Self-amplifying RNA encoding influenza virus antigens

Abstract

Disclosed herein are self-amplifying RNA (saRNA) molecules encoding influenza virus antigens and methods of use thereof.

Description

[0001] Cross-references to applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 359,857 filed on July 10, 2022, U.S. Provisional Application No. 63 / 431,462 filed on December 9, 2022, and U.S. Provisional Application No. 63 / 484,745 filed on February 13, 2023, each of which is incorporated herein by reference in its entirety.

[0003] field

[0004] The present invention relates to compositions and methods for the preparation, manufacture and therapeutic use of RNA vaccines, wherein the RNA vaccines comprise polynucleotide molecules encoding one or more influenza antigens such as hemagglutinin antigens.

[0005] background

[0006] 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) proteins.

[0007] The genome of influenza A virus consists of eight molecules (seven for influenza C virus) of linear, negative polarity, single-stranded RNA, which encode several polypeptides, including: RNA-guided RNA polymerase proteins (PB2, PB1, and PA) and nucleoprotein (NP), which form the nucleocapsid; matrix proteins (M1, M2, which are also surface-exposed proteins embedded in the viral membrane); two surface glycoproteins, which extend from the lipoprotein envelope: hemagglutinin (HA) and neuraminidase (NA); and nonstructural proteins (NS1 and NS2).

[0008] Hemagglutinin is the major envelope glycoprotein of influenza A and B viruses, while hemagglutinin esterase (HE) of influenza C virus is a protein homologous to HA.

[0009] The challenge of using traditional vaccines for treatment and prevention against influenza and other infections is that the vaccines are limited in breadth, providing protection only against closely related subtypes. In addition, the length of time required to complete the current standard influenza virus vaccine production process hinders the rapid development and production of adaptive vaccines in a pandemic scenario.

[0010] There is a need for improved compositions, preferably immunogenic compositions, against influenza.

[0011] Overview

[0012] Provided herein are unmet needs for improved compositions, preferably immunogenic compositions, for influenza and others. In one aspect, the present disclosure relates to a composition comprising a self-amplifying RNA (saRNA), the self-amplifying RNA (saRNA) comprising: a 5' cap; a 5' untranslated region (5'UTR); a coding region for a nonstructural protein derived from an alphavirus; a first subgenomic promoter derived from an alphavirus; a first open reading frame encoding a first target gene derived from influenza virus hemagglutinin (HA); a second subgenomic promoter derived from an alphavirus; a second open reading frame encoding a second target gene derived from influenza virus; a 3' untranslated region (3'UTR); and a 3' poly A sequence.

[0013] In another aspect, the present disclosure relates to a composition comprising a self-amplifying RNA (saRNA), the self-amplifying RNA (saRNA) comprising: a 5' cap; a 5' untranslated region (5'UTR); a coding region for a non-structural protein derived from an alphavirus; a subgenomic promoter derived from an alphavirus; an open reading frame encoding a gene of interest derived from an influenza virus; a 3' untranslated region (3'UTR); and a 3' poly A sequence; wherein at least 5% of the total population of specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0014] In preferred embodiments, the saRNA polynucleotide has clinical grade purity. In some embodiments, the purity of the RNA polynucleotide is between about 60% and about 100%. In some embodiments, the purified RNA polynucleotide has 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 integrity, as determined by known methods, such as capillary electrophoresis. In some embodiments, the total RNA molecules in the composition equal to any one of the following, at least any one of the following, at most any one of the following, or between any two of the following are full-length RNA transcripts: 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%. A "full-length" RNA molecule is an RNA molecule that includes a 5' cap and a poly A tail. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The following drawings constitute part of this 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.

[0017] Figure 1 – Functional anti-HA antibodies elicited by immunization of mice with saRNA encoding influenza HA and / or NA formulated with LNPs, as measured by HAI; Figure 1 Depicted are 3 weeks after priming and 2 weeks after boosting; female Balb / c mice were immunized IM at day 0 with 20 ng LNP-formulated bicistronic and monocistronic saRNA vaccine formulations, a total of 40 ng (20 ng each) of saRNA-HA + saRNA-NA in a 1: 1 mixture, and 200 ng of modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA. At day 21 (3 weeks after immunization), antibody responses to A / Wisconsin / 588 / 2019 were measured by HAI or 1-day MNT assays. HAI titers (geometric means with geometric SD) are reported.

[0018] Figure 2 – Neutralizing antibodies elicited by immunization of mice with saRNA encoding influenza HA and / or NA formulated with LNPs, as measured by 1-day MNT; Figure 2 Depicted are 3 weeks after priming and 2 weeks after boosting; female Balb / c mice were immunized IM on day 0 with 20 ng LNP-formulated bicistronic and monocistronic saRNA vaccine formulations, a total of 40 ng (20 ng each) of saRNA-HA + saRNA-NA in a 1:1 mixture, and 200 ng of modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA. Antibody responses to A / Wisconsin / 588 / 2019 were measured by HAI or 1-day MNT assays at day 21 (3 weeks after immunization). 50% neutralization titers (geometric means with geometric SD) are reported.

[0019] Figure 3 – Neutralizing antibodies elicited by immunization of mice with saRNA encoding influenza HA and / or NA formulated with LNPs, as measured by 3-day MNT

[0020] Figure 4 – Functional anti-NA antibodies elicited by immunization of mice with saRNA encoding influenza HA and / or NA formulated with LNPs, as measured by NAI; Figure 4Depicted are 3 weeks after priming and 2 weeks after boosting; female Balb / c mice were immunized IM at day 0 with 20 ng LNP-formulated bicistronic and monocistronic saRNA vaccine formulations, a total of 40 ng (20 ng each) of saRNA-HA + saRNA-NA in a 1: 1 mixture, and 200 ng of modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) NA. Antibody responses to A / Wisconsin / 588 / 2019 were measured by NAI at day 21 (3 weeks after immunization). Geometric mean titers with geometric SD are reported.

[0021] Figure 5 – Serum cytokines and chemokines 24 hours after immunization of Balb / c mice with influenza saRNA-HA vaccine formulations with different amounts of modified nucleosides

[0022] Figure 6 – Functional HAI and neutralizing antibodies elicited by immunization of Balb / c mice with saRNA-HA vaccine formulations containing different amounts of modified nucleosides formulated with LNPs

[0023] Figure 7 – Serum cytokines and chemokines 24 hours after immunization of C57BL6 / J mice with influenza saRNA-HA vaccine formulations with different amounts of modified nucleosides

[0024] Figure 8 - Functional HAI and neutralizing antibodies elicited by immunization of C57NL6 / J mice with saRNA-HA vaccine formulations formulated with LNPs with different amounts of modified nucleosides; Female Balb / c mice were immunized IM with 20ng LNP-formulated tetravalent saRNA (composed of 4 bicistronic constructs encoding HA and NA from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage) and B / Phuket / 3073 / 2013 (B / Yamagata lineage)), or 2.4μg of a licensed adjuvanted tetravalent inactivated vaccine (QIV; FluAd) on day 0. Antibody responses to each vaccine component were measured by HAI or 1-day MNT assay on day 42 (2 weeks after the second dose). HAI and 50% neutralization titers (geometric means with geometric SD) are reported.

[0025] Fig. 9- Functional HAI and neutralizing antibodies elicited by immunization of mice with tetravalent bicistronic saRNA formulated with LNPs encoding HA and NA from four seasonal influenza virus strains

[0026] Fig.10 - Functional NAI antibodies elicited by immunization of mice with tetravalent bicistronic saRNA formulated with LNPs encoding HA and NA from 4 seasonal influenza virus strains; Female Balb / c mice were immunized IM with 20ng LNP-formulated tetravalent saRNA consisting of 4 bicistronic constructs encoding HA and NA from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage), or 2.4μg of a licensed adjuvanted tetravalent inactivated vaccine (QIV; FluAd) on day 0. Antibody responses to each vaccine component were measured by NAI on day 42 (2 weeks after the second dose). NAI titers (geometric mean with geometric SD) were reported for 3 of the 4 strains. Due to technical issues using the NAI assay for this strain, H3N2 NAI titers could not be reported for both saRNA and QIV.

[0027] Fig.11 – Geometric mean titers and 95% CI: HAI – vaccine formulations 1, 2 and control – immunogenicity evaluable population

[0028] Abbreviations: GMT = geometric mean titer; HAI = hemagglutination inhibition; QIV = quadrivalent influenza vaccine, Vax Prep = vaccine preparation.

[0029] Note: V1 = 1 day before vaccination; V3 = 1 week; V4 = 2 weeks; V5 = 4 weeks.

[0030] Note: Dots indicate individual antibody levels.

[0031] Note: The number / GMT within each bar represents the number of participants with valid and definitive assay results for the specified assay at a given sampling time point, and the corresponding geometric mean titer. The mean of two samples collected on day 1 prior to vaccination was used for GMT calculations.

[0032] Note: Licensed QIV-15A includes participants in studies receiving a licensed QIV and their VRD data are tested concurrently with the VRD data for the Test Cl group.

[0033] Note: Licensed QIV-18 includes participants in studies receiving licensed QIVs, and their VRD data are tested concurrently with VRD data for groups C2-C5 and C7.

[0034] Note: Placebo includes participants in studies who received a placebo at the time of randomization.

[0035] Fig.12 – Geometric mean titers and 95% CI: HAI – vaccine formulations 3, 4, 7 and control – evaluable immunogenicity population

[0036] Abbreviations: GMT = geometric mean titer; HAI = hemagglutination inhibition; QIV = quadrivalent influenza vaccine, Vax Prep = vaccine preparation.

[0037] Note: V1 = 1 day before vaccination; V3 = 1 week; V4 = 2 weeks; V5 = 4 weeks.

[0038] Note: Dots indicate individual antibody levels.

[0039] Note: The number / GMT within each bar represents the number of participants with valid and definitive assay results for the specified assay at a given sampling time point, and the corresponding geometric mean titer. The mean of two samples collected on day 1 prior to vaccination was used for GMT calculations.

[0040] Note: Licensed Q1V-18 includes participants in studies receiving licensed QIVs whose VRD data were tested concurrently with VRD data for groups C2-C5 and C7.

[0041] Note: Placebo includes participants in studies who received a placebo at the time of randomization.

[0042] Fig.13 – Geometric mean titers and 95% CI: HAI – vaccine formulations 5, 6 and control – immunogenicity evaluable population

[0043] Abbreviations: GMT = geometric mean titer; HAI = hemagglutination inhibition; QIV = quadrivalent influenza vaccine, Vax Prep = vaccine preparation.

