embodiments, a LNP of the invention comprises an N: P ratio of from about 2:1 to about 30:1. In some embodiments, a LNP of the invention comprises an N: P ratio of about 6:1. In some embodiments, a LNP of the invention comprises an N: P ratio of about 3:1, 4:1, or 5:1.

[0217] In some embodiments, the characteristics of a LNP may depend on the absolute or relative amounts of its components. For instance, a LNP including a higher molar fraction of a phospholipid may have different characteristics than a LNP including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the lipid nanoparticle. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0218] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin (SM). Further examples of a phospholipid moiety for the lipid nanoparticle include a lipid that is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidyl serine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), diemcoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1, 2-dilauroyl-sn-glycero-3 -pho sphoethanolamine (DLPE); l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. In some embodiments, the lipid nanoparticle includes sphingomyelin. In some embodiments, the nanoparticle composition comprising a plurality of lipid nanoparticles, wherein the lipid nanoparticles comprise: (a) a sphingomyelin of about 5 to 40 mol percent of the total lipid present in the nanoparticle composition; (b) a cationic lipid; (c) a steroid; (d) a polymer conjugated lipid; and (e) a nucleic acid. In some embodiments, the sphingomyelin is about 10 to 40 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 to 30 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 to 25 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 to 20 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 to 15 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 15 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 20 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 to 20 mol percent of the total lipid present in the nanoparticle composition, and wherein the cationic lipid is about 40 to 50 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 to 15 mol percent of the total lipid present in the nanoparticle composition, and wherein the cationic lipid is about 45 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 to 15 mol percent of the total lipid present in the nanoparticle composition, and wherein the cationic lipid is about 40 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 mol percent of the total lipid present in the nanoparticle composition, and wherein the cationic lipid is about 50 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 10 mol percent of the total lipid present in the nanoparticle composition, and wherein the cationic lipid is about 45 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is about 15 mol percent of the total lipid present in the nanoparticle composition, and wherein the cationic lipid is about 45 mol percent of the total lipid present in the nanoparticle composition. In some embodiments, the sphingomyelin is a sphingomyelin compound. In some embodiments, the sphingomyelin is selected from SM-01, SM-02, SM-03, SM-04, SM-05, SM-06 and SM-07. In some embodiments, the molar percentage of sphingomyelin in the total lipid present in the nanoparticle composition is the same as the molar percentage of DSPC in the total lipid present in a reference nanoparticle composition. In some embodiments, the sphingomyelin is of about 5 to 40 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin is of about 10 to 40 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin is of about 10 to 30 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin is of about 10 to 25 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin is of about 10 to 20 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin is of about 10 to 15 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin is of about 10 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin is of about 15 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin is of about 20 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin is of about 5 mol percent, about 6 mol percent, about 7 mol percent, about 8 mol percent, about 9 mol percent, about 10 mol percent, about 11 mol percent, about 11.5 mol percent, about 12 mol percent, about 12.5 mol percent, about 3 mol percent, about 3.5 mol percent, about 14 mol percent, about 14.5 mol percent, about 15 mol percent, about 15.5 mol percent, about 16 mol percent, about 16.5 mol percent, about 17 mol percent, about 17.5 mol percent, about 18 mol percent, about 18.5 mol percent, about 19 mol percent, about 19.5 mol percent, about 20 mol percent, about 21 mol percent, about 22 mol percent, about 23 mol percent, about 24 mol percent, about 25 mol percent, about 30 mol percent, about 35 mol percent, or about 40 mol percent of the total lipid present in the composition. In some embodiments, the sphingomyelin in the composition is a sphingomyelin compound having the following structure:

[0219]

[0220] wherein R is an alkyl or alkenyl. In one embodiment, R is a C n-C 23 alkyl. In one embodiment, R is a C 11-C 19 alkyl. In one embodiment, R is a C 13-C 19 alkyl. In one embodiment, R is a C 15-C 19 alkyl. In one embodiment, R is a C u alkyl (e.g., - (CH 2) 10-CH 3). In one embodiment, R is a C 13 alkyl (e.g., - (CH 2) 12-CH 3). In one embodiment, R is aC u alkyl (e.g., - (CH 2) 13-CH 3). In one embodiment, R is a C 15 alkyl (e.g., - (CH 2) u-CH 3). In one embodiment, R is a

[0221] C is alkyl (e.g., - (CH 2) 15-CH 3). In one embodiment, R is a C 7 alkyl (e.g., - (CH 2) le-CH 3). In one embodiment, R is a C u alkyl (e.g., - (CH 2) 17-CH 3). In one embodiment, R is a C 19 alkyl (e.g., - (CH 2) 18-CH 3). In one embodiment, R is a C 20 alkyl (e.g., - (CH 2) 19-CH 3). In one embodiment, R is a C 21 alkyl (e.g., - (CH 2) 20-CH 3). In one embodiment, R is a C 22 alkyl (e.g., - (CH 2) 21-CH 3). In some embodiments, R is a C 23 alkyl (e.g., - (CH 2) 22-CH 3). In one embodiment, the alkyl is a straight alkyl. In one embodiment, the alkyl is a branched alkyl. In some embodiments, the alkyl is unsubstituted. In some embodiments, the sphingomyelin provided herein is selected from the SM-01, SM-02, SM-03, SM-06 and SM-07 molecules shown in Table 51. In some embodiments, R is a C n-C 23 alkenyl. In one embodiment, R is a C 13-C 19 alkenyl. In one embodiment, R is a C 15-C 19 alkenyl. In one embodiment, R is a C 11 alkenyl. In one embodiment, R is a C 13 alkenyl. In one embodiment, R is a C u alkenyl. In one embodiment, R is a C 15 alkenyl. In one embodiment, R is a C alkenyl. In one embodiment, R is a C 17 alkenyl. In one embodiment, R is a C alkenyl. In one embodiment, R is a C 19 alkenyl. In one embodiment, R is a C 20 alkenyl. In one embodiment, R is a

[0222] C 21 alkenyl. In one embodiment, R is a C 22 alkenyl. In one embodiment, R is a C 23 alkenyl. In one embodiment, the alkenyl has one double bond. In one embodiment, the double bond has a Z-configuration. In one embodiment, the double bond is at 9-position of the alkenyl R group. In one embodiment, the alkenyl is a straight alkenyl. In one embodiment, the alkenyl is a branched alkenyl. In one embodiment, the alkenyl is unsubstituted.

[0223] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. In some embodiments, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. In some embodiments, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidyl-ethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. In some embodiments, a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC.

[0224] Lipid nanoparticles may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a LNP. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a LNP, such as particle size, polydispersity index, and zeta potential.

[0225] The mean size of a LNP may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may 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 mean size of a LNP may 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 mean size of a LNP may be from about 70 nm to about 100 nm. In a particular embodiment, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm.

[0226] A LNP may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index 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 a LNP may be from about 0.10 to about 0.20.

[0227] The zeta potential of a LNP may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may 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.

[0228] The efficiency of encapsulation of a therapeutic and / or prophylactic describes the amount of therapeutic and / or prophylactic that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and / or prophylactic in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and / or prophylactic (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a therapeutic and / or prophylactic may be at least 50%, for example 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 may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0229] A LNP may optionally comprise one or more coatings. For example, a LNP may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[0230] Formulations comprising amphiphilic polymers and lipid nanoparticles may be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may include one or more amphiphilic polymers and one or more lipid nanoparticles. For example, a pharmaceutical composition may include one or more amphiphilic polymers and one or more lipid nanoparticles including one or more different therapeutics and / or prophylactics. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents 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 accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a LNP or the one or more amphiphilic polymers in the formulation of the disclosure. An excipient or accessory ingredient may be incompatible with a component of a LNP or the amphiphilic polymer of the formulation if its combination with the component or amphiphilic polymer may result in any undesirable biological effect or otherwise deleterious effect.

[0231] In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a LNP. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia. Relative amounts of the one or more amphiphilic polymers, the one or more lipid nanoparticles, the one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more lipid nanoparticles. As another example, a pharmaceutical composition may comprise between 0.1% and 15% (wt / vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% w / v).

[0232] In certain embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and / or shipment (e.g., being stored at a temperature of 4 °C or lower, such as a temperature 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 example, the pharmaceutical composition comprising one or more amphiphilic polymers and one or more lipid nanoparticles is a solution or solid (e.g., via lyophilization) that is refrigerated for storage and / or shipment at, for example, about -20 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, or -80 °C. In certain embodiments, the disclosure also relates to a method of increasing stability of the lipid nanoparticles by adding an effective amount of an amphiphilic polymer and by storing the lipid nanoparticles and / or pharmaceutical compositions thereof at a temperature of 4 °C or lower, such as a temperature 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).

[0233] The chemical properties of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure may be characterized by a variety of methods. In some embodiments, electrophoresis (e.g., capillary electrophoresis) or chromatography (e.g., reverse phase liquid chromatography) may be used to examine the mRNA integrity.

[0234] The efficacy of the product is dependent on expression of the delivered RNA, which requires a sufficiently intact RNA molecule. RNA integrity is a measure of RNA quality that quantitates intact RNA. The method is also capable of detecting potential degradation products. RNA integrity is preferably determined by capillary gel electrophoresis. The initial specification is set to ensure sufficient RNA integrity in drug product preparations. In some embodiments, the RNA polynucleotide has an integrity of at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the RNA polynucleotide has an integrity of or greater than about 95%. In some embodiments, the RNA polynucleotide has an integrity of or greater than about 98%. In some embodiments, the RNA polynucleotide has an integrity of or greater than about 99%.