[0044] Note: VI = 1 day before vaccination; V3 = 1 week; V4 = 2 weeks; V5 = 4 weeks.

[0045] Note: Dots indicate individual antibody levels.

[0046] Note: The number / GMT within each bar represents the number of participants with valid and definitive assay results for the specified assay at a given sampling time point, and the corresponding geometric mean titer. The mean of two samples collected on day 1 prior to vaccination was used for GMT calculations.

[0047] Note: Licensed QIV-18 includes participants in studies receiving licensed QIVs, and their VRD data are tested concurrently with VRD data for groups C2-C5 and C7.

[0048] Note: Licensed QIV-15B includes participants in studies receiving a licensed QIV, and their VRD data are tested concurrently with the VRD data for group C6.

[0049] Note: Placebo includes participants in studies who received a placebo at the time of randomization.

[0050] Fig.14 – Functional anti-HA antibodies elicited in mice after one dose of HA / NA encoding saRNA-LNPs, Quad modRNA, or FluAd as measured by HAI

[0051] Fig.15 – Functional anti-NA antibodies elicited in mice after one dose of HA / NA encoding saRNA-LNPs or FluAd as measured by NAI

[0052] Fig.16 – Virus neutralizing antibodies elicited in mice after one dose of HA / NA encoding saRNA-LNPs, Quad modRNA, or FluAd, measured by 1-day MNT

[0053] Fig.17 – Functional anti-HA antibodies elicited in mice after two doses of HA / NA encoding saRNA-LNPs, Quad modRNA, or FluAd as measured by HAI

[0054] Fig.18 – Functional anti-NA antibodies elicited in mice after two doses of HA / NA encoding saRNA-LNPs or FluAd as measured by NAI

[0055] Fig.19 – Virus neutralizing antibodies elicited in mice after two doses of HA / NA encoding saRNA-LNPs, Quad modRNA, or FluAd, as measured by 1-day MNT

[0056] Details

[0057] Embodiments of the present disclosure provide compositions comprising self-amplifying RNA (saRNA) polynucleotides encoding influenza virus antigens.Influenza virus RNA vaccines as provided herein can be used to induce a balanced immune response, including both cellular and humoral immunity.

[0058] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa.In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0059] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating specific embodiments of the present disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from the detailed description.

[0060] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error associated with the measurement or quantitative method.

[0061] When used in conjunction with the term "comprising," the use of the word "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0062] The phrase "and / or" means "and" or "or". For example, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, "and / or" serves as an inclusive "or".

[0063] The words "comprising" (and any forms of comprising, such as "comprise" and "comprises"), "having" (and any forms of having, such as "have" and "has"), "including" (and any forms of including, such as "includes" and "include"), or "contain" (and any forms of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0064] The phrase "substantially all" is defined as "at least 95%"; if substantially all members of a group have a property, then at least 95% of the members of the group have that property. In some cases, substantially all means that equal to any one, at least any one, or any two of the members of the group have that property.

[0065] Compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of any ingredient or step disclosed throughout the specification." Compositions and methods that "consist essentially of any disclosed ingredient or step" limit the scope of the claim to the specified materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure.

[0066] A. Self-amplifying RNA (saRNA)

[0067] In some embodiments, the RNA molecule, such as the first RNA molecule, is saRNA. "saRNA", "self-amplifying RNA" and "replicon" refer to RNA with self-replication ability. Self-amplifying RNA molecules can be produced by using replication elements derived from one or more viruses, such as alpha viruses, and replacing structural viral polypeptides with nucleotide sequences encoding target polypeptides. Self-amplifying RNA molecules are generally positive-strand molecules that can be directly translated after delivery to cells, and this translation provides RNA-dependent RNA polymerases, which then produce both antisense transcripts and sense transcripts from the delivered RNA. The delivered RNA results in the production of multiple sub-RNAs. These sub-RNAs and colinear subgenomic transcripts can be self-translated to provide in situ expression of the encoded target gene, such as a viral antigen, or can be transcribed to provide further transcripts with the same meaning as the delivered RNA, which are translated to provide in situ expression of a target protein, such as an antigen. The overall result of this transcription sequence is the amplification of the number of saRNAs introduced, and thus the encoded target gene, such as a viral antigen, can become the main polypeptide product of the cell.

[0068] In some embodiments, the self-amplifying RNA includes at least one or more genes selected from any one of viral replicase, viral protease, viral helicase and other non-structural viral proteins. In some embodiments, the self-amplifying RNA may also include 5'- and 3' end traction replication sequences (tractive replication sequence), and optionally a heterologous sequence encoding a desired amino acid sequence (e.g., a target antigen). A subgenomic promoter for instructing the expression of a heterologous sequence may be included in the self-amplifying RNA. Optionally, a heterologous sequence (e.g., a target antigen) may be fused in frame with other coding regions in the self-amplifying RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0069] In some embodiments, the self-amplifying RNA molecule is not encapsulated in a virus-like particle. The self-amplifying RNA molecule described herein can be designed so that the self-amplifying RNA molecule cannot induce the generation of infectious viral particles. This can be achieved, for example, by omitting one or more viral genes encoding structural proteins, which are necessary for producing viral particles in the self-amplifying RNA. For example, when the self-amplifying RNA molecule is based on an alpha virus, 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 can be omitted.

[0070] In some embodiments, the self-amplifying RNA molecules described herein encode (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-amplifying RNA molecule and (ii) a target polypeptide, such as a viral antigen. In some embodiments, the polymerase can be an alphavirus replicase, for example, including any of the alphavirus proteins nsP1, nsP2, nsP3, nsP4 and any combination thereof. In some embodiments, the self-amplifying RNA molecules described herein may include one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine). In some embodiments, the self-amplifying RNA molecules do not include modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine).

[0071] The saRNA construct may encode at least one nonstructural protein (NSP) that is disposed 5' or 3' to the sequence encoding at least one peptide or polypeptide of interest. In some embodiments, the sequence encoding at least one NSP is disposed 5' to the sequence 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 nonstructural protein encoded by the RNA construct may be an RNA polymerase nsP4. In some embodiments, the saRNA construct encodes nsP1, nsP2, nsP3, and nsP4. As known in the art, nsP1 is a membrane anchor for the viral capping enzyme and replication complex (RC). nsP2 is an RNA helicase and a protease responsible for ns polyprotein processing. nsP3 interacts with several host proteins and may regulate protein poly- and mono-ADP ribosylation. nsP4 is a core viral RNA-dependent RNA polymerase. In some embodiments, the polymerase may be an alphavirus replicase, for example, comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4.

[0072] Although the natural alphavirus genome encodes structural virion proteins other than 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 result in the production of its own genomic RNA copies in the cell, but does not result in the production of RNA including virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike wild-type alphaviruses, self-amplifying RNA molecules cannot continue themselves in an infectious form. Alphavirus structural proteins necessary for continuation in wild-type viruses may not be present in the self-amplifying RNA of the present disclosure, and their positions may be replaced by genes encoding the immunogen of interest, so that the subgenomic transcripts encode the immunogen rather than the structural alphavirus virion proteins.

[0073] In some embodiments, the self-amplifying RNA molecule can 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 can have additional (e.g., downstream) open reading frames, for example, to encode further antigens or to encode additional polypeptides.

[0074] In some embodiments, the second RNA or saRNA molecule further comprises (1) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.

[0075] Optionally, the self-amplifying RNA molecules described herein can also be designed to induce the production of attenuated or virulent infectious viral particles, or to produce viral particles capable of a single subsequent round of infection.

[0076] In some embodiments, the saRNA molecule is based on an alphavirus. Alphaviruses include a group of genetically, structurally, and serologically related arthropod-borne Togaviridae viruses. Exemplary viruses and subtypes of viruses within the genus Alphavirus include Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. Like this, the self-amplifying RNA described herein can be incorporated with an RNA replicase derived from any of Semliki Forest virus (SFV), Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), Ross River virus (RRV), or other viruses belonging to the alphaviridae family. In some embodiments, the self-amplifying RNA described herein can be incorporated with a sequence derived from a mutant or wild-type viral sequence, for example, the attenuated TC83 mutant of VEEV has been used in saRNA.

[0077] Alphavirus-based saRNA is (+) chain saRNA, which can be translated after delivery to cells, which results in the translation of replicase (or replicase-transcriptase). Replicase is translated as a polyprotein that automatically cuts to provide a replication complex, and the replication complex produces a genome (-) chain copy of the (+) chain delivery RNA. These (-) chain transcripts can be transcribed by themselves to give more copies of the (+) chain parent RNA, and to give subgenomic transcripts encoding the desired gene product. The translation of subgenomic transcripts therefore results in the in situ expression of the desired gene product by infected cells. Suitable alphavirus saRNA can use replicase from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus or its mutant variants.

[0078] In some embodiments, the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, such as a positive-strand RNA virus, and in particular a picornavirus, a flavivirus, a rubella virus, a pestivirus, a hepatitis virus, a calicivirus, or a coronavirus. Suitable wild-type alphavirus sequences are well known and available from sequence collections, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Geta virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus (ATCC VR-1277), Marburg (ATCC VR-370), Mukumbu virus (ATCC VR-580, ATCC VR-1244), Ndum (ATCC VR-371), Pixona virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373), and VR-373, ATCC VR-1246), Semliki Forest Virus (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 Encephalitis Virus (ATCC VR-69, ATCC VR-923, ATCC VR-1250ATCC VR-1249, ATCC VR-532), Western Equine Encephalitis Virus (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Vodaro River (ATCC VR-926), and Y-62-33 (ATCC VR-375). In some aspects, one or more alphaviruses in the list can be excluded.

[0079] 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 can be equal to any one of 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, at least any one, at most any one or any two. In some cases, the self-amplifying RNA can 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 8 kb. 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.

[0080] In some embodiments, the self-amplifying RNA molecule can encode a single polypeptide antigen, or optionally, two or more polypeptide antigens that are linked together (e.g., tandemly linked) in a manner that each sequence retains its identity when expressed as an amino acid sequence. The polypeptide generated by the self-amplifying RNA can then be produced as a fusion polypeptide, or modified in a manner that results in a separate polypeptide or peptide sequence. In some embodiments, the saRNA molecule can encode one or more polypeptides of interest, such as an antigen or more than one antigen, such as two, three, four, five, six, seven, eight, nine, ten or more polypeptides. Alternatively or in addition, a saRNA molecule can also encode more than one polypeptide of interest, such as an antigen, such as a bicistronic or tricistronic RNA molecule encoding different antigens or the same antigen.