[0235] In preferred embodiments, the RNA polynucleotide has a 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, wherein the purified mRNA has a clinical grade purity without further purification. In some embodiments, the clinical grade purity is achieved through a method including tangential flow filtration (TFF) purification. In some embodiments, the clinical grade purity is achieved without the further purification selected from high performance liquid chromatography (HPLC) purification, ligand or binding based purification, and / or ion exchange chromatography. In some embodiments, the method of producing the RNA polynucleotides removes long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA residual solvent and / or residual salt. In some embodiments, the short abortive transcript contaminants comprise less than 15 bases. In some embodiments, the short abortive transcript contaminants comprise about 8-12 bases. In some embodiments, the method of the invention also removes RNAse inhibitor.

[0236] In some embodiments, the purified RNA polynucleotide comprises 5% or less, 4% or less, 3% or less, 2% or less, 1 % or less or is substantially free of protein contaminants as determined by capillary electrophoresis. In some embodiments, the purified RNA polynucleotide comprises less than 5%, less than 4%, less than 3%, less than 2%, less than 1 %, or is substantially free of salt contaminants determined by high performance liquid chromatography (HPLC). In some embodiments, the purified RNA polynucleotide comprises 5% or less, 4% or less, 3% or less, 2% or less, 1 % or less or is substantially free of short abortive transcript contaminants determined by known methods, such as, e.g., high performance liquid chromatography (HPLC). In some embodiments, the purified RNA polynucleotide has integrity of 60% or greater, 70% or greater, 80% or greater, 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater as determined by a known method, such as, e.g., capillary electrophoresis.

[0237] Modified nucleobases

[0238] Modified nucleobases which may be incorporated into modified nucleosides and nucleotides and be present in the RNA molecules include, for example, m5C (5- methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2- thiouridine), Um (2'-0-methyluridine), mlA (1 -methyladenosine); m2A (2- methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6- glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2- methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6- threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-0-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (l,2'-0- dimethylinosine); m3C (3-methylcytidine); Cm (2T-0-methylcytidine); s2C (2- thiocytidine); ac4C (N4-acetylcytidine); £5C (5-fonnylcytidine); m5Cm (5,2-0- dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); mIG (1- methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-0- methylguanosine); m22G (N2, N2-dimethylguanosine); m2Gm (N2,2'-0- dimethylguanosine); m22Gm (N2, N2,2'-0-trimethylguanosine); Gr(p) (2'-0- ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl- queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7- aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-0-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-methoxycarbonyl methyluridine); mcm5Um (5-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-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-0-methyluridine); cmnm5U (5- carboxymethylaminomethyluridine); cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6, N6-dimethyladenosine); Tm (2'-0-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-0-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6, T-0-dimethyladenosine); rn62Am (N6, N6,0-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2, N2,7- trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-0-methylcytidine); mIGm (l,2'-0-dimethylguanosine); m'Am (1,2-0- dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG- 14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(Ci-C6)-alkyluracil, 5- methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(Ci-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-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or2'-O-methyl-U. In some aspects, one or more of the modified nucleosides in the list may be excluded.

[0239] Additional exemplary modified nucleotides include any one of N-1-methylpseudouridine; pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine. In some embodiments, the modified nucleotide is N-1 -methylpseudouridine.

[0240] In some embodiments, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0241] In some embodiments, the RNA molecule includes 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-methyl uridine. In some embodiments, the modified or unnatural nucleotides are 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-methyl uridine. In some embodiments, the modified or unnatural nucleotides are selected from the group consisting of 5-methyluridine, N1-methylpseudouridine, 5-methoxyuridine, and 5-methylcytosine.

[0242] In some embodiments, at least 10% of a total population of a particular nucleotide in the saRNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, essentially all of the particular nucleotide population in the molecule has been replaced with one or more modified or unnatural nucleotides.

[0243] In some embodiments, at least a portion, or all of a total population of a particular nucleotide in the saRNA molecule has been replaced with two modified or unnatural nucleotides. In some embodiments, the two modified or unnatural nucleotides are provided in a ratio equal to any one of, at least any one of, at most any one of, or between any two of 1:99 to 99:1, including 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 range derivable therein.

[0244] In some embodiments, at least 10% of a total population of a first particular nucleotide in a saRNA molecule as disclosed herein has been replaced with one or more modified or unnatural nucleotides, and at least 10% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 25% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 25% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, essentially all of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.

[0245] In some embodiments, at least 25% of a total population of uridine nucleotides in the saRNA molecule has been replaced with N1 -methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 75% of a total population of uridine nucleotides in the molecule has been replaced with N1 -methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 5-methoxyuridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methyluridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 5-methyluridine. In some embodiments, at least 50% of a total population of cytosine nucleotides in the molecule has been replaced with 5-methylcytosine. In some embodiments, essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 2-thiouridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 2-thiouridine.

[0246] In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with N1 -methylpseudouridine and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methoxyuridine and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methyluridine and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine.

[0247] In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with about 75% 5-methoxyuridine and about 25% N1 -methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with about 25% 5-methoxyuridine and about 75% N1 -methylpseudouridine.

[0248] UTRs

[0249] The 5' untranslated regions (UTR) is a regulatory region of DNA situated at the 5' end of a protein coding sequence that is transcribed into mRNA but not translated into protein. 5' UTRs may contain various regulatory elements, e.g., 5' cap structure, stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in the control of translation initiation. The 3' UTR, situated downstream of a protein coding sequence, may 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 specific tissue (e.g., lymphoid tissue), to which the mRNA expression is targeted. In some embodiments, the UTR increases protein synthesis. Without being bound by mechanism or theory, the UTR may increase protein synthesis by increasing the time that the mRNA remains in translating polysomes (message stability) and / or the rate at which ribosomes initiate translation on the message (message translation efficiency). According, the UTR sequence may prolong protein synthesis in a tissue-specific manner. In some embodiments, the 5' UTR and the 3' UTR sequences are computationally derived. In some embodiments, the 5' UTR and the 3' UTRs are derived from a naturally abundant mRNA in a tissue. The tissue may be, for example, liver, a stem cell, or lymphoid tissue. The lymphoid tissue may include, for example, any one of a lymphocyte (e.g., a B-lymphocyte, a helper T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, or a natural killer cell), a macrophage, a monocyte, a dendritic cell, a neutrophil, an eosinophil and a reticulocyte. In some embodiments, the 5' UTR and the 3' UTR are derived from an alphavirus. In some embodiments, the 5' UTR and the 3' UTR are from a wild-type alphavirus. Examples of alphaviruses are described below.

[0250] In some embodiments, the first RNA molecule includes a 5' UTR and the 3' UTR derived from a naturally abundant mRNA in a tissue. In some embodiments, the first RNA molecule includes a 5' UTR and the 3' UTR derived from an alphavirus. In some embodiments, the second RNA or the saRNA molecule includes a 5' UTR and the 3' UTR derived from an alphavirus. In some embodiments, the second RNA or the saRNA molecule includes a 5' UTR and the 3' UTR from a wild-type alphavirus. In some embodiments, the RNA molecule includes a 5’ cap.

[0251] Open reading frame (ORF)

[0252] The 5' and 3' UTRs may be operably linked to an ORF, which may be a sequence of codons that is capable of being translated into a polypeptide of interest. As stated above, the RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs).

[0253] In some embodiments, the ORF encodes a non-structural viral gene. In some embodiments, the ORF further includes one or more subgenomic promoters. In some embodiments, the RNA molecule includes 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 B2. In some embodiments, the cis-acting regulatory element is immediately downstream (5’-3’) of a guanine that is immediately downstream of B2. In some embodiments, the cis-acting regulatory element is an AU-rich element. In some embodiments, the AU-rich element is au, auaaaagau, auaaaaagau (SEQ ID NO: 183), auag, auauauauau (SEQ ID NO: 184), auauauau, auauauauauau (SEQ ID NO:185), augaugaugau (SEQ ID NO:186), augau, auaaaagaua (SEQ ID NO:187), or auaaaagaug (SEQ ID NO:188). In some embodiments, the second RNA or the saRNA molecule may include (i) an ORF encoding a replicase which may transcribe RNA from the second RNA or the saRNA molecule and (ii) an ORF encoding at least one an antigen or polypeptide of interest. The polymerase may be an alphavirus replicase e.g., including any one of the non-structural alphavirus proteins nsP1, nsP2, nsP3 and nsP4, or a combination thereof. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP1. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP2. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP4. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsP1, nsP2, and nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule includes any combination of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule does not include nsP4.

[0254] In some embodiments, an open reading frame of an RNA (e.g., saRNA) composition is codon-optimized. In some embodiments, the open reading frame which the influenza polypeptide or fragment thereof is encoded is codon-optimized.

[0255] 5’ cap

[0256] In some embodiments, the saRNA molecule described herein includes a 5’ cap. In some embodiments, the 5'-cap moiety is a natural 5'-cap.

[0257] A “natural 5'-cap” is defined as a cap that includes 7-methylguanosine connected to the 5’ end of an mRNA molecule through a 5' to 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 with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which may be incorporated during RNA synthesis (e.g., co-transcriptional capping) or may be enzymatically engineered after RNA transcription (e.g., post- transcriptional capping), wherein “N” is any ribonucleotide. In some embodiments, the 5’ end of the RNA molecule is capped with a modified ribonucleotide via an enzymatic reaction after RNA transcription. In some embodiments, capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule. An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) that includes mRNA triphosphatase, guanylyl- transferase, and guanine-7-methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures. Cap 0 structure can help maintaining the stability and translational efficacy of the RNA molecule. The 5' cap of the RNA molecule may be further modified by a 2 '-O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2 '-O] N), which may further increase translation efficacy. In some embodiments, the RNA molecule may be enzymatically capped at the 5' end using Vaccinia guanylyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine to yield cap 0 structure. An inverted 7-methylguanosine cap is added via a 5' to 5' triphosphate bridge. Alternatively, use of a 2'0-methyltransferase with Vaccinia guanylyltransferase yields the cap 1 structure where in addition to the cap 0 structure, the 2'OH group is methylated on the penultimate nucleotide. S-adenosyl-L-methionine (SAM) is a cofactor utilized as a methyl transfer reagent. Non-limiting examples of 5' cap structures are those which, among other things, have enhanced binding of cap binding polypeptides, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5' decapping, as compared to synthetic 5'cap structures known in the art (or to a wild-type, natural or physiological 5'cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-O-methyltransferase enzyme may create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine includes an N7 methylation and the 5'-terminal nucleotide of the mRNA includes a 2'-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., 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 a N7-methyl guanosine connected to the 5' nucleotide through a 5' to 5' triphosphate linkage, typically referred to as m7G cap or m7Gppp. In the cell, the cap 0 structure can help provide for efficient translation of the mRNA that carries the cap. An additional methylation on the 2'0 position of the initiating nucleotide generates Cap 1, or refers to as m7GpppNm-, wherein Nm denotes any nucleotide with a 2'0 methylation. In some embodiments, the 5' terminal cap includes a cap analog, for example, a 5' terminal cap may 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 capping region may include a single cap or a series of nucleotides forming the cap. In this embodiment the capping region may be equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the cap is absent. In some embodiments, the first and second operational regions may be equal to any one of, at least any one of, at most any one of, or between any two of 3 to 40, e.g., 5-30, 10-20, 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 fewer nucleotides in length and may comprise, in addition to a Start and / or Stop codon, one or more signal and / or restriction sequences.

[0258] In some embodiments, the 5’ Cap is represented by Formula I:

[0259]

[0260] where R1and R2are each independently H or Me, and B1and B2are each independently guanine, adenine, or uracil. In some embodiments, B1and B2are naturally-occurring bases. In some embodiments, R1is methyl and R2is hydrogen. In some embodiments, B1is guanine. In some embodiments, B1is adenine. In some embodiments, B2is adenine. In some embodiments, B2is uracil. In some embodiments, B2is uracil and at least 5% of a total population of uracil nucleotides in the molecule that are downstream of B2have been replaced with one or more modified or unnatural nucleotides.

[0261] In some embodiments, the nucleotide immediately downstream (5’ to 3’ direction) of the 5’ Cap comprises guanine. In some embodiments, B1is adenine and B2is uracil. In some embodiments, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen. In some instances, the saRNA does not comprise a 5’ Cap. In some instances, the 5’ Cap is not represented by Formula I. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen; this embodiment corresponds to CleanCap AU, and the inclusion of B2= uracil, while optionally substituting uracil nucleotides downstream of B2, has been shown to improve saRNA functionality in some embodiments. 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 total RNA molecules are full length. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus. In some embodiments, the alphavirus is Semliki Forest virus.

[0262] In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with N1-methylpseudouridine, and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5- methoxyuridine, and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methyluridine, and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, essentially all uridine nucleotides in the molecule have been replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, essentially all uridine nucleotides in the molecule have been replaced with about 75% 5-methoxyuridine and about 25% N1 -methylpseudouridine. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen, essentially all uridine nucleotides in the molecule have been replaced with about 25% 5-methoxyuridine and about 75% N1 -methylpseudouridine.

[0263] In some embodiments, a 5' terminal cap is 7mG(5')ppp(5')NlmpNp. In some preferred OH O'X

[0264] embodiments, the 5’ cap comprises:

[0265]

[0266] OH OH. in some embodiments, the 5’ cap comprises CLEANCAP® Reagent AG (31OMe) for co-transcriptional capping of mRNA, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG,

[0267]

[0268] . In alternative embodiments, the 5’ cap comprises CLEANCAP® AU for Self-Amplifying mRNA, CLEANCAP® Reagent AU for co-transcriptional

[0269]

[0270] capping of mRNA, m7G(5')ppp(5')(2'OMeA)pU,

[0271] Poly-A tail

[0272] As used herein, “poly A tail” refers to a stretch of consecutive adenine residues, which may be attached to the 3’ end of the RNA molecule. The poly-A tail may increase the half-life of the RNA molecule. Poly-A tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyperpolyadenylation may signal for RNA degradation. Exemplary designs include a poly-A tails of about 40 adenine residues to about 80 adenine residues. In some embodiments, the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly A tail sequence. In some embodiments, such as for the second RNA or the saRNA molecule, the RNA molecule further includes a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. A “full length” RNA molecule is one that includes a 5’-cap and a poly A tail.

[0273] In some embodiments, the poly A tail includes 5-400 nucleotides in length. The poly A tail nucleotide length may be equal to any one of, at least any one of, at most any one of, or between any two 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. In some embodiments, the RNA molecule includes a poly A tail that includes 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 includes a poly A tail includes a sequence of greater than 30 adenosine nucleotides (“As”). In some embodiments, the RNA molecule includes a poly A tail that includes about 40 As. In some embodiments, the RNA molecule includes a poly A tail that includes 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’ poly-A tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. In some embodiments, the RNA molecule includes at least 20 consecutive adenosine residues and at most 40 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 40 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 80 consecutive adenosine residues.

[0274] Composition

[0275] In some instances, the compositions described herein include at least one saRNA as described herein. Some embodiments of the present disclosure provide influenza virus (influenza) vaccines (or compositions or immunogenic compositions) that include 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 to influenza).

[0276] In some embodiments, equal to any one of, at least any one of, at most any one of, or between any two of 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 are capped.

[0277] In some embodiments, equal to any one of, at least any one of, at most any one of, or between any two of 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 are full length RNA transcripts. Purity may be determined as described herein, e.g., via reverse phase HPLC or Bioanalyzer chip-based electrophoresis and measure by, e.g., peak area of full-length RNA molecule relative to total peak. In some embodiments, a fragment analyzer (FA) may be used to quantify and purify the RNA. The fragment analyzer automates capillary electrophoresis and HPLC.

[0278] In some embodiments, the composition is substantially free of one or more impurities or contaminants including the linear DNA template and / or reverse complement transcription products and, for instance, includes RNA molecules that are equal to any one of, at least any one of, at most any one of, or between any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure; at least 98% pure, or at least 99% pure.

[0279] In some embodiments, the composition comprises an amount of the first RNA molecule that is greater than the amount of the second RNA molecule. In some embodiments, the composition comprises an amount of the first RNA molecule that is at least about 1 to 2 times greater than the amount of the second RNA molecule. In some embodiments, the composition comprises an amount of the first RNA molecule that is at least about 1 to 100 times greater than the amount of the second RNA molecule.

[0280] In some embodiments, the composition further includes a pharmaceutically acceptable carrier. In some embodiments, the composition further includes a pharmaceutically acceptable vehicle.

[0281] In some embodiments, the composition further includes a lipid-based delivery system, which delivers an RNA molecule to the interior of a cell, where it can then replicate and / or express the encoded polypeptide of interest. The delivery system may have adjuvant effects which enhance the immunogenicity of an encoded antigen. In some embodiments, the composition further includes neutral lipids, cationic lipids, cholesterol, and polyethylene glycol (PEG), and forms nanoparticles that encompass the RNA molecules. In some embodiments, the composition further includes any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion. In some embodiments, the RNA molecule is encapsulated in, bound to or adsorbed on any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion, or a combination thereof.

[0282] In some instances, the compositions described herein include at least two RNA molecules: a first RNA molecule and a second RNA molecule as described herein. To protect against more than one strain of influenza, a combination vaccine composition may be administered that includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first influenza virus or organism and further includes a second RNA molecule encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second influenza virus or organism. RNA can be co-formulated, for example, in a single lipid nanoparticle (LNP) or can be formulated in separate LNPs for co-administration.

[0283] In some embodiments, the second RNA molecule includes any one of a 5’ cap, a 5’ UTR, an open reading frame, a 3’ UTR, and a poly A sequence, or any combination thereof. In some embodiments, the second RNA molecule includes a 5’ cap moiety. In some embodiments, the second RNA molecule includes a 5’ UTR and a 3’UTR. In some embodiments, the second RNA molecule includes a 5’UTR, an open reading frame, a 3’UTR, and does not further include a 5’ cap. In some embodiments, the second RNA molecule includes a 5’ cap moiety, 5’ UTR, coding region, 3’ UTR, and a 3’ poly A sequence. In some embodiments, the second RNA molecule includes a 5’ cap moiety, 5’ UTR, noncoding region, 3’ UTR, and a 3’ poly A sequence. In some embodiments, the second RNA molecule includes a noncoding region and does not further comprise any one of a 5’ cap moiety, 5’ UTR, 3’ UTR, and a 3’ poly A sequence. In some embodiments, the second RNA molecule includes a 5’ cap moiety, a 5’ untranslated region (5’ UTR), a modified nucleotide, an open reading frame, a 3’ untranslated region (3’ UTR), and a 3’ poly A sequence.