[0081] As used herein, the term "connection" refers to a first amino acid sequence or polynucleotide sequence covalently or non-covalently connected to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence can be directly connected or juxtaposed to the second amino acid or polynucleotide sequence, or alternatively the intervening sequence can covalently connect the first sequence to the second sequence. The term "connection" not only means the fusion of the first RNA molecule at the 5' end or 3' end with the RNA molecule, but also includes inserting the entire first RNA molecule into any two nucleotides in the second RNA molecule. The first second RNA molecule can be connected to the second RNA molecule by a phosphodiester bond or a joint. The joint can be, for example, a polynucleotide.

[0082] In some embodiments, the self-amplifying RNA described herein can encode one or more polypeptide antigens including a series of epitopes. In some embodiments, the self-amplifying RNA described herein can encode epitopes capable of eliciting a helper T cell response or a cytotoxic T cell response or both.

[0083] In some embodiments, the saRNA molecules are purified, e.g., such as by filtration, which can occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration.

[0084] Some embodiments of the present disclosure relate to compositions comprising a self-amplifying RNA molecule comprising a 5' cap, a 5' untranslated region, a coding region comprising a sequence encoding an RNA-dependent RNA polymerase (also known as a "replicase"), a subgenomic promoter, such as a subgenomic promoter derived from an alphavirus, an open reading frame encoding a gene of interest (e.g., an antigen derived from an influenza virus), a 3' untranslated region, and a 3' poly A sequence. In some embodiments, at least 5% of the total population of specific nucleotides in the saRNA molecule has been replaced by one or more modified or non-natural nucleotides.

[0085] In some embodiments, the saRNA molecule does not include modified nucleotides, for example, does not include modified nucleobases, and all nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C, except for an optional 5' cap, which can include, for example, 7-methylguanosine, which is further described below. In some embodiments, the RNA can include a 5' cap comprising 7'-methylguanosine, and the first 1, 2, or 3 5' ribonucleotides can be methylated at the 2' position of the ribose.

[0086] The efficacy of the product depends on the expression of the delivered saRNA, which requires sufficiently intact RNA molecules. RNA integrity is a measure of RNA quality that quantifies intact RNA. The method is also capable of detecting potential degradation products. RNA integrity is preferably determined by capillary gel electrophoresis. The initial specifications are set to ensure sufficient RNA integrity in the drug product formulation. In some embodiments, the RNA polynucleotide has at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% integrity. In some embodiments, the RNA polynucleotide has about 95% or greater than about 95% integrity. In some embodiments, the RNA polynucleotide has about 98% or greater than about 98% integrity. In some embodiments, the RNA polynucleotide has about 99% or greater than about 99% integrity.

[0087] In preferred embodiments, saRNA polynucleotides have clinical grade purity. In some embodiments, the purity of RNA polynucleotides is between about 60% and about 100%. In some embodiments, the purity of RNA polynucleotides is between about 80% and 99%. In some embodiments, the purity of RNA polynucleotides is between about 90% and about 99%. In some embodiments, wherein the purified mRNA has clinical grade purity without further purification. In some embodiments, clinical grade purity is achieved by a method comprising tangential flow filtration (TFF) purification. In some embodiments, clinical grade purity is achieved without further purification, and the further purification is selected from high performance liquid chromatography (HPLC) purification, ligand or binding-based purification and / or ion exchange chromatography. In some embodiments, the method for producing RNA polynucleotides removes long abortion RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual solvents and / or residual salts. In some embodiments, short abortion transcript contaminants contain less than 15 bases. In some embodiments, short abortion transcript contaminants contain about 8-12 bases. In some embodiments, the methods of the invention also remove RNase inhibitors.

[0088] In some embodiments, the purified saRNA polynucleotides contain 5% or less, 4% or less, 3% or less, 2% or less, 1% or less protein contaminants, or are substantially free of protein contaminants as determined by capillary electrophoresis. In some embodiments, the purified RNA polynucleotides contain less than 5%, less than 4%, less than 3%, less than 2%, less than 1% salt contaminants, or are substantially free of salt contaminants as determined by high performance liquid chromatography (HPLC). In some embodiments, the purified RNA polynucleotides contain 5% or less, 4% or less, 3% or less, 2% or less, 1% or less short aborted transcript contaminants, or are substantially free of short aborted transcript contaminants, as determined by known methods, such as high performance liquid chromatography (HPLC). In some embodiments, the purified RNA polynucleotides have 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 integrity as determined by known methods, such as capillary electrophoresis.

[0089] B. Modified Nucleobases

[0090] Modified nucleobases that can be incorporated into modified nucleosides and nucleotides and are present in 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-N6-isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); adenosine); g6A (N6-glycylaminoformyladenosine); t6A (N6-threonylaminoformyladenosine); ms2t6A (2-methylthio-N6-threonylaminoformyladenosine); m6t6A (N6-methyl-N6-threonylaminoformyladenosine); hn6A (N6-hydroxynorvalylaminoformyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine); Ar(p) (2'-0-ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (1,2'-0-dimethylinosine); m3C (3-methylcytidine); Cm (2T-0-methylcytidine); s2C (2-thio Cytidine); ac4C (N4-acetylcytidine); ￡5C (5-fonnylcytidine); m5Cm (5,2-0-dimethylcytidine); ac4Cm (N4-acetyl 2TO methylcytidine); k2C (lysine); mlG (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 (whiting glycoside); o2yW (peroxywhiting glycoside) guanosine); OHyW (hydroxy wyoside); OHyW* (undermodified hydroxy wyoside); imG (wyoside); mimG (methylguanosine); Q (quercetin); oQ (epoxyquercetin); galQ (galactosylquercetin); manQ (mannosylquercetin); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaurine); 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-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl -2-thiouridine); mnm5U (5-methylaminomethyl uridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-seleno-uridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-0-methyl uridine); cmnm5U (5-carboxymethylaminomethyl uridine); cnmm5Um (5-carboxymethyl 1-aminomethyl-2-0-methyl uridine); cmnm5s2U (5-carboxymethyl 1-aminomethyl-2-0-methyl uridine) methylaminomethyl-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); m2'2'7 G (N2,N2,7-trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-0-methylcytidine); mlGm (l,2'-0-dimethylguanosine); m'Am (1,2-0-dimethyladenosine) iminomethyluridine (irinomethyluridine); tm5s2U (S-taurine methyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine);ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxoadenine, its 7-substituted derivatives, 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-fluoro Cytosine, 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 or 2'-O-methyl-U. In some aspects, one or more modified nucleosides in the list can be excluded. ;

[0091] 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.

[0092] In some embodiments, the RNA molecules can include phosphoramidite, phosphorothioate, and / or methylphosphonate linkages.

[0093] In some embodiments, the RNA molecule includes a modified nucleotide selected from any one of the following: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. In some embodiments, the modified or non-natural nucleotide is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deazapseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. In some embodiments, the modified or non-natural nucleotide is selected from 5-methyluridine, N1-methylpseudouridine, 5-methoxyuridine, and 5-methylcytosine.

[0094] In some embodiments, at least 10% of the total population of specific nucleotides in the saRNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the total population of specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, substantially all of the specific nucleotide population in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0095] In some embodiments, at least a portion or all of the total population of specific nucleotides in the saRNA molecule has been replaced with two modified or non-natural nucleotides. In some embodiments, the two modified or non-natural nucleotides are provided in a ratio equal to any one, at least any one, at most any one, 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; and 99:1, or any range derivable therein.

[0096] In some embodiments, at least 10% of the total population of the first specific nucleotides in the saRNA molecules disclosed herein have been replaced by one or more modified or non-natural nucleotides, and at least 10% of the total population of the second specific nucleotides in the molecule have been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotides in the molecule have been replaced by one or more modified or non-natural nucleotides, and at least 25% of the total population of the second specific nucleotides in the molecule have been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotides in the molecule have been replaced by one or more modified or non-natural nucleotides, and at least 50% of the total population of the second specific nucleotides in the molecule have been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotides in the molecule have been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of the second specific nucleotides in the molecule have been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and at least 25% of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and at least 50% of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of a first specific nucleotide in the molecule has been replaced by one or more modified or non-natural nucleotides, and at least 50% of the total population of a second specific nucleotide in the molecule has been replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 50% of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, substantially all of the total population of the first specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of the second specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0097] In some embodiments, at least 25% of the total population of uridine nucleotides in the saRNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule has been replaced with N1-methylpseudouridine. In some embodiments, at least 75% of the total population of uridine nucleotides in the molecule has been replaced with N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule has been replaced with 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced with 5-methoxyuridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule has been replaced with 5-methyluridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced with 5-methyluridine. In some embodiments, at least 50% of the total population of cytosine nucleotides in the molecule has been replaced with 5-methylcytosine. In some embodiments, substantially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule have been replaced with 2-thiouridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced with 2-thiouridine.

[0098] In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule has been replaced by N1-methyl pseudouridine, and substantially all cytosine nucleotides in the molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule has been replaced by 5-methoxyuridine, and substantially all cytosine nucleotides in the molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the molecule has been replaced by 5-methyluridine, and substantially all cytosine nucleotides in the molecule have been replaced by 5-methylcytosine.

[0099] In some embodiments, substantially all uridine nucleotides in the molecule have been replaced by about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced by about 75% 5-methoxyuridine and about 25% N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced by about 25% 5-methoxyuridine and about 75% N1-methylpseudouridine.

[0100] C.UTR

[0101] The 5' untranslated region (UTR) is a DNA regulatory region located at the 5' end of a protein coding sequence, which is transcribed into mRNA but not translated into protein. The 5'UTR may contain various regulatory elements, such as a 5' cap structure, a stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in the control of translation initiation. The 3'UTR located downstream of the protein coding sequence may be involved in regulatory processes, including transcript cleavage, stability and polyadenylation, translation, and mRNA localization. In some embodiments, UTR is derived from mRNA naturally abundant in a specific tissue (e.g., lymphoid tissue), and mRNA expression targets the specific tissue. In some embodiments, UTR increases protein synthesis. Without being bound by mechanism or theory, UTR may increase protein synthesis by increasing the time that mRNA stays in a translation polysome (messenger stability) and / or the rate at which ribosomes initiate translation on messengers thereunder (messenger translation efficiency). Accordingly, UTR sequences may extend protein synthesis in a tissue-specific manner. In some embodiments, 5'UTR and 3'UTR sequences are calculated. In some embodiments, 5'UTR and 3'UTR are derived from mRNA naturally abundant in tissue. The tissue can be, for example, liver, stem cell or lymphoid tissue. Lymphoid tissue can include, for example, lymphocytes (e.g., B lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or natural killer cells), macrophages, monocytes, dendritic cells, neutrophils, eosinophils and reticulocytes. In some embodiments, 5'UTR and 3'UTR are derived from alpha viruses. In some embodiments, 5'UTR and 3'UTR are from wild-type alpha viruses. Examples of alpha viruses are described below.