[0284] Some aspects of the disclosure are directed to a composition comprising (i) first RNA molecule encoding a gene of interest derived from influenza; and (ii) a second RNA molecule comprising a modified or unnatural nucleotide In some instances, the first RNA molecule is any one of the saRNA molecules described herein. In some instances, the first RNA molecule comprises a 5’ Cap, a 5’ untranslated region, a coding region for a nonstructural protein comprising a RNA replicase, a subgenomic promoter, an open reading frame encoding a gene of interest, a 3’ untranslated region, and a 3’ poly A sequence. In some instances, at least 5% of a total population of a particular nucleotide in the first RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some instances, the RNA molecule comprises natural, unmodified nucleotides and does not include a modified or unnatural nucleotide. In some instances, the 5’ Cap is represented by Formula I, where R1 and R2 are each independently H or Me, B1 and B2 are each independently guanine, adenine, or uracil, a 5’ untranslated region, a coding region for a nonstructural protein derived from an alphavirus, a subgenomic promoter, such as one derived from an alphavirus, an open reading frame encoding a gene of interest, a 3’ untranslated region, and a 3’ poly A sequence. In some embodiments, B1 and B2 are naturally-occurring bases. In some embodiments, R1 is methyl and R2 is hydrogen. In some embodiments, B1 is guanine. In some embodiments, B1 is adenine. In some embodiments, B2 is adenine. In some embodiments, B2 is uracil. In some embodiments, the nucleotide immediately downstream (5’ to 3’ direction) of the 5’ Cap comprises guanine.

[0285] In some embodiments, B1is adenine and B2is uracil. In some embodiments, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen. In some embodiments, the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B1is adenine, B2is uracil, R1is methyl, and R2is hydrogen; this embodiment corresponds to CLEANCAP AU (Trilink), and the inclusion of B2= uracil, while optionally substituting uracil nucleotides downstream of B2, which has been shown to provide increased saRNA functionality in some embodiments.

[0286] In some embodiments, at least 10% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, essentially all of a particular nucleotide population in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, the one or more modified or unnatural replacement nucleotides comprise 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 a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 10% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 25% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 25% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides.

[0287] In some embodiments, at least 25% of a total population of uridine nucleotides in the first RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 75% of a total population of uridine nucleotides in the first RNA molecule has been replaced with N1 -methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1 -methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 5-methoxyuridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with 5-methyluridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with 5-methyluridine. In some embodiments, at least 50% of a total population of cytosine nucleotides in the first RNA molecule has been replaced with 5-methylcytosine. In some embodiments, essentially all cytosine nucleotides in the first RNA molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with 2-thiouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with 2-thiouridine.

[0288] In some embodiments, at least 25% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1 -methylpseudouridine. In some embodiments, at least 75% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with N1 -methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with 5-methoxyuridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methyluridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with 5-methyluridine. In some embodiments, at least 50% of a total population of cytosine nucleotides in the second RNA molecule has been replaced with 5- methylcytosine. In some embodiments, essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 2-thiouridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with 2-thiouridine.

[0289] In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1-methylpseudouridine and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methoxyuridine and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methyluridine and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine.

[0290] In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with about 50% 5-methoxyuridine and about 50% N1 -methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with about 75% 5-methoxyuridine and about 25% N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with about 25% 5-methoxyuridine and about 75% N1-methylpseudouridine.

[0291] In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1 -methylpseudouridine and at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1 -methylpseudouridine and essentially all uridine nucleotides in the second RNA molecule have been replaced with N1 -methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine and at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methyluridine, and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine and essentially all uridine nucleotides in the second RNA molecule have been replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. Methods of use

[0292] The RNA compositions may be utilized to treat and / or prevent an influenza virus of various genotypes, strains, and isolates. Some embodiments provide methods of preventing or treating influenza viral infection comprising administering to a subject any of the RNA compositions described herein. In some embodiments, the antigen specific immune response comprises a T cell response. In some embodiments, the antigen specific immune response comprises a B cell response. In some embodiments, the antigen specific immune response comprises both a T cell response and a B cell response. In some embodiments, the method of producing an antigen specific immune response involves a single administration of the RNA composition. In some embodiments, the RNA composition is administered to the subject by intradermal, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration. In some embodiments, the nanoparticle has a net neutral charge at a neutral pH value. In some embodiments, the RNA (e.g., mRNA) vaccine is multivalent.

[0293] In some embodiments, the RNA polynucleotides or portions thereof may encode one or more polypeptides or fragments thereof of an influenza strain as an antigen.

[0294] Some aspects of the disclosure are directed to a method of inducing an immune response in a subject, comprising administering to the subject in need thereof an effective amount of a composition as disclosed herein. Some aspects of the disclosure are directed to a method of vaccinating a subject, comprising administering to the subject in need thereof an effective amount of a composition as disclosed herein. Some aspects of the disclosure are directed to a method comprising administering to the subject in need thereof an effective amount of a composition as disclosed herein. In some embodiments, a composition as disclosed herein elicits an immune response comprising an antibody response. In some embodiments, a composition as disclosed herein elicits an immune response comprising a T cell response.

[0295] Nucleic Acids

[0296] In certain embodiments, nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding polypeptides, such as antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, mRNA, saRNA, and complementary sequences of the foregoing described herein. Nucleic acids that encode an epitope to which antibodies may bind. Nucleic acids encoding fusion proteins that include these polypeptides are also provided. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids). The term “polynucleotide” refers to a nucleic acid molecule that can be recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

[0297] In this respect, the term “gene” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar polypeptide.

[0298] In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising equal to any one of, at least any one of, at most any one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide. In some embodiments, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 95% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

[0299] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, equal to any one of, at least any one of, at most any one of, or between any two of 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, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

[0300] Lipid delivery

[0301] In some embodiments, the saRNA composition comprises lipids. The lipids and saRNA may together form nanoparticles. In some embodiments, the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure is about 20% or higher, about 25% or higher, about 30% or higher, about 35% or higher, about 40% or higher, about 45% or higher, about 50% or higher, about 55% or higher, about 60% or higher, about 65% or higher, about 70% or higher, about 75% or higher, about 80% or higher, about 85% or higher, about 90% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, or about 99% or higher.

[0302] In some embodiments, the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure is higher than the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method by about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 folds or more, about 2 folds or more, about 3 folds or more, about 4 folds or more, about 5 folds or more, about 10 folds or more, about 20 folds or more, about 30 folds or more, about 40 folds or more, about 50 folds or more, about 100 folds or more, about 200 folds or more, about 300 folds or more, about 400 folds or more, about 500 folds or more, about 1000 folds or more, about 2000 folds or more, about 3000 folds or more, about 4000 folds or more, about 5000 folds or more, or about 10000 folds or more.

[0303] In some embodiments, the Txo% of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure is about 12 months or longer, about 15 months or longer, about 18 months or longer, about 21 months or longer, about 24 months or longer, about 27 months or longer, about 30 months or longer, about 33 months or longer, about 36 months or longer, about 48 months or longer, about 60 months or longer, about 72 months or longer, about 84 months or longer, about 96 months or longer, about 108 months or longer, about 120 months or longer.

[0304] In some embodiments, the Txo% of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure is longer than the Txo% of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method by about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 folds or more, about 2 folds or more, about 3 folds or more, about 4 folds or more, about 5 folds or more.

[0305] In some embodiments, the T 1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure is about 12 months or longer, about 15 months or longer, about 18 months or longer, about 21 months or longer, about 24 months or longer, about 27 months or longer, about 30 months or longer, about 33 months or longer, about 36 months or longer, about 48 months or longer, about 60 months or longer, about 72 months or longer, about 84 months or longer, about 96 months or longer, about 108 months or longer, about 120 months or longer.

[0306] In some embodiments, the T 1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure is longer than the T 1 / 2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method by about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 folds or more, about 2 folds or more, about 3 folds or more, about 4 folds or more, about 5 folds or more

[0307] As used herein, “Tx” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to degrade to about X of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For example, “T8o%” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to degrade to about 80% of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For another example, “T 1 / 2” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to degrade to about 1 / 2 of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation.

[0308] Lipid nanoparticles may include a lipid component and one or more additional components, such as a therapeutic and / or prophylactic, such as a nucleic acid. A LNP may be designed for one or more specific applications or targets. The elements of a LNP may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a LN P may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combination of elements. The efficacy and tolerability of a LNP formulation may be affected by the stability of the formulation.

[0309] The lipid component of a LNP may include, for example, a cationic lipid, a phospholipid (such as an unsaturated lipid, e.g., DOPE or DSPC), a PEG lipid, and a structural lipid. The elements of the lipid component may be provided in specific fractions.

[0310] In some embodiments, the LNP further comprises a phospholipid, a PEG lipid, a structural lipid, or any combination thereof. Suitable phospholipids, PEG lipids, and structural lipids for the methods of the present disclosure are further disclosed herein.

[0311] In some embodiments, the lipid component of a LNP includes a cationic lipid, a phospholipid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid component of the lipid nanoparticle includes 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 % of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the lipid nanoparticle includes about 35 mol % to about 55 mol % compound of cationic lipid, 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 % of PEG lipid. In a particular embodiment, the lipid component includes about 50 mol % said cationic lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another embodiment, the lipid component includes about 40 mol % said cationic lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and / or the structural lipid may be cholesterol.