[0102] In some embodiments, the first RNA molecule comprises a 5'UTR and a 3'UTR derived from a naturally abundant mRNA in a tissue. In some embodiments, the first RNA molecule comprises a 5'UTR and a 3'UTR derived from an alphavirus. In some embodiments, the second RNA or saRNA molecule comprises a 5'UTR and a 3'UTR derived from an alphavirus. In some embodiments, the second RNA or saRNA molecule comprises a 5'UTR and a 3'UTR derived from a wild-type alphavirus. In some embodiments, the RNA molecule comprises a 5' cap.

[0103] D. Open reading frame (ORF)

[0104] The 5' and 3' UTRs may be operably linked to an ORF, which may be a codon sequence that can be translated into a polypeptide of interest.As described above, an RNA molecule may include one (monocistronic), two (bicistronic) or more (polycistronic) open reading frames (ORFs).

[0105] In some embodiments, the ORF encodes a nonstructural viral gene. In some embodiments, the ORF further comprises one or more subgenomic promoters. In some embodiments, the RNA molecule comprises a subgenomic promoter operably linked to the ORF. In some embodiments, the subgenomic promoter comprises a cis-acting regulatory element. In some embodiments, the cis-acting regulatory element is adjacent to the B 2 In some embodiments, the cis-acting regulatory element is immediately downstream (5'-3') of the guanine that is immediately downstream (5'-3') of B 2 Downstream of. In some embodiments, the cis-acting regulatory element is an AU-rich element. In some embodiments, the AU-rich element is au, auaaaagau, auaaaaagau, auag, auauauauau, auauauau, auauauauauau, augaugaugau, augau, auaaaagaua or auaaaagaug. In some embodiments, the second RNA or saRNA molecule may include (i) an ORF encoding a replicase that may transcribe RNA from the second RNA or saRNA molecule, and (ii) an ORF encoding at least one antigen or polypeptide of interest. The polymerase may be an alphavirus replicase, for example, including any one or a combination of non-structural alphavirus proteins nsP1, nsP2, nsP3 and nsP4. In some embodiments, the RNA molecule includes an alphavirus non-structural protein nsP1. In some embodiments, the RNA molecule includes an alphavirus non-structural protein nsP2. In some embodiments, the RNA molecule includes an alphavirus non-structural protein nsP3. In some embodiments, the RNA molecule includes an alphavirus non-structural 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.

[0106] In some embodiments, the open reading frame of the RNA (e.g., saRNA) composition is codon optimized. In some embodiments, the open reading frame encoding an influenza polypeptide or fragment thereof is codon optimized.

[0107] E. Gene encoding antigenic polypeptide

[0108] In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or an immunogenic fragment thereof. In some embodiments, the hemagglutinin protein is H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18 or an immunogenic fragment thereof. In some embodiments, the hemagglutinin protein does not include a head domain. In some embodiments, the hemagglutinin protein includes a portion of a head domain. In some embodiments, the hemagglutinin protein does not include a cytoplasmic domain. In some embodiments, the hemagglutinin protein includes a portion of a cytoplasmic domain. In some embodiments, the truncated hemagglutinin protein includes a portion of a transmembrane domain.

[0109] Some embodiments provide influenza vaccines, which include one or more RNA polynucleotides and a pharmaceutically acceptable carrier or excipient having an open reading frame encoding a hemagglutinin protein formulated in 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 a neuraminidase (NA) protein. In some embodiments, the hemagglutinin protein is derived from a strain of influenza A virus or influenza B virus or a combination thereof. In some embodiments, the influenza virus is selected from H1N1, H3N2, H7N9 and H10N8.

[0110] In some embodiments, the virus is a strain of influenza A or influenza B or a combination 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 a fragment thereof. In some embodiments, the hemagglutinin protein is H7 or H10 or a fragment thereof. In some embodiments, the hemagglutinin protein comprises a part of the head domain (HA1). In some embodiments, the hemagglutinin protein comprises a part of the cytoplasmic domain. In some embodiments, a truncated hemagglutinin protein. In some embodiments, the protein is a truncated hemagglutinin protein comprising a part of a transmembrane domain. In some embodiments, the virus is selected from H7N9 and H10N8. Protein fragments, functional protein domains and homologous proteins are also considered to be within the scope of the polypeptide of interest. For example, provided herein are any protein fragments of a reference protein (meaning a polypeptide sequence that is at least one amino acid residue shorter than a reference polypeptide sequence but is otherwise identical) that are 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids in length.

[0111] In some embodiments, at least one antigenic polypeptide is one of the defined antigenic subdomains of HA referred to as 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), nonstructural protein 1 (NS1), and nonstructural protein 2 (NS2).

[0112] In some embodiments, at least one antigenic polypeptide is HA or a derivative 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.

[0113] In some embodiments, at least one antigenic polypeptide is HA or a derivative 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.

[0114] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an influenza virus protein or an immunogenic fragment thereof.

[0115] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding multiple influenza virus proteins or immunogenic fragments thereof.

[0116] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one HA1, HA2, or a combination of both).

[0117] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one HA1, HA2, or a combination of both, any one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or any or all combinations thereof), and at least one other RNA (e.g., saRNA) polynucleotide having an open reading frame encoding a protein selected from the group consisting of: an HA protein, an NP protein, a NA protein, an M1 protein, an M2 protein, an NS1 protein, and an NS2 protein obtained from an influenza virus.

[0118] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one, any one, or any or all combinations of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18), and at least two other RNA (e.g., saRNA) polynucleotides having two open reading frames encoding two proteins selected from the group consisting of: an HA protein, an NP protein, a NA protein, an M1 protein, an M2 protein, an NS1 protein, and an NS2 protein obtained from an influenza virus.

[0119] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one of any one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or any or all combinations thereof), and at least three other RNA (e.g., saRNA) polynucleotides having three open reading frames encoding three proteins selected from the group consisting of: an HA protein, an NP protein, a NA protein, an M protein, an M2 protein, an NS1 protein, and an NS2 protein obtained from an influenza virus.

[0120] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one of any one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or any or all combinations thereof), and at least four other RNA (e.g., saRNA) polynucleotides having four open reading frames encoding four proteins selected from the group consisting of: an HA protein, an NP protein, a NA protein, an M1 protein, an M2 protein, an NS1 protein, and an NS2 protein obtained from an influenza virus.

[0121] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof (e.g., at least one of any one of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or any or all combinations thereof), and at least five other RNA (e.g., saRNA) polynucleotides having five open reading frames encoding five proteins selected from the group consisting of: an HA protein, an NP protein, a NA protein, an M1 protein, an M2 protein, an NS1 protein, and an NS2 protein obtained from an influenza virus.

[0122] In some embodiments, the saRNA composition comprises at least one RNA (e.g., saRNA) polynucleotide having an open reading frame encoding an HA protein or an immunogenic fragment thereof obtained from an influenza virus (e.g., at least one of any of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and / or H18, or any or all combinations thereof), an HA protein, an NP protein or an immunogenic fragment thereof, an NA protein or an immunogenic fragment thereof, an M1 protein or an immunogenic fragment thereof, an M2 protein or an immunogenic fragment thereof, an NS1 protein or an immunogenic fragment thereof, and an NS2 protein or an immunogenic fragment thereof.

[0123] In some embodiments, the influenza RNA composition includes a saRNA encoding an antigen fusion protein. Therefore, the encoded one or more antigens may include two or more proteins (e.g., proteins and / or protein fragments) linked together. Alternatively, the protein to which the protein antigen is fused does not promote a strong immune response to itself, but to a strong immune response to the influenza antigen. In some embodiments, the antigen fusion protein retains the functional properties from each source protein.

[0124] F.5' Cap

[0125] In some embodiments, the saRNA molecules described herein include a 5' cap. In some embodiments, the 5' cap portion is a native 5' cap.

[0126] "Natural 5' cap" is defined as a cap comprising a 7-methylguanosine bonded to the 5' end of an mRNA molecule by a 5' to 5' triphosphate bond. In some embodiments, the 5' cap portion is a 5' cap analog. In some embodiments, the 5' end of the RNA is capped with a modified ribonucleotide having the structure m7G(5')ppp(5')N (cap 0 structure) or a derivative thereof, which can be incorporated during RNA synthesis (e.g., co-transcriptional capping) or can be enzymatically modified 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 of the RNA molecule, such as tangential flow filtration. Exemplary enzymatic reactions for capping can include the use of a vaccinia virus capping enzyme (VCE), which includes an mRNA triphosphatase, a guanylate transferase, and a guanine-7-methyltransferase, which catalyzes the construction of a N7-monomethylated cap 0 structure. Cap 0 structure can help maintain the stability and translation efficacy of RNA molecules. The 5' cap of RNA molecules can be further modified by 2'-O-methyltransferase, which leads to the generation of cap 1 structure (m7Gppp[m2'-O]N), which can further increase translation efficacy. In some embodiments, RNA molecules can be enzymatically capped at the 5' end using vaccinia guanylate transferase, guanosine triphosphate and S-adenosine-L-methionine to produce cap 0 structure. Reverse 7-methylguanosine cap is added via 5' to 5' triphosphate bridge. Alternatively, the use of 2'O-methyltransferase and vaccinia guanylate transferase produces cap 1 structure, wherein in addition to cap 0 structure, 2'OH group is methylated on the penultimate nucleotide. S-adenosine-L-methionine (SAM) is a cofactor used as a methyl transfer reagent. A non-limiting example of a 5' cap structure is a structure that has enhanced binding of a cap-bound polypeptide, increased half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' decapping, compared to synthetic 5' cap structures known in the art (or wild-type, natural or physiological 5' cap structures). For example, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase may produce a classic 5'-5'-triphosphate bond 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 group. Such a structure is referred to as a cap 1 structure. Compared to other 5' cap analog structures known in the art, for example, the cap results in higher translational capacity and cellular stability, as well as reduced activation of cellular proinflammatory cytokines. 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 an N7-methylguanosine bonded to the 5' nucleotide by a 5' to 5' triphosphate bond, commonly referred to as an m7G cap or m7Gppp. In cells, the cap 0 structure can help provide efficient translation of the mRNA carrying the cap. Additional methylation at the 2'O position of the start nucleotide generates cap 1, or is referred to as m7GpppNm-, where Nm represents any nucleotide with 2'O methylation. In some embodiments, the 5' end cap includes a cap analog, for example, the 5' end cap may include a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methylguanosine, 2'fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNAguanosine, and 2-azidoguanosine. In some embodiments, the capping region may include a single cap or a series of nucleotides forming a cap. In this embodiment, the length of the capping region can be equal to any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at least any one, at most any one, or between any two, or at least 2 or 10 or fewer nucleotides. In some embodiments, the cap is absent. In some embodiments, the length of the first operational region and the second operational region can be equal to any one, at least any one, at most any one, or between any two of 3 to 40 nucleotides, such as 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 less nucleotides, and may include one or more signal and / or restriction sequences in addition to the start and / or stop codons.