[0312] The amount of a therapeutic and / or prophylactic in a LNP may depend on the size, composition, desired target and / or application, or other properties of the lipid nanoparticle as well as on the properties of the therapeutic and / or prophylactic. For example, the amount of an RNA useful in a LNP may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic and / or prophylactic (i.e. pharmaceutical substance) and other elements (e.g., lipids) in a LNP may also vary. In some embodiments, the wt / wt ratio of the lipid component to a therapeutic and / or prophylactic in a LNP may be from about 5: 1 to about 60: 1, such as 5: 1, 6: 1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1,30:1,35:1, 40: 1, 45: 1, 50: 1, and 60: 1. For example, the wt / wt ratio of the lipid component to a therapeutic and / or prophylactic may be from about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20: 1. The amount of a therapeutic and / or prophylactic in a LNP may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0313] In some embodiments, the ionizable lipid is a compound of Formula (I):

[0314]

[0315] or their N-oxides, or salts or isomers thereof, wherein:

[0316] Ri 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 selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, a C3-6 carbocycle, -(CH2)nQ, - (CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -0(CH2)nN(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)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -0C(0)N(R)2J -N(R)C(0)0R, -N(0R)C(0)R, -N(0R)S(0)2R, -N(0R)C(0)0R, -N(0R)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)0R, and -C(R)N(R)2C(0)0R, and 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 Re 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-, an aryl group, and a heteroaryl group, in which M” is a bond, C1-13 alkyl orC2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; Re is selected from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H, CN, NO2, Ci-6 alkyl, -OR, -S(0)2R, -S(0)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-is alkyl, C2-is alkenyl, -R*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 Ci-i2 alkyl and C2-i2 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein when R4 is -(CH2)nQ, - (CH2)nCHQR, -CHQR, or -CQ(R)2, then (i) Q is not -N(R)2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2. In some embodiments, the ionizable lipid is SM-102. In some embodiments, the ionizable lipid is ALC-0315. In some embodiments,

[0317]

[0318] the ionizable lipid is:

[0319] In some embodiments, the compounds have the following structure (I):

[0320] (IE)

[0321]

[0322] O O

[0323] 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; G1 and G2 are each independently unsubstituted 01-012 alkylene or 01-012 alkenylene; G3 is 01-024 alkylene, 01-024 alkenylene, 03-08 cycloalkylene, 03-08 cycloalkenylene; Ra is H or 01-012 alkyl; R1 and R2 are each independently 06-024 alkyl or 06-024 alkenyl; R3 is H, 0R5, ON, — C(=O)OR4, — OC(=O)R4 or — NR5C(=O)R4; R4 is 01-012 alkyl; R5 is H or 01-06 alkyl; and x is 0, 1 or 2. In a preferred embodiment, the ionizable lipid is:

[0324]

[0325] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some embodiments, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. As used herein, the term “PEG lipid” refers to polyethylene glycol (PEG) -modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerCI4 or PEG-CerC20), PEG- modified dialkylamines and PEG-modified l,2-diacyloxypropan-3 -amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, a PEG lipid can be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-modified lipids are a modified form of PEG DMG. In some embodiments, the PEG-modified lipid is PEG lipid with the formula (IV):

[0326] N

[0327]

[0328] (IV) wherein R8 and R9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

[0329] Formulation

[0330] In one aspect, the disclosure relates to an immunogenic composition including: (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen, said antigen including 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, said second antigen including at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the first and second RNA polynucleotides are formulated in a lipid nanoparticle (LNP). In some embodiments, the first and second antigens include hemagglutinin (HA), or an immunogenic fragment or variant thereof. In some embodiments, the first antigen includes an HA from a different subtype of influenza virus to the influenza virus antigenic polypeptide or an immunogenic fragment thereof of the second antigen. In some embodiments, the composition further includes (iii) a third antigen including at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the third antigen is from influenza virus but is from a different strain of influenza virus to both the first and second antigens. In some embodiments, the first, second and third RNA polynucleotides are formulated in a lipid nanoparticle.

[0331] In some embodiments, the composition comprises an octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide associated with two type A viruses and two type B viruses that are predicted to be prevalent in a relevant jurisdiction. In some embodiments, an octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide derived from HA from an influenza type A virus, RNA encoding an antigenic polypeptide derived from HA from an influenza type A virus, RNA encoding an antigenic polypeptide derived from HA from an influenza type B virus, RNA encoding an antigenic polypeptide derived from HA from an influenza type B virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from an influenza type A virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from an influenza type A virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from an influenza type B virus, and RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from an influenza type B virus. In some embodiments, an octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide derived from HA from an influenza type A virus, RNA encoding an antigenic polypeptide derived from HA from an influenza type A virus, RNA encoding an antigenic polypeptide derived from HA from an influenza type B virus, RNA encoding an antigenic polypeptide derived from HA from an influenza type B virus, RNA encoding an antigenic polypeptide derived from NA from an influenza type A virus, RNA encoding an antigenic polypeptide derived from NA from an influenza type A virus, RNA encoding an antigenic polypeptide derived from NA from an influenza type B virus, and RNA encoding an antigenic polypeptide derived from NA from an influenza type B virus. In some embodiments, an 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, an octavalent influenza vaccine comprises RNA associated with influenza types that are predicted to be prevalent in a relevant jurisdiction (e.g., HA polypeptides associated with the H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses that are predicted to be prevalent in a relevant geography). The RNA (e.g., mRNA) vaccines may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. The RNA vaccines may be utilized to treat and / or prevent an influenza virus of various genotypes, strains, and isolates. The RNA vaccines typically have superior properties in that they produce much larger antibody titers and produce responses earlier than commercially available anti-viral therapeutic treatments. While not wishing to be bound by theory, it is believed that the RNA vaccines, as mRNA polynucleotides, are better designed to produce the appropriate protein conformation upon translation as the RNA vaccines co-opt natural cellular machinery. Unlike traditional vaccines, which are manufactured ex vivo and may trigger unwanted cellular responses, RNA (e.g., mRNA) vaccines are presented to the cellular system in a more native fashion.

[0332] In one aspect, a method of purifying an RNA polynucleotide synthesized by in vitro transcription is provided. The method includes ultrafiltration and diafiltration. In some embodiments, the method does not comprise a chromatography step. In some embodiments, the purified RNA polynucleotide is substantially free of contaminants comprising short abortive RNA species, long abortive RNA species, double- stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcription enzymes, residual solvent and / or residual salt. In some embodiments, the residual plasmid DNA is < 500 ng DNA / mg RNA. In some embodiments, purity of the purified mRNA is between about 60% and about 100%. In another aspect, a method of producing an RNA polynucleotide-encapsulated LNP is provided. The method includes buffer exchanging the LNPs. The method further includes concentrating the LNPs via flat sheet cassette membranes. In preferred embodiments, the UFDF process does not utilize hollow fiber membranes.

[0333] There may be situations in which persons are at risk for infection with more than one strain of influenza virus. RNA (e.g., mRNA) therapeutic vaccines are particularly amenable to combination vaccination approaches due to a number of factors including, but not limited to, speed of manufacture, ability to rapidly tailor vaccines to accommodate perceived geographical threat, and the like. Moreover, because the vaccines utilize the human body to produce the antigenic protein, the vaccines are amenable to the production of larger, more complex antigenic proteins, allowing for proper folding, surface expression, antigen presentation, etc. in the human subject. To protect against more than one strain of influenza, a combination vaccine can be administered that includes RNA (e.g., mRNA) encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first influenza virus or organism and further includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second influenza virus or organism. RNA (e.g., mRNA) can be co-formulated, for example, in a single lipid nanoparticle (LNP) or can be formulated in separate LNPs for coadministration.

[0334] Some embodiments of the present disclosure provide influenza virus (influenza) vaccines (or compositions or immunogenic compositions) that include at least one RNA 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 to influenza).

[0335] In some embodiments, the at least one antigenic polypeptide is one of the defined antigenic subdomains of HA, termed 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).

[0336] In some embodiments, the at least one antigenic polypeptide is HA or derivatives thereof comprising antigenic sequences from HA1 and / or HA2, and at least one antigenic polypeptide selected from HA, NA, NP, M1, M2, NS1 and NS2.

[0337] In some embodiments, the at least one antigenic polypeptide is HA or derivatives thereof comprising antigenic sequences from HA1 and / or HA2 and at least two antigenic polypeptides selected from HA, NA, NP, M1, M2, NS1 and NS2.

[0338] In some embodiments, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza virus protein, or an immunogenic fragment thereof.

[0339] In some embodiments, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding multiple influenza virus proteins, or immunogenic fragments thereof.

[0340] In some embodiments, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g., at least one HA1, HA2, or a combination of both).

[0341] In some embodiments, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g., at least one HA1, HA2, or a combination of both, of any one of or a combination of any or all 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., mRNA) polynucleotide having an open reading frame encoding a protein selected from a HA protein, NP protein, a NA protein, a M1 protein, a M2 protein, a NS1 protein and a NS2 protein obtained from influenza virus.

[0342] In some embodiments, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g., at least one any one of or a combination of any or all 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 RNAs (e.g., mRNAs) polynucleotides having two open reading frames encoding two proteins selected from a HA protein, NP protein, a NA protein, a M1 protein, a M2 protein, a NS1 protein and a NS2 protein obtained from influenza virus.

[0343] In some embodiments, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g., at least one of any one of or a combination of any or all of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18) and at least three other RNAs (e.g., mRNAs) polynucleotides having three open reading frames encoding three proteins selected from a HA protein, NP protein, a NA protein, a M protein, a M2 protein, a NS1 protein and a NS2 protein obtained from influenza virus. In some embodiments, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g., at least one of any one of or a combination of any or all of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18) and at least four other RNAs (e.g., mRNAs) polynucleotides having four open reading frames encoding four proteins selected from a HA protein, NP protein, a NA protein, a M1 protein, a M2 protein, a NS1 protein and a NS2 protein obtained from influenza virus.

[0344] In some embodiments, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g., at least one of any one of or a combination of any or all of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18) and at least five other RNAs (e.g., mRNAs) polynucleotides having five open reading frames encoding five proteins selected from a HA protein, NP protein, a NA protein, a M1 protein, a M2 protein, a NS1 protein and a NS2 protein obtained from influenza virus.