[0127] In some embodiments, the 5' cap is represented by Formula I:

[0128]

[0129] Where R 1 and R 2 are each independently H or Me, and B 1 and B 2 Each is independently guanine, adenine or uracil. In some embodiments, B 1 and B 2 is a naturally occurring base. In some embodiments, R 1 is methyl and R 2 In some embodiments, B 1 In some embodiments, B 1 In some embodiments, B 2In some embodiments, B 2 In some embodiments, B 2 is uracil, and the molecule is located at B 2 At least 5% of the total population of downstream uracil nucleotides has been replaced by one or more modified or non-natural nucleotides.

[0130] In some embodiments, the nucleotide immediately downstream (5' to 3' direction) of the 5' cap comprises guanine. 1 Adenine and B 2 In some embodiments, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen. In some cases, the saRNA does not comprise a 5' cap. In some cases, 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, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen; this embodiment corresponds to CleanCap AU, and B 2 = uracil, optionally substituted with B 2 Downstream uracil nucleotides have 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.

[0131] In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap comprises a guanine, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, at least 50% of the total population of uridine nucleotides in the molecule have been replaced by N1-methylpseudouridine, and substantially all of the cytosine nucleotides in the molecule have been replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap comprises guanine, B 1 Adenine, B2 is uracil, R 1 is methyl, and R 2 is hydrogen, at least 50% of the total population of uridine nucleotides in the molecule have been replaced by 5-methoxyuridine, and substantially all of the cytosine nucleotides in the molecule have been replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap comprises guanine, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, at least 50% of the total population of uridine nucleotides in the molecule have been replaced by 5-methyluridine, and substantially all of the cytosine nucleotides in the molecule have been replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap comprises guanine, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, and substantially all of the uridine nucleotides in the molecule have been replaced by about 50% 5-methoxyuridine and about 50% N1-methyl pseudouridine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap comprises guanine, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, and substantially all of the uridine nucleotides in the molecule have been replaced by about 75% 5-methoxyuridine and about 25% N1-methyl pseudouridine. In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap comprises guanine, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, and essentially all of the uridine nucleotides in the molecule have been replaced by about 25% 5-methoxyuridine and about 75% N1-methylpseudouridine.

[0132] In some embodiments, the 5' terminal cap is 7mG(5')ppp(5')NlmpNp. In some preferred embodiments, the 5' cap comprises: In some embodiments, the 5' cap comprises a Reagent AG(3'OMe), m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, In an alternative embodiment, the 5' cap comprises a AU, for co-transcriptional capping of mRNA ReagentAU、m7G(5')ppp(5')(2'OMeA)pU、

[0133]

[0134] G. Poly A tail

[0135] As used herein, "poly A tail" refers to a continuous adenine residue that can be attached to the 3' end of an RNA molecule. The poly A tail can increase the half-life of an RNA molecule. The poly A tail can play a key regulatory role in enhancing translation efficiency and regulating mRNA quality control and degradation efficiency. Short sequences or high polyadenylation can send signals for RNA degradation. Exemplary designs include poly A tails of about 40 adenine residues to about 80 adenylic acid 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, for example, for a second RNA or saRNA molecule, the RNA molecule further includes a poly A polymerase recognition sequence (e.g., AAUAAA) close to its 3' end. A "full-length" RNA molecule is a molecule including a 5' cap and a poly A tail.

[0136] In some embodiments, the poly A tail comprises 5-400 nucleotides in length. The poly A tail nucleotide length can be equal to any one of the following, at least any one of the following, at most any one of the following, or between any two of the following: 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 including a sequence of about 25 to about 400 adenosine nucleotides, about 50 to about 400 adenosine nucleotides, about 50 to about 300 adenosine nucleotides, about 50 to about 250 adenosine nucleotides, about 60 to about 250 adenosine nucleotides, or about 40 to about 100 adenosine nucleotides. In some embodiments, the RNA molecule includes a poly A tail including a sequence of more than 30 adenosine nucleotides ("A"). In some embodiments, the RNA molecule includes a poly A tail including about 40 A's. In some embodiments, the RNA molecule includes a poly A tail including about 80 A's. As used herein, the term "about" refers to a deviation of ±10% of the value to which it is attached. In some embodiments, the 3' poly A tail has a segment of at least 10 consecutive adenosine residues and up to 300 consecutive adenosine residues. In some embodiments, the RNA molecule includes at least 20 consecutive adenosine residues and up to 40 consecutive adenosine residues. In some embodiments, the RNA molecule comprises about 40 consecutive adenosine residues. In some embodiments, the RNA molecule comprises about 80 consecutive adenosine residues.

[0137] H. Composition

[0138] In some cases, the compositions described herein include at least one saRNA as described herein. Some embodiments of the present disclosure provide an influenza virus (flu) vaccine (or composition or immunogenic composition) comprising at least one saRNA polynucleotide having an open reading frame encoding at least one influenza antigen polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response against influenza).

[0139] In some embodiments, the total RNA molecules (capped and uncapped) in the composition are capped: 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%.

[0140] In some embodiments, the total RNA molecules in the composition equal to any one, at least any one, at most any one, or between any two are full-length RNA transcripts: 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%. Purity can be determined as described herein, for example, via reversed-phase HPLC or electrophoresis based on a Bioanalyzer chip, and measured, for example, by the peak area of ​​the full-length RNA molecule relative to the total peak. In some embodiments, a fragment analyzer (FA) can be used for quantitative and purified RNA. Fragment analyzers automate capillary electrophoresis and HPLC.

[0141] In some embodiments, the composition is substantially free of one or more impurities or contaminants including linear DNA templates and / or reverse complementary transcription products, and, for example, includes RNA molecules having a purity equal to any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, at least any one, at most any one, or between any two; a purity of at least 98% or a purity of at least 99%.

[0142] In some embodiments, the amount of the first RNA molecule included in the composition is greater than the amount of the second RNA molecule. In some embodiments, the amount of the first RNA molecule included in the composition is at least about 1 to 2 times the amount of the second RNA molecule. In some embodiments, the amount of the first RNA molecule included in the composition is at least about 1 to 100 times the amount of the second RNA molecule.

[0143] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a pharmaceutically acceptable vehicle.

[0144] In some embodiments, the composition further includes a lipid-based delivery system that delivers the RNA molecule to the interior of the cell, where it can then replicate and / or express the encoded polypeptide of interest. The delivery system can have an adjuvant effect that enhances the immunogenicity of the encoded antigen. In some embodiments, the composition further includes a neutral lipid, a cationic lipid, cholesterol, and polyethylene glycol (PEG), and forms nanoparticles comprising the RNA molecule. In some embodiments, the composition further includes any one of cationic lipids, liposomes, lipid nanoparticles, polyplexes, cochleates, virosomes, immunostimulatory complexes, microparticles, microspheres, nanospheres, unilamellar vesicles, multilamellar vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsions, polycationic peptides, and cationic nanoemulsions. In some embodiments, the RNA molecule is encapsulated in, associated with, or adsorbed on any of the following: a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a lipid roll, a virosome, an immunostimulatory complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsion body, a polycationic peptide, and a cationic nanoemulsion, or a combination thereof.

[0145] In some cases, the compositions described herein include at least two RNA molecules: a first saRNA molecule and a second RNA molecule as described herein. In order to protect against more than one influenza strain, a combination vaccine composition can be administered that includes RNA (e.g., saRNA) 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 (e.g., saRNA) can be co-formulated, for example, in a single lipid nanoparticle (LNP), or can be formulated in separate LNPs for co-administration.

[0146] 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 portion. 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 portion, a 5' UTR, a coding region, a 3' UTR, and a 3' poly A sequence. In some embodiments, the second RNA molecule includes a 5' cap portion, a 5' UTR, a non-coding region, a 3' UTR, and a 3' poly A sequence. In some embodiments, the second RNA molecule includes a non-coding region and does not further include any one of a 5' cap portion, a 5' UTR, a 3' UTR, and a 3' poly A sequence. In some embodiments, the second RNA molecule includes a 5' cap portion, a 5' untranslated region (5'UTR), modified nucleotides, an open reading frame, a 3' untranslated region (3'UTR), and a 3' poly A sequence.

[0147] Some aspects of the present disclosure relate to a composition comprising (i) a first RNA molecule encoding a target gene derived from influenza; and (ii) a second RNA molecule comprising modified or non-natural nucleotides. In some cases, the first RNA molecule is any saRNA molecule described herein. In some cases, the first RNA molecule comprises a 5' cap, a 5' untranslated region, a coding region for a nonstructural protein containing an RNA replicase, a subgenomic promoter, an open reading frame encoding a target gene, a 3' untranslated region, and a 3' poly A sequence. In some cases, at least 5% of the total population of specific nucleotides in the first RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some cases, the saRNA molecule comprises natural, unmodified nucleotides and does not include modified or non-natural nucleotides. In some cases, the 5' cap is represented by Formula I, wherein 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 a subgenomic promoter 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.

[0148] In some embodiments, B 1 Adenine and B 2 In some embodiments, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 In some embodiments, the nucleotide immediately downstream (5' to 3') of the 5' cap comprises guanine, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen; this embodiment corresponds to CLEANCAP AU (Trilink), and B 2 = uracil, optionally substituted with B 2 Downstream uracil nucleotides, which in some embodiments have been shown to provide increased saRNA functionality.

[0149] In some embodiments, at least 10% of the total population of a specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of a specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of a specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the total population of a specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, substantially all of the specific nucleotide population in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, one or more modified or non-natural replacement nucleotides include two modified or non-natural nucleotides provided in a ratio ranging from 1:99 to 99:1 or any derivable range thereof. In some embodiments, at least 10% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 10% of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 25% of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 50% of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 10% of the total population of first specific nucleotides in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of second specific nucleotides in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides.In some embodiments, at least 25% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 25% of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 50% of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 25% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and at least 75% of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 50% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides. In some embodiments, at least 75% of the total population of the first specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides, and substantially all of the total population of the second specific nucleotide in the first RNA molecule or the second RNA molecule has been replaced by one or more modified or non-natural nucleotides.