[0345] In some embodiments, a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein or an immunogenic fragment thereof (e.g., at least one of any one of or a combination of any or all of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18), HA protein, a NP protein or an immunogenic fragment thereof, a NA protein or an immunogenic fragment thereof, a M1 protein or an immunogenic fragment thereof, a M2 protein or an immunogenic fragment thereof, a NS1 protein or an immunogenic fragment thereof and a NS2 protein or an immunogenic fragment thereof obtained from influenza virus. In some embodiments, an influenza RNA composition includes an saRNA encoding an antigenic fusion protein. Thus, the encoded antigen or antigens may include two or more proteins (e.g., protein and / or protein fragment) joined together. Alternatively, the protein to which a protein antigen is fused does not promote a strong immune response to itself, but rather to the influenza antigen. Antigenic fusion proteins, in some embodiments, retain the functional property from each original protein.

[0346] Some embodiments of the present disclosure provide the following novel influenza virus polypeptide sequences: H1HA10-Foldon_ANgly1; H1HA10TM-PR8 (H1 A / Puerto Rico / 8 / 34 HA); H1HA10-PR8-DS (H1 A / Puerto Rico / 8 / 34 HA; pH1HA10-Cal04-DS (H1 A / California / 04 / 2009 HA); Pandemic H1HA10 from California 04; pH1HA10-ferritin; HA10; Pandemic H1HA10 from California 04; Pandemic H1HA10 from California 04 strain / without foldon and with K68C / R76C mutation for trimerization; H1HA10 from A / Puerto Rico / 8 / 34 strain, without foldon and with Y94D / N95L mutation for trimerization; H1HA10 from A / Puerto Rico / 8 / 34 strain, without foldon and with K68C / R76C mutation for trimerization; H1N1 A / Viet Nam / 850 / 2009; H3N2 A / Wisconsin / 67 / 2005; H7N9 (A / Anhui / 1 / 2013); H9N2 A / Hong Kong / 1073 / 99; H10N8 A / JX346 / 2013. Some embodiments of the present disclosure provide influenza virus (influenza) vaccines that include at least one RNA polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide or an immunogenic fragment of the novel influenza virus polypeptide sequences described above (e.g., an immunogenic fragment capable of inducing an immune response to influenza). In some embodiments, an influenza vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide comprising a modified sequence that is at least 75% (e.g., any number between 75% and 100%, inclusive, e.g., 70%, 80%, 85%, 90%, 95%, 99%, and 100%) identity to an amino acid sequence of the novel influenza virus sequences described above. The modified sequence can be at least 75% (e.g., any number between 75% and 100%, inclusive, e.g., 70%, 80%, 85%, 90%, 95%, 99%, and 100%) identical to an amino acid sequence of the novel influenza virus sequences described above.

[0347] Some embodiments of the present disclosure provide an isolated nucleic acid comprising a sequence encoding the novel influenza virus polypeptide sequences described above; an expression vector comprising the nucleic acid; and a host cell comprising the nucleic acid. The present disclosure also provides a method of producing a polypeptide of any of the novel influenza virus sequences described above. A method may include culturing the host cell in a medium under conditions permitting nucleic acid expression of the novel influenza virus sequences described above, and purifying from the cultured cell or the medium of the cell a novel influenza virus polypeptide. The present disclosure also provides antibody molecules, including full length antibodies and antibody derivatives, directed against the novel influenza virus sequences.

[0348] In some embodiments, an open reading frame of a RNA (e.g., mRNA) vaccine is codon-optimized. In some embodiments, the open reading frame which the influenza polypeptide or fragment thereof is encoded is codon-optimized. Some embodiments provide use of an influenza vaccine that includes at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide or an immunogenic fragment thereof, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracil in the open reading frame have a chemical modification, optionally wherein the vaccine is formulated in a lipid nanoparticle. In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some preferred embodiments, a chemical modification is a N1-methyl pseudouridine.

[0349] In some embodiments, a RNA (e.g., mRNA) vaccine further comprising an adjuvant. In some embodiments, at least one RNA polynucleotide encodes at least one influenza antigenic polypeptide that attaches to cell receptors.

[0350] In some embodiments, at least one RNA polynucleotide encodes at least one influenza antigenic polypeptide that causes fusion of viral and cellular membranes. In some embodiments, at least one RNA polynucleotide encodes at least one influenza antigenic polypeptide that is responsible for binding of the virus to a cell being infected.

[0351] Some embodiments of the present disclosure provide a vaccine that includes at least one ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide, at least one 5' terminal cap and at least one chemical modification, formulated within a lipid nanoparticle.

[0352] In some embodiments, a 5' terminal cap is 7mG(5')ppp(5')NlmpNp. In some preferred embodiments, the 5’ cap comprises:

[0353] OH

[0354]

[0355] OH 6H. in some embodiments, at least one chemical modification is selected from pseudouridine, N1 -methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyl uridine. In some embodiments, the chemical modification is in the 5-position of the uracil. In some embodiments, the chemical modification is a N1-methylpseudouridine. In some embodiments, the chemical modification is a N1 -ethylpseudouridine.

[0356] In some embodiments, a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, a cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, a cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z.15Z) — N, N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), and N, N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530).

[0357] Some embodiments of the present disclosure provide a vaccine that includes at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) of the uracil in the open reading frame have a chemical modification, optionally wherein the vaccine is formulated in a lipid nanoparticle (e.g., a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid).

[0358] In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a N1-methyl pseudouridine. In some embodiments, 100% of the uracil in the open reading frame have a N1-methyl pseudouridine in the 5-position of the uracil.

[0359] In some embodiments, an open reading frame of a RNA (e.g., mRNA) polynucleotide encodes at least one influenza antigenic polypeptides. In some embodiments, the open reading frame encodes at least two, at least five, or at least ten antigenic polypeptides. In some embodiments, the open reading frame encodes at least 100 antigenic polypeptides. In some embodiments, the open reading frame encodes 1-100 antigenic polypeptides.

[0360] In some embodiments, a vaccine comprises at least two RNA (e.g., mRNA) polynucleotides, each having an open reading frame encoding at least one influenza antigenic polypeptide. In some embodiments, the vaccine comprises at least five or at least ten RNA (e.g., mRNA) polynucleotides, each having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof. In some embodiments, the vaccine comprises at least 100 RNA (e.g., mRNA) polynucleotides, each having an open reading frame encoding at least one antigenic polypeptide. In some embodiments, the vaccine comprises 2-100 RNA (e.g., mRNA) polynucleotides, each having an open reading frame encoding at least one antigenic polypeptide.

[0361] Also provided herein is an influenza RNA (e.g., mRNA) vaccine of any one of the foregoing paragraphs formulated in a nanoparticle (e.g., a lipid nanoparticle).

[0362] In some embodiments, the nanoparticle has a mean diameter of 50-200 nm. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of about 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.

[0363] In some embodiments, the nanoparticle has a polydispersity value of less than 0.4 (e.g., less than 0.3, 0.2 or 0.1).

[0364] In some embodiments, the nanoparticle has a net neutral charge at a neutral pH value. In some embodiments, the RNA (e.g., mRNA) vaccine is multivalent.

[0365] Some embodiments of the present disclosure provide methods of inducing an antigen specific immune response in a subject, comprising administering to the subject any of the RNA (e.g., mRNA) vaccine as provided herein in an amount effective to produce an antigen-specific immune response. In some embodiments, the RNA (e.g., mRNA) vaccine is an influenza vaccine. In some embodiments, the RNA (e.g., mRNA) vaccine is a combination vaccine comprising a combination of influenza vaccines (a broad spectrum influenza vaccine).

[0366] In some embodiments, an antigen-specific immune response comprises a T cell response or a B cell response.

[0367] In some embodiments, a method of producing an antigen-specific immune response comprises administering to a subject a single dose (no booster dose) of an influenza RNA (e.g., mRNA) vaccine of the present disclosure.

[0368] In some embodiments, a method further comprises administering to the subject a second (booster) dose of an influenza RNA (e.g., mRNA) vaccine. Additional doses of an influenza RNA (e.g., mRNA) vaccine may be administered.

[0369] In some embodiments, the subjects exhibit a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) following the first dose or the second (booster) dose of the vaccine. Seroconversion is the time period during which a specific antibody develops and becomes detectable in the blood. After seroconversion has occurred, a virus can be detected in blood tests for the antibody. During an infection or immunization, antigens enter the blood, and the immune system begins to produce antibodies in response. Before seroconversion, the antigen itself may or may not be detectable, but antibodies are considered absent. During seroconversion, antibodies are present but not yet detectable. Any time after seroconversion, the antibodies can be detected in the blood, indicating a prior or current infection.

[0370] In some embodiments, an influenza RNA (e.g., mRNA) vaccine is administered to a subject by intradermal injection, intramuscular injection, or by intranasal administration. In some embodiments, an influenza RNA (e.g., mRNA) vaccine is administered to a subject by intramuscular injection.

[0371] Some embodiments, of the present disclosure provide methods of inducing an antigen specific immune response in a subject, including administering to a subject an influenza RNA (e.g., mRNA) vaccine in an effective amount to produce an antigen specific immune response in a subject. Antigen-specific immune responses in a subject may be determined, in some embodiments, by assaying for antibody titer (for titer of an antibody that binds to an influenza antigenic polypeptide) following administration to the subject of any of the influenza RNA (e.g., mRNA) vaccines of the present disclosure. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control.

[0372] In some embodiments, the anti-antigenic polypeptide antibody titer produced in a subject is increased at least 2 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased at least 5 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased 2-10 times relative to a control.

[0373] In some embodiments, the control is an anti-antigenic polypeptide antibody titer produced in a subject who has not been administered a RNA (e.g., mRNA) vaccine of the present disclosure. In some embodiments, the control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated or inactivated influenza, or wherein the control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a recombinant or purified influenza protein vaccine. In some embodiments, the control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered an influenza virus-like particle (VLP) vaccine.