[0150] In some embodiments, at least 25% of the total population of uridine nucleotides in the first RNA molecule has been replaced by N1-methylpseudouridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the first RNA molecule has been replaced by N1-methylpseudouridine. In some embodiments, at least 75% of the total population of uridine nucleotides in the first RNA molecule has been replaced by N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by N1-methylpseudouridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the first RNA molecule has been replaced by 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides in the molecule have been replaced by 5-methoxyuridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the first RNA molecule has been replaced by 5-methyluridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by 5-methyluridine. In some embodiments, at least 50% of the total population of cytosine nucleotides in the first RNA molecule has been replaced by 5-methylcytosine. In some embodiments, substantially all cytosine nucleotides in the first RNA molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the first RNA molecule have been replaced by 2-thiouridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced by 2-thiouridine.

[0151] In some embodiments, at least 25% of the total population of uridine nucleotides in the second RNA molecule has been replaced by N1-methyl pseudouridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by N1-methyl pseudouridine. In some embodiments, at least 75% of the total population of uridine nucleotides in the second RNA molecule has been replaced by N1-methyl pseudouridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by N1-methyl pseudouridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by 5-methoxyuridine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by 5-methyluridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by 5-methyluridine. In some embodiments, at least 50% of the total population of cytosine nucleotides in the second RNA molecule has been replaced by 5-methylcytosine. In some embodiments, substantially all cytosine nucleotides in the second RNA molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule have been replaced by 2-thiouridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by 2-thiouridine.

[0152] In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by N1-methyl pseudouridine, and substantially all cytosine nucleotides in the second RNA molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by 5-methoxyuridine, and substantially all cytosine nucleotides in the second RNA molecule have been replaced by 5-methylcytosine. In some embodiments, at least 50% of the total population of uridine nucleotides in the second RNA molecule has been replaced by 5-methyluridine, and substantially all cytosine nucleotides in the second RNA molecule have been replaced by 5-methylcytosine.

[0153] In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by about 75% 5-methoxyuridine and about 25% N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides in the second RNA molecule have been replaced by about 25% 5-methoxyuridine and about 75% N1-methylpseudouridine.

[0154] In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine, and at least 50% of the total population of uridine nucleotides in the second RNA molecule have been replaced with N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine, and substantially all uridine nucleotides in the second RNA molecule have been replaced with N1-methylpseudouridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine, and at least 50% of the total population of uridine nucleotides in the second RNA molecule have been replaced with 5-methoxyuridine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine, at least 50% of the total population of uridine nucleotides in the second RNA molecule have been replaced with 5-methyluridine, and substantially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, substantially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine, and substantially all uridine nucleotides in the second RNA molecule have been replaced with about 50% 5-methoxyuridine and about 50% N1-methylpseudouridine.

[0155] I. How to use

[0156] The saRNA composition can be used to treat and / or prevent influenza viruses of various genotypes, strains, and isolates. Some embodiments provide a method for preventing or treating influenza virus infection, comprising administering any saRNA composition described herein to a subject. 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 for producing an antigen-specific immune response involves a single administration of a saRNA composition. In some embodiments, the saRNA composition is administered to a subject by intradermal, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration.

[0157] In some embodiments, the RNA (e.g., saRNA) polynucleotides or portions thereof may encode one or more polypeptides of an influenza strain or fragments thereof as antigens.

[0158] Some aspects of the present disclosure relate to methods for inducing an immune response in a subject, comprising administering an effective amount of a composition as disclosed herein to a subject in need thereof. Some aspects of the present disclosure relate to methods for vaccinating a subject, comprising administering an effective amount of a composition as disclosed herein to a subject in need thereof. Some aspects of the present disclosure relate to methods comprising administering an effective amount of a composition as disclosed herein to a subject in need thereof. In some embodiments, a composition as disclosed herein induces an immune response comprising an antibody response. In some embodiments, a composition as disclosed herein induces an immune response comprising a T cell response.

[0159] Some embodiments of the present disclosure provide a method of inducing an antigen-specific immune response in a subject, comprising administering to the subject any RNA (e.g., saRNA) composition as provided herein, in an amount effective to produce an antigen-specific immune response. In some embodiments, the RNA (e.g., saRNA) composition is an influenza vaccine. In some embodiments, the RNA (e.g., saRNA) composition is a combination vaccine comprising a combination of influenza vaccines (broad-spectrum influenza vaccines).

[0160] In some embodiments, the antigen-specific immune response comprises a T cell response or a B cell response. In some embodiments, the method of generating an antigen-specific immune response comprises administering to the subject a single dose (without a booster dose) of an influenza RNA (e.g., saRNA) composition of the present disclosure. In some embodiments, the method further comprises administering to the subject a second (boost) dose of an influenza RNA (e.g., saRNA) composition. Additional doses of influenza RNA (e.g., saRNA) compositions may be administered.

[0161] In some embodiments, the subject exhibits a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) after the first dose or the second (boost) dose of the vaccine. Seroconversion is the period of time during which specific antibodies develop and become detectable in the blood. After seroconversion has occurred, the virus can be detected in a blood test for antibodies. During infection or immunity, the antigen enters the blood and the immune system begins to produce antibodies in response. Before seroconversion, the antigen itself may be detectable or may be undetectable, but the antibody is considered absent. During seroconversion, the antibody is present but not yet detected. At any time after seroconversion, antibodies can be detected in the blood, indicating a previous infection or a current infection.

[0162] In some embodiments, the influenza RNA (e.g., saRNA) composition is administered to a subject by intradermal injection, intramuscular injection, or intranasal administration. In some embodiments, the influenza RNA (e.g., saRNA) composition is administered to a subject by intramuscular injection.

[0163] Some embodiments of the present disclosure provide a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject an effective amount of an influenza RNA (e.g., saRNA) composition to produce an antigen-specific immune response in the subject. In some embodiments, the antigen-specific immune response in the subject can be determined by measuring the antibody titer (titer for antibodies bound to influenza antigen polypeptides) after administering any influenza RNA (e.g., saRNA) composition of the present disclosure to the subject. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject increases by at least 1 log relative to the control. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject increases by 1-3 log relative to the control.

[0164] In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject is increased to at least 2 times relative to the control. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject is increased to at least 5 times relative to the control. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject is increased to at least 10 times relative to the control. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject is increased to 2-10 times relative to the control.

[0165] In some embodiments, the control is the anti-antigen polypeptide antibody titer produced in a subject that has not been administered an RNA (e.g., saRNA) composition of the present disclosure. In some embodiments, the control is the anti-antigen polypeptide antibody titer produced in a subject that has been administered live attenuated influenza or inactivated influenza, or wherein the control is the anti-antigen polypeptide antibody titer produced in a subject that has been administered a recombinant or purified influenza protein vaccine.

[0166] In some embodiments, the RNA (e.g., saRNA) composition is formulated in an effective amount to generate an antigen-specific immune response in a subject.

[0167] In some embodiments, the effective amount is a total dose of 1 μg to 1000 μg, or 1 μg to 100 μg of saRNA. In some embodiments, the effective amount is a total dose of 30 μg. In some embodiments, the effective amount is a total dose of 10 μg administered to a subject twice. In some embodiments, the effective amount is a total dose of 10 μg administered to a subject twice. In some embodiments, the effective amount is a total dose of 15 μg administered to a subject twice. In some embodiments, the effective amount is a total dose of 30 μg administered to a subject twice.

[0168] In some embodiments, the method comprises administering to the subject a saRNA composition described herein at a dose of 10 μg / kg to 400 μg / kg. In some embodiments, the dose of the saRNA polynucleotide is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg, 100- 200μg, 120-250μg, 150-250μg, 180-280μg, 200-300μg, 50-300μg, 80-300μg, 100-300μg, 40-300μg, 50-350μg, 100-350μg, 200-350μg, 300-350μg, 320-400μg, 40-380μg, 40-100μg, 100-400μg, 200-400μg, or 300-400μg / dose. In some embodiments, the saRNA composition is administered to the subject by intradermal injection or intramuscular injection. In some embodiments, the saRNA composition is administered to the subject on day zero. In some embodiments, the second dose of the saRNA composition is administered to the subject on day 21.

[0169] In some embodiments, the subject is about 5 years old or less. For example, the subject can be about 1 year old to about 5 years old (e.g., about 1, 2, 3, 5 or 5 years old), or about 6 months to about 1 year old (e.g., about 6, 7, 8, 9, 10, 11 or 12 months). In some embodiments, the subject is about 12 months or less (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 less.

[0170] In some embodiments, the subject is born at full term (e.g., about 37-42 weeks). In some embodiments, the subject is, for example, born prematurely 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 be born at about 32 weeks of gestation or earlier. In some embodiments, the subject is born prematurely between about 32 weeks of gestation to about 36 weeks. In such subjects, RNA (e.g., mRNA) vaccines may be administered in later life, for example, at about 6 months to about 5 years old or more.

[0171] In some embodiments, the subject is a young adult between about 20 and about 50 years old (eg, about 20, 25, 30, 35, 40, 45, or 50 years old).

[0172] In some embodiments, the subject is an elderly subject who is about 60 years old, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85, or 90 years old).

[0173] In some embodiments, the subject has been exposed to influenza (eg, C. trachomatis); the subject is infected with influenza (eg, C. trachomatis); or the subject is at risk of being infected with influenza (eg, C. trachomatis).

[0174] In some embodiments, the subject has been exposed to a betacoronavirus (e.g., SARS-CoV-2); the subject is infected with a betacoronavirus (e.g., SARS-CoV-2); or the subject is at risk of being infected by a betacoronavirus (e.g., SARS-CoV-2).

[0175] In some embodiments, the subject has received at least one dose of an immunogenic composition against a betacoronavirus (e.g., SARS-CoV-2), such as selected from Any of the Pfizer-BioNTech COVID-19 vaccine, Moderna mRNA-1273 COVID-19 vaccine, and Janssen COVID-19 vaccine; The subject has received at least two doses of an immunogenic composition against a betacoronavirus (e.g., SARS-CoV-2); The subject is currently receiving at least one dose of an immunogenic composition against a betacoronavirus (e.g., SARS-CoV-2), such as selected from Any of the Pfizer-BioNTech COVID-19 vaccine, Moderna mRNA-1273 COVID-19 vaccine, and Janssen COVID-19 vaccine; or the subject is at risk of infection with a betacoronavirus (e.g., SARS-CoV-2) and is being administered an immunogenic composition against a betacoronavirus (e.g., SARS-CoV-2), such as selected from the group consisting of concomitantly, simultaneously, or within 12-48 hours of any of the immunogenic compositions against influenza disclosed herein Any of the Pfizer-BioNTech COVID-19 vaccine, Moderna mRNA-1273 COVID-19 vaccine, and Janssen COVID-19 vaccine.

[0176] In some embodiments, the subject is immunocompromised (has a compromised immune system, eg, suffers from an immune disorder or an autoimmune disorder).

[0177] Aspects of the present disclosure provide saRNA compositions comprising one or more saRNA polynucleotides having an open reading frame encoding a first antigen polypeptide, wherein the saRNA polynucleotide is present in a formulation for in vivo administration to a host that confers an antibody titer superior to an acceptable percentage of human subjects for the seroprotection standard for a first antigen (e.g., HA). In some embodiments, the antibody titer produced by the saRNA compositions of the present 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 saRNA compositions is greater than an adjuvanted protein vaccine. In yet other embodiments, the neutralizing antibody titer produced by the saRNA composition 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. The neutralization titer is typically expressed as the highest serum dilution required to achieve a 50% reduction in the number of plaques.

[0178] J. Nucleic Acids

[0179] In certain embodiments, the nucleic acid sequence may be present in various situations, such as: an isolated segment and a recombinant vector of a recombinant polynucleotide incorporating a sequence or encoding a polypeptide, such as one or two chains of an antigen or antibody, or a fragment, derivative, mutant protein or variant thereof, a polynucleotide sufficient for use as a hybridization probe, a PCR primer or sequencing primer for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, an antisense nucleic acid for inhibiting the expression of a polynucleotide, mRNA, saRNA, and the aforementioned complementary sequences described herein. Nucleic acids encoding epitopes to which antibodies can bind. Nucleic acids encoding fusion proteins including these polypeptides are also provided. Nucleic acids may be single-stranded or double-stranded and may contain RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0180] The term "polynucleotide" refers to a nucleic acid molecule that can be recombinant or separated from a total genomic nucleic acid. Included within the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), and recombinant vectors include, for example, plasmids, cosmids, phages, viruses, and the like. In some aspects, a polynucleotide includes a regulatory sequence that is substantially separated from its naturally occurring gene or protein coding sequence. A polynucleotide can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA or synthetic), its analogs or combinations thereof. Additional coding sequences or non-coding sequences can, but need not be, present in a polynucleotide.

[0181] In this regard, the term "gene" is used to refer to nucleic acids (including any sequence required for correct transcription, post-translational modification or positioning) encoding proteins, polypeptides or peptides. As will be appreciated by those skilled in the art, the term encompasses genomic sequences, expression cassettes, cDNA sequences and smaller modified nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins and mutants. The nucleic acid encoding all or part of a polypeptide may contain all or part of the adjacent nucleic acid sequences encoding such polypeptides. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid containing a variation, the nucleic acid having a slightly different nucleotide sequence, but nevertheless encoding the same or substantially similar polypeptide.

[0182] In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein; using the methods described herein (e.g., BLAST analysis using standard parameters), compared to the polynucleotide sequences provided herein, comprising sequences equal to any one, at least any one, at most any one, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity. In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 90% identity to the amino acid sequence described herein over the entire sequence length; or a nucleotide sequence complementary to the isolated polynucleotide. In some embodiments, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 95% identity to the amino acid sequence described herein over the entire sequence length; or a nucleotide sequence complementary to the isolated polynucleotide.

[0183] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 14. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 15. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 16. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 17. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 19. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 20. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 22.

[0184] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 12; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 13; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 14; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 15; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: NO:16 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:17; a polynucleotide sequence encoding a polypeptide selected from the group consisting of HA, NA, NP, M1, M2, NS1 and NS2; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:19; a polynucleotide sequence encoding a polypeptide selected from the group consisting of HA, NA, NP, M1, M2, NS1 and NS2; NO:21 has a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity; and a poly (A) tail comprising at least 20 consecutive adenines.

[0185] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 12; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 13; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 14; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 15; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: NO:16 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:17; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:18; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:19; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:11; NO:20 has a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:21; and a poly(A) tail comprising at least 20 consecutive adenines.

[0186] In some embodiments, the polynucleotide comprises a 5'UTR sequence having SEQ ID NO: 12; a polynucleotide sequence having SEQ ID NO: 13; a polynucleotide sequence having SEQ ID NO: 14; a sequence having SEQ ID NO: 15; a polynucleotide sequence having SEQ ID NO: 16; a polynucleotide sequence having SEQ ID NO: 17; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1 and NS2; a polynucleotide sequence having SEQ ID NO: 19; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1 and NS2; a polynucleotide sequence having SEQ ID NO: 21; and a poly (A) tail comprising at least 20 consecutive adenines.

[0187] In some embodiments, the polynucleotide comprises a 5'UTR sequence having SEQ ID NO: 12; a polynucleotide sequence having SEQ ID NO: 13; a polynucleotide sequence having SEQ ID NO: 14; a sequence having SEQ ID NO: 15; a polynucleotide sequence having SEQ ID NO: 16; a polynucleotide sequence having SEQ ID NO: 17; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 18; a polynucleotide sequence having SEQ ID NO: 19; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 20; a polynucleotide sequence having SEQ ID NO: 21; and a poly A tail comprising at least 20 consecutive adenines.

[0188] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 23. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 24. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 25. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 26. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 27. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 28. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 29. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 30. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 31. In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:32.

[0189] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:23; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:24; a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:25; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:26; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:27; NO:27 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:28; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:29; a polynucleotide sequence encoding a polypeptide selected from the group consisting of HA, NA, NP, M1, M2, NS1 and NS2; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:29; a polynucleotide sequence encoding a polypeptide selected from the group consisting of HA, NA, NP, M1, M2, NS1 and NS2; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: NO:31 has a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity; and a poly (A) tail comprising at least 20 consecutive adenines.

[0190] In some embodiments, the polynucleotide comprises a 5'UTR sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:23; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:24; a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:25; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:26; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:27; NO:27 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:28; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:29; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:30; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:31; NO:31 has a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity; and a poly (A) tail comprising at least 20 consecutive adenines.

[0191] In some embodiments, the polynucleotide comprises a 5'UTR sequence of SEQ ID NO: SEQ ID NO: 23; a polynucleotide sequence having SEQ ID NO: 24; a polynucleotide sequence having SEQ ID NO: 25; a polynucleotide sequence having SEQ ID NO: 26; a polynucleotide sequence having SEQ ID NO: 27; a polynucleotide sequence having SEQ ID NO: 28; a polynucleotide sequence having SEQ ID NO: 29; a polynucleotide sequence encoding a polypeptide selected from HA, NA, NP, M1, M2, NS1 and NS2; a polynucleotide sequence having SEQ ID NO: 31; and a poly (A) tail comprising at least 20 consecutive adenines.

[0192] In some embodiments, the polynucleotide comprises a 5'UTR sequence of SEQ ID NO: SEQ ID NO: 23; a polynucleotide sequence having SEQ ID NO: 24; a polynucleotide sequence having SEQ ID NO: 25; a polynucleotide sequence having SEQ ID NO: 26; a polynucleotide sequence having SEQ ID NO: 27; a polynucleotide sequence having SEQ ID NO: 28; a polynucleotide sequence having SEQ ID NO: 29; a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 30; a polynucleotide sequence having SEQ ID NO: 31; and a poly A tail comprising at least 20 consecutive adenines. Regardless of the length of the coding sequence itself, the nucleic acid segment can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding fragments, etc., so that their overall length can vary greatly. The nucleic acids can be of any length. Their length can be, for example, equal to any one, at least any one, at most any one 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, and / or can contain one or more additional sequences, such as regulatory sequences, and / or can be part of a larger nucleic acid, such as a vector. It is therefore contemplated that nucleic acid fragments of almost any length can be used, with the total length being limited by the ease of preparation and the use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence with an additional heterologous coding sequence, for example to allow purification, transport, secretion, post-translational modification of the polypeptide, or for therapeutic benefit such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is different from the modified polypeptide.

[0193] K. Lipid Delivery

[0194] In some embodiments, the saRNA composition comprises lipids. The lipids and saRNA can form nanoparticles together. The lipids can encapsulate mRNA in the form of lipid nanoparticles (LNPs) to help the cell entry and stability of RNA / lipid nanoparticles.

[0195] Lipid nanoparticle may comprise lipid component and one or more other components, for example therapeutic agent and / or preventive agent.LNP may be designed for one or more specific applications or targets.The element of LNP may be selected based on specific application or target and / or based on effect, toxicity, expense, ease of use, availability or other characteristics of one or more elements.Similarly, may select the specific formulation of LNP for specific application or target according to effect and toxicity of the specific combination of for example element.Effect and tolerability of LNP preparation may be subject to the influence of formulation stability.

[0196] Lipid nanoparticles can be designed for one or more specific applications or targets. For example, LNPs can be designed to deliver therapeutic and / or prophylactic agents such as RNA to specific cells, tissues, organs, systems or groups thereof in the body of a mammal.

[0197] The physicochemical properties of lipid nanoparticles can be changed to increase the selectivity for specific body targets. For example, the pore size can be adjusted based on different organs. The therapeutic agent and / or preventive agent included in the LNP can also be selected based on the desired delivery target. For example, the therapeutic agent and / or preventive agent can be selected for specific indications, conditions, diseases or illnesses and / or for delivery to specific cells, tissues, organs, systems or their groups (for example, limited or specific delivery). In certain embodiments, LNP may include mRNA encoding a target polypeptide, which can be translated intracellularly to produce a target polypeptide. Such compositions can be designed to be specifically delivered to specific organs. In some embodiments, the compositions can be designed to be specifically delivered to mammal liver. In some embodiments, the compositions can be designed to be specifically delivered to lymph nodes. In some embodiments, the compositions can be designed to be specifically delivered to mammal spleen.

[0198] LNP may include one or more components described herein. In some embodiments, LNP preparations of the present disclosure include at least one lipid nanoparticle component. Lipid nanoparticles may include lipid components and one or more other components, such as therapeutic and / or prophylactic agents, such as nucleic acids. LNP may be designed for one or more specific applications or targets. The elements of LNP may be selected based on specific applications or targets and / or based on efficacy, toxicity, cost, ease of use, availability or other characteristics of one or more elements. Similarly, the specific preparation of LNP may be selected for specific applications or targets according to efficacy and toxicity of a specific combination of, for example, elements. The efficacy and tolerability of LNP preparations may be affected by formulation stability.