[0374] A RNA (e.g., mRNA) vaccine of the present disclosure is administered to a subject in an effective amount (an amount effective to induce an immune response). In some embodiments, the effective amount is a dose equivalent to an at least 2-fold, at least 4-fold, at least 10-fold, at least 100-fold, at least 1000-fold reduction in the standard of care dose of a recombinant influenza protein vaccine, wherein the anti-antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant influenza protein vaccine, a purified influenza protein vaccine, a live attenuated influenza vaccine, an inactivated influenza vaccine, or an influenza VLP vaccine. In some embodiments, the effective amount is a dose equivalent to 2-1000-fold reduction in the standard of care dose of a recombinant influenza protein vaccine, wherein the anti-antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant influenza protein vaccine, a purified influenza protein vaccine, a live attenuated influenza vaccine, an inactivated influenza vaccine, or an influenza VLP vaccine.

[0375] In some embodiments, the control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a virus-like particle (VLP) vaccine comprising structural proteins of influenza.

[0376] In some embodiments, the RNA (e.g., mRNA) vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.

[0377] In some embodiments, the effective amount is a total dose of 25 pg to 1000 pg, or 50 pg to 1000 pg. In some embodiments, the effective amount is a total dose of 100 pg. In some embodiments, the effective amount is a dose of 25 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 pg administered to the subject a total of two times. In some embodiments, the effective amount is a total dose of 1 µg to 1000 µg, or 1 pg to 100 pg of saRNA. In some embodiments, the effective amount is a total dose of 30 pg. In some embodiments, the effective amount is a dose of 10 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 10 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 15 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 30 pg administered to the subject a total of two times. In some embodiments, the method includes administering to the subject a saRNA composition described herein at dosage of between 10 pg / kg and 400 pg / kg is administered to the subject.

[0378] In some embodiments the dosage of the saRNA polynucleotide is 1-5 pg, 5-10 pg, 10-15 pg, 15-20 pg, 10-25 pg, 20-25 pg, 20-50 pg, 30-50 pg, 40-50 pg, 40-60 pg, 60-80 pg, 60-100 pg, 50-100 pg, 80-120 pg, 40-120 pg, 40-150 pg, 50-150 pg, 50-200 pg, 80-200 pg, 100-200 pg, 120-250 pg, 150-250 pg, 180-280 pg, 200-300 pg, 50-300 pg, 80-300 pg, 100-300 pg, 40-300 pg, 50-350 pg, 100-350 pg, 200-350 pg, 300-350 pg, 320-400 pg, 40-380 pg, 40-100 pg, 100-400 pg, 200-400 pg, or 300-400 pg per 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 zero. In some embodiments, a second dose of the saRNA composition is administered to the subject on day twenty-one.

[0379] In some embodiments, the efficacy (or effectiveness) of a RNA (e.g., mRNA) vaccine is greater than 60%. In some embodiments, the RNA (e.g., mRNA) polynucleotide of the vaccine at least one Influenza antigenic polypeptide.

[0380] Vaccine efficacy may be assessed using standard analyses. For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas:

[0381] Efficacy=(ARU-ARV) / ARUx100; and Efficacy=(1-RR)x100.

[0382] Likewise, vaccine effectiveness may be assessed using standard analyses. Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination:

[0383] Effectiveness=(1-OR)x100. In some embodiments, the efficacy (or effectiveness) of a RNA (e.g., mRNA) vaccine is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0384] In some embodiments, the vaccine immunizes the subject against Influenza for up to 2 years. In some embodiments, the vaccine immunizes the subject against Influenza for more than 2 years, more than 3 years, more than 4 years, or for 5-10 years.

[0385] In some embodiments, the subject is about 5 years old or younger. For example, the subject may be between the ages of about 1 year and about 5 years (e.g., about 1, 2, 3, 5 or 5 years), or between the ages of about 6 months and about 1 year (e.g., about 6, 7, 8, 9, 10, 11 or 12 months). In some embodiments, the subject is about 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months or 1 month). In some embodiments, the subject is about 6 months or younger. In some embodiments, the RNA composition is administered to the subject by intradermal or intramuscular injection. In some embodiments, the RNA composition is administered to the subject on day zero. In some embodiments, a second dose of the RNA composition is administered to the subject on day twenty-one.

[0386] In some embodiments, the subject was born full term (e.g., about 37-42 weeks). In some embodiments, the subject was born prematurely, for example, at 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 may have been born at about 32 weeks of gestation or earlier. In some embodiments, the subject was born prematurely between about 32 weeks and about 36 weeks of gestation. In such subjects, a RNA (e.g., mRNA) vaccine may be administered later in life, for example, at the age of about 6 months to about 5 years, or older.

[0387] In some embodiments, the subject is a young adult between the ages of about 20 years and about 50 years (e.g., about 20, 25, 30, 35, 40, 45 or 50 years old).

[0388] 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).

[0389] In some embodiments, the subject has been exposed to influenza (e.g., C. trachomatis); the subject is infected with influenza (e.g., C. trachomatis); or subject is at risk of infection by influenza (e.g., C. trachomatis).

[0390] In some embodiments, the subject has been exposed to betacoronavirus (e.g., SARS-CoV-2); the subject is infected with betacoronavirus (e.g., SARS-CoV-2); or subject is at risk of infection by betacoronavirus (e.g., SARS-CoV-2).

[0391] In some embodiments, the subject has received at least one dose of an immunogenic composition against betacoronavirus (e.g., SARS-CoV-2), e.g., selected from any one of COMIRNATY®, the Pfizer-BioNTech COVID-19 vaccine, the Moderna mRNA-1273 COVID-19 vaccine, and the Janssen COVID-19 vaccine; the subject has received at least two doses of an immunogenic composition against betacoronavirus (e.g., SARS-CoV-2); the subject is receiving at least one dose of an immunogenic composition against betacoronavirus (e.g., SARS-CoV-2), e.g., selected from any one of COMIRNATY®, the Pfizer-BioNTech COVID-19 vaccine, the Moderna mRNA-1273 COVID-19 vaccine, and the Janssen COVID-19 vaccine; or the subject is being administered an immunogenic composition against betacoronavirus (e.g., SARS-CoV-2), e.g., selected from any one of COMIRNATY®, the Pfizer-BioNTech COVID-19 vaccine, the Moderna mRNA-1273 COVID-19 vaccine, and the Janssen COVID-19 vaccine at risk of infection by betacoronavirus (e.g., SARS-CoV-2) concomitantly, simultaneously, or within 12-48 hours of any one of the immunogenic compositions against influenza disclosed herein.

[0392] In some embodiments, the subject is immunocompromised (has an impaired immune system, e.g., has an immune disorder or autoimmune disorder).

[0393] In some embodiments the nucleic acid vaccines described herein are chemically modified. In other embodiments the nucleic acid vaccines are unmodified.

[0394] Yet other aspects provide compositions for and methods of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first virus antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and wherein an adjuvant is not co-formulated or co-administered with the vaccine.

[0395] In other aspects the invention is a composition for or method of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide wherein a dosage of between 10 pg / kg and 400 pg / kg of the nucleic acid vaccine is administered to the subject. In some embodiments the dosage of the RNA polynucleotide is 1-5 pg, 5-10 pg, 10-15 pg, 15-20 pg, 10-25 pg, 20-25 pg, 20-50 pg, 30-50 pg, 40-50 pg, 40-60 pg, 60-80 pg, 60-100 pg, 50-100 pg, 80-120 pg, 40-120 pg, 40-150 pg, 50-150 pg, 50-200 pg, 80-200 pg, 100-200 pg, 120-250 pg, 150-250 pg, 180-280 pg, 200-300 pg, 50-300 pg, 80-300 pg, 100-300 pg, 40-300 pg, 50-350 pg, 100-350 pg, 200-350 pg, 300-350 pg, 320-400 pg, 40-380 pg, 40-100 pg, 100-400 pg, 200-400 pg, or 300-400 pg per dose. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day twenty-one.

[0396] In some embodiments, a dosage of 25 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 50 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 75 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 150 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 200 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100-fold higher level in the local lymph node in comparison with the distal lymph node. In other embodiments the nucleic acid vaccine is chemically modified and in other embodiments the nucleic acid vaccine is not chemically modified.

[0397] Aspects of the disclosure provide a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and a pharmaceutically acceptable carrier or excipient, wherein an adjuvant is not included in the vaccine. In some embodiments, the stabilization element is a histone stem-loop. In some embodiments, the stabilization element is a nucleic acid sequence having increased GC content relative to wild type sequence.

[0398] Aspects of the disclosure provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is present in the formulation for in vivo administration to a host, which confers an antibody titer superior to the criterion for seroprotection for the first antigen for an acceptable percentage of human subjects. In some embodiments, the antibody titer produced by the mRNA vaccines of the disclosure is a neutralizing antibody titer. In some embodiments the neutralizing antibody titer is greater than a protein vaccine. In other embodiments the neutralizing antibody titer produced by the mRNA vaccines of the disclosure is greater than an adjuvanted protein vaccine. In yet other embodiments the neutralizing antibody titer produced by the mRNA vaccines of the disclosure is 1,000-10,000, 1,200-10,000, 1,400-10,000, 1,500-10,000, 1,000-5,000, 1,000-4,000, 1,800-10,000, 2000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000-5,000, 3,000-4,000, or 2,000-2,500. A neutralization titer is typically expressed as the highest serum dilution required to achieve a 50% reduction in the number of plaques.