[0199] In some embodiments, for example, a polymer can be included in the LNP and / or used to encapsulate or partially encapsulate the LNP. The polymer can be biodegradable and / or biocompatible. The polymer can be selected from, but not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, and polyarylates. For example, the 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), poly(D,L-lactide-co-PEO). , L-lactide co-PPO co-D,L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, 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 (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyethylene ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinyl pyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, hydroxypropyl celluloses, carboxymethyl celluloses, acrylic acid polymers such as poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(methyl methacrylate), acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, poly(p-dioxanone) and copolymers thereof, 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), derivatives thereof and polyglycerol.

[0200] Surface-altering agents can include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dioctadecyldimethylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocysteine, eprazone, mesna, ambroxol, sobriosol, domiol, letosteine, sitoronin, tiopronin, gelsolin, thymosin beta 4, dornase alfa, netexin, and erdosteine), and DNA enzymes (e.g., rhDNase). The surface-altering agent can be disposed within the nanoparticle and / or on the surface of the LNP (e.g., by coating, adsorption, covalent bonding, or other processes).

[0201] LNP may also comprise one or more functionalized lipids.For example, lipid may be functionalized with an alkyne group, which may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions.Especially, lipid bilayers may be functionalized with one or more groups in this manner, which groups may be used to promote membrane penetration, cell recognition or imaging.The surface of LNP may also be conjugated with one or more useful antibodies.Functional groups and conjugates that may be used for targeted cell delivery, imaging and membrane penetration are well known in the art.

[0202] In addition to these components, the lipid nanoparticles may also include any substance that can be used in a pharmaceutical composition. For example, the lipid nanoparticles may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, surface active agents, buffers, preservatives and other types.

[0203] Surfactants and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, Polyoxyethylene sorbitan Polyoxyethylene sorbitan monooleate Sorbitan monopalmitate Sorbitan monostearate Sorbitan tristearate Glyceryl monooleate, Sorbitan monooleate ), polyoxyethylene esters (e.g., polyoxyethylene monostearate Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ), polyoxyethylene ethers (e.g., lauryl alcohol polyoxyethylene ether ), poly(vinyl pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Cetrimide bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium and / or combinations thereof.

[0204] The example of preservative may include but is not limited to antioxidant, chelating agent, free radical scavenger, antimicrobial preservative, antifungal preservative, alcohol preservative, acidic preservative and / or other preservative. The example of antioxidant includes but is not limited to alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium pyrosulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium pyrosulfite and / or sodium sulfite. The example of chelating agent includes 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. The example of antimicrobial preservative includes but is not limited to benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol and / or thimerosal. The example of antifungal preservative includes but is not limited to butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and / or sorbic acid. The example of alcohol preservative includes but is 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 hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT Methylparaben, 115. NEOLONE TM 、KATHON TMand / or Exemplary free radical scavengers include butylated hydroxytoluene (BHT or butylated hydroxytoluene) or deferoxamine.

[0205] Examples of buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, monopotassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, monosodium phosphate, sodium phosphate mixtures, tromethamine, sulfamate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, Tris buffer, and / or combinations thereof.

[0206] In some embodiments, the preparation comprising LNP may further include salts, such as chloride salts. In some embodiments, the preparation comprising LNP may further include sugars, such as disaccharides. In some embodiments, the preparation further includes sugars, but does not include salts, such as chloride salts. In some embodiments, LNP may further include one or more small hydrophobic molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0207] The characteristics of LNP may depend on its components. For example, LNPs including cholesterol as structural lipids may have different characteristics from LNPs including different structural lipids. As used herein, the term "structural lipid" refers to sterols and lipids containing sterol moieties. As defined herein, "sterols" are steroidal subgroups consisting of steroidal 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.

[0208] In some embodiments, the characteristic of LNP may depend on the absolute amount or relative amount of its components. For example, the LNP comprising a higher mole fraction of phospholipids may have a different characteristic from the LNP comprising a lower mole fraction of phospholipids. The characteristic may also depend on the preparation method and conditions of the lipid nanoparticles and be different. Generally speaking, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0209] Phospholipid part can be selected from the non-restrictive group such as being made up of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin.Fatty acid part can be selected from the non-restrictive group such as being made up of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid.Specific phospholipid can promote the fusion with film.In some embodiments, cationic phospholipid can interact with one or more negatively charged phospholipids of film (for example, cell membrane or intracellular membrane).The fusion of phospholipid and film can allow one or more elements (for example, therapeutic agent) of the composition (for example, LNP) containing lipid to pass through film, allows for example one or more elements to be delivered to target tissue. Non-natural phospholipid species are also contemplated, including natural species with modifications and substitutions including branching, oxidation, cyclization and alkynes. In some embodiments, phospholipids can be functionalized or cross-linked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, the alkyne group can undergo copper-catalyzed cycloaddition after being exposed to azide. Such reactions can be used to functionalize the lipid bilayer of nanoparticle compositions to promote membrane penetration or cell recognition, or to conjugate nanoparticle compositions with useful components such as targeting or imaging moieties (e.g., dyes). Phospholipids include, but are not limited to, glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and phosphatidic acid. Phospholipids also include phospho-sphingolipids, such as sphingomyelin. In some embodiments, phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC.

[0210] Lipid nanoparticles can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of LNP. Dynamic light scattering or potentiometric determination (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of LNP, such as particle size, polydispersity index and zeta potential.

[0211] The average size of the LNPs may be between 10 nm and 100 nm, for example, as measured by dynamic light scattering (DLS). For example, the average size may be between about 40 nm and about 150 nm, for example, about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of LNP may be about 50nm to about 100nm, about 50nm to about 90nm, about 50nm to about 80nm, about 50nm to about 70nm, about 50nm to about 60nm, about 60nm to about 100nm, about 60nm to about 90nm, about 60nm to about 80nm, about 60nm to about 70nm, about 70nm to about 100nm, about 70nm to about 90nm, about 70nm to about 80nm, about 80nm to about 100nm, about 80nm to about 90nm, or about 90nm to about 100nm. In certain embodiments, the average size of LNP may be about 70nm to about 100nm. In a specific embodiment, the average size may be about 80nm. In other embodiments, the average size may be about 100nm.

[0212] In some embodiments, the polydispersity index of LNP may be about 0 to about 0.25, for example, 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 LNP may be about 0.10 to about 0.20.

[0213] The zeta potential of LNP can be used to indicate the zeta potential of the composition. For example, the zeta potential can describe the surface charge of LNP. Lipid nanoparticles with relatively low positive or negative charge are generally desirable because higher charged species may not interact with cells, tissues and other elements in the body. In some embodiments, the zeta potential of the LNP may be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0214] The encapsulation efficiency of therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent encapsulated with LNP or otherwise combined with LNP after preparation relative to the initial amount provided. Encapsulation efficiency is expected to be very high (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by the following: before and after decomposing lipid nanoparticles with one or more organic solvents or detergents, the amount of therapeutic and / or prophylactic agent in the solution containing lipid nanoparticles is compared. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. For lipid nanoparticles described herein, the encapsulation efficiency of therapeutic and / or prophylactic agent may be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0215] The LNP may optionally include one or more coatings. For example, the LNP may be formulated in a capsule, film, or tablet having a coating. The capsule, film, or tablet comprising the composition described herein may have any useful size, tensile strength, hardness, or density.

[0216] The formulation comprising an amphiphilic polymer and a lipid nanoparticle may be formulated as a pharmaceutical composition in whole or in part. The pharmaceutical composition may include one or more amphiphilic polymers and one or more lipid nanoparticles. For example, the pharmaceutical composition may include one or more amphiphilic polymers and one or more lipid nanoparticles, and the lipid nanoparticles include one or more different therapeutic agents and / or preventive agents. The pharmaceutical composition may further include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for the preparation and manufacture of pharmaceutical compositions and medicaments can be obtained, 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 auxiliary ingredients may be used in any pharmaceutical composition, unless any conventional excipient or auxiliary ingredient may be incompatible with one or more components of the LNP or one or more amphiphilic polymers in the formulation of the present disclosure. An excipient or adjunct ingredient may be incompatible with a component of the LNP or the amphiphilic polymer of the formulation if the combination of the excipient or adjunct ingredient with the component or the amphiphilic polymer may result in any undesirable biological effect or other deleterious effect.

[0217] In some embodiments, one or more excipients or auxiliary components may constitute more than 50% of the total mass or volume of the pharmaceutical composition including LNP. For example, one or more excipients or auxiliary components may constitute 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In some embodiments, the purity of the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%. In some embodiments, the excipient is approved for use in humans and for veterinary use. In some embodiments, the excipient is approved by the United States Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (United States Pharmacopoeia) (USP), the European Pharmacopoeia (European Pharmacopoeia) (EP), the British Pharmacopoeia (British Pharmacopoeia) and / or the International Pharmacopoeia (International Pharmacopoeia). The relative amounts of one or more amphiphilic polymers, one or more lipid nanoparticles, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, and further depend on the route by which the composition is to be administered. For example, a pharmaceutical composition may contain 0.1% to 100% (wt / wt) of one or more lipid nanoparticles. As another example, a pharmaceutical composition may contain 0.1% to 15% (wt / vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% ​​w / v).

[0218] In certain embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or shipment (e.g., stored at 4°C or lower, e.g., between about -150°C and about 0°C or at a temperature between about -80°C and about -20°C (e.g., -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, a 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 shipment, e.g., at about -2 In some embodiments, the present disclosure also relates to a method of increasing the stability of lipid nanoparticles by adding an effective amount of an amphiphilic polymer and storing the lipid nanoparticles and / or pharmaceutical compositions thereof at a temperature of 4°C or less, such as between about -150°C and about 0°C or between about -80°C and about -20°C, for example, at a temperature of 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).

[0219] In some embodiments, the lipid component of LNP includes cationic lipid, phospholipid, PEG lipid and structural lipid.In certain embodiments, the lipid component of 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 % PEG lipid, and condition is that total mol % is no more than 100%.In some embodiments, the lipid component of lipid nanoparticle includes about 35 mol % to about 55 mol % cationic lipid compound, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid and about 0 mol % to about 10 mol % PEG lipid.In a specific embodiment, lipid component includes about 50 mol % described cationic lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid and about 1.5 mol % PEG lipid. In another embodiment, lipid component comprises about 40 mole % described cationic lipid, about 20 mole % phospholipid, about 38.5 mole % structural lipid and about 1.5 mole % PEG lipid.In some embodiments, phospholipid may be DOPE or DSPC.In other embodiments, PEG lipid may be PEG-DMG and / or structural lipid may be cholesterol.

[0220] In some embodiments, the ionizable lipid is a compound of formula (I):

[0221]

[0222] or its N-oxide, or its salt or isomer, wherein: Ri is selected from C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" and -R"M'R'; R2 and R3 are independently selected from H, C