[0399] Also provided are nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is present in a formulation for in vivo administration to a host for eliciting a longer lasting high antibody titer than an antibody titer elicited by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide. In some embodiments, the RNA polynucleotide is formulated to produce a neutralizing antibodies within one week of a single administration. In some embodiments, the adjuvant is selected from a cationic peptide and an immunostimulatory nucleic acid. In some embodiments, the cationic peptide is protamine. Aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally no modified nucleotides, the open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is present in the formulation for in vivo administration to a host such that the level of antigen expression in the host significantly exceeds a level of antigen expression produced by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide.

[0400] Other aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally no modified nucleotides, the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10-fold less RNA polynucleotide than is required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dosage of 25-100 micrograms.

[0401] Aspects of the disclosure also provide a unit of use vaccine, comprising between 10 ug and 400 ug of one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally no modified nucleotides, the open reading frame encoding a first antigenic polypeptide, and a pharmaceutically acceptable carrier or excipient, formulated for delivery to a human subject. In some embodiments, the vaccine further comprises a cationic lipid nanoparticle.

[0402] Aspects of the disclosure provide methods of creating, maintaining or restoring antigenic memory to a virus strain in an individual or population of individuals comprising administering to said individual or population an antigenic memory booster nucleic acid vaccine comprising (a) at least one RNA polynucleotide, said polynucleotide comprising at least one chemical modification or optionally no modified nucleotides and two or more codon-optimized open reading frames, said open reading frames encoding a set of reference antigenic polypeptides, and (b) optionally a pharmaceutically acceptable carrier or excipient. In some embodiments, the vaccine is administered to the individual via a route selected from the group consisting of intramuscular administration, intradermal administration, and subcutaneous administration. In some embodiments, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition. In some embodiments, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition in combination with electroporation.

[0403] In some aspects, methods of inducing an antigen specific immune response in a subject are provided. The method includes administering to the subject an influenza RNA composition in an amount effective to produce an antigen specific immune response. In some embodiments, an antigen specific immune response comprises a T cell response or a B cell response. In some embodiments, an antigen specific immune response comprises a T cell response and a B cell response. In some embodiments, a method of producing an antigen specific immune response involves a single administration of the vaccine. In some embodiments, a method further includes administering to the subject a booster dose of the vaccine. In some embodiments, a vaccine is administered to the subject by intradermal or intramuscular injection.

[0404] Aspects of the disclosure provide methods of vaccinating a subject comprising administering to the subject a single dosage of between 25 ug / kg and 400 ug / kg of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide in an effective amount to vaccinate the subject.

[0405] Other aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification, the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10-fold less RNA polynucleotide than is required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dosage of 25-100 micrograms.

[0406] Other aspects provide nucleic acid vaccines comprising an LNP formulated RNA polynucleotide having an open reading frame comprising no nucleotide modifications (unmodified), the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10-fold less RNA polynucleotide than is required for an unmodified mRNA vaccine not formulated in a LNP to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotide is present in a dosage of 25-100 micrograms.

[0407] The data presented in the Examples demonstrate significant enhanced immune responses using the formulations of the disclosure. Both chemically modified and unmodified RNA vaccines are useful according to the invention. Surprisingly, in contrast to prior art reports that it was preferable to use chemically unmodified mRNA formulated in a carrier to produce vaccines, it is described herein that chemically modified mRNA-LNP vaccines required a much lower effective mRNA dose than unmodified mRNA, i.e., tenfold less than unmodified mRNA when formulated in carriers other than LNP. Both the chemically modified and unmodified RNA vaccines of the disclosure produce better immune responses than mRNA vaccines formulated in a different lipid carrier.

[0408] In other aspects the invention encompasses a method of treating an elderly subject age 60 years or older comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a virus antigenic polypeptide in an effective amount to vaccinate the subject.

[0409] In other aspects the invention encompasses a method of treating a young subject age 17 years or younger comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a virus antigenic polypeptide in an effective amount to vaccinate the subject.

[0410] In other aspects the invention encompasses a method of treating an adult subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a virus antigenic polypeptide in an effective amount to vaccinate the subject.

[0411] In some aspects the invention is a method of vaccinating a subject with a combination vaccine including at least two nucleic acid sequences encoding antigens wherein the dosage for the vaccine is a combined therapeutic dosage wherein the dosage of each individual nucleic acid encoding an antigen is a sub therapeutic dosage. In some embodiments, the combined dosage is 25 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 100 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments the combined dosage is 50 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 75 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 150 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 400 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject.

[0412] In preferred aspects, vaccines of the disclosure (e.g., LNP-encapsulated mRNA vaccines) produce prophylactically- and / or therapeutically efficacious levels, concentrations and / or titers of antigen-specific antibodies in the blood or serum of a vaccinated subject. As defined herein, the term antibody titer refers to the amount of antigen-specific antibody produces in s subject, e.g., a human subject. In exemplary embodiments, antibody titer is expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result. In exemplary embodiments, antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody titer is determined or measured by neutralization assay, e.g., by microneutralization assay. In certain aspects, antibody titer measurement is expressed as a ratio, such as 1:40, 1:100, etc.

[0413] In exemplary embodiments of the disclosure, an efficacious vaccine produces an antibody titer of greater than 1:40, greater that...

Claims

CLAIMS1. An RNA polynucleotide that encodes a polypeptide derived from hemagglutinin of an influenza B virus, wherein the polypeptide comprises a deletion consisting of LLKERGF (SEQ ID NO: 124) when compared to the amino acid sequence of a hemagglutinin of the respective wild-type influenza B virus from which the polypeptide was derived.

2. The RNA polynucleotide according to claim 1, comprising at least 80% identity to any one of SEQ ID NOs: 116-123.

3. The RNA polynucleotide according to claim 1, comprising at least 80% identity to any one of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, and SEQ ID NO: 123.

4. The RNA polynucleotide according to claim 1, further comprising at least one poly(A) sequence comprising 30 to 200 adenosine nucleotides.

5. The RNA polynucleotide according to claim 1, comprising at least 80% identity to any one of SEQ ID NOs: 116-123 and SEQ ID NOs: 125-182.

6. An RNA polynucleotide encoding a polypeptide comprising at least 80% identity to any one of amino acid sequences SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, and SEQ ID NO: 123.

7. An RNA polynucleotide encoding a polypeptide comprising at least 80% identity to any one of amino acid sequences SEQ ID NO: 125; SEQ ID NO: 126; SEQ ID NO: 127; SEQ ID NO: 128; SEQ ID NO: 129; SEQ ID NO: 130; SEQ ID NO: 131; SEQ ID NO: 132; SEQ ID NO: 133; SEQ ID NO: 134; SEQ ID NO: 135; SEQ ID NO: 136; SEQ ID NO: 137; SEQ ID NO: 138; SEQ ID NO: 139; SEQ ID NO: 140; SEQ ID NO: 141; SEQ ID NO: 142; SEQ ID NO: 143; SEQ ID NO: 144; SEQ ID NO: 145; SEQ ID NO: 146; SEQ ID NO: 147; SEQ ID NO: 148; SEQ ID NO: 149; SEQ ID NO: 150; SEQ ID NO: 151; SEQ ID NO: 152; SEQ ID NO: 153; SEQ ID NO: 154; SEQ ID NO: 155; SEQ ID NO: 156; SEQ ID NO: 157; SEQ ID NO: 158; SEQ ID NO: 159; SEQ ID NO: 160; SEQ ID NO: 161; SEQ ID NO: 162; SEQ ID NO: 163; SEQ ID NO: 164; SEQ ID NO: 165; SEQ ID NO: 166; SEQ ID NO: 167; SEQ ID NO: 168; SEQ ID NO: 169; SEQ ID NO: 170; SEQ ID NO: 171; SEQ ID NO: 172; SEQ ID NO: 173; SEQ ID NO: 174; SEQ ID NO: 175; SEQ ID NO: 176; SEQ ID NO: 177; SEQ ID NO: 178; SEQ ID NO: 179; SEQ ID NO: 180; SEQ ID NO: 181; and SEQ ID NO: 182.

8. The RNA polynucleotide according to any one of claims 1-7, further comprising at least one poly(A) sequence comprising 30 to 200 adenosine nucleotides.

9. The RNA polynucleotide according to any one of claims 1-8, further comprising at least one untranslated region selected from at least one heterologous 5-UTR and at least one heterologous 3-UTR.

10. The RNA polynucleotide according to any one of claims 1-9, further comprising a nucleotide analog.

11. The RNA polynucleotide according to any one of claims 1-10, wherein the RNA comprises a 1-methylpseudouridine substitution.

12. The RNA polynucleotide according to any one of claims 1-11, comprising a 5'-cap structure, which comprises a structure selected from the group consisting of m7G, capO, cap1, cap2, a modified capO, and a modified cap1 structure.

13. A composition comprising an RNA according to any one of claims 1-12.

14. The composition according to claim 13, further comprising at least one pharmaceutically acceptable carrier, wherein the RNA is complexed or associated with lipids, wherein the lipids comprise a cationic lipid, a neutral lipid, and a sterol.

15. The composition according to any one of claims 13-14, further comprising a second RNA polynucleotide comprising an open reading frame encoding a second antigen, wherein the first and second RNA polynucleotides are complexed or associated with lipids.

16. The composition according to any one of claims 13-15, wherein the second antigen comprises hemagglutinin (HA) or an immunogenic fragment or variant thereof.

17. The composition according to claim 16, wherein the first and second antigens each comprise an HA or an immunogenic fragment thereof from different subtypes of influenza virus.

18. A polypeptide comprising an amino acid sequence having a deletion consisting of LLKERGF (SEQ ID NO: 124) compared to the amino acid sequence of a hemagglutinin of the respective wild-type influenza B virus from which the polypeptide was derived.

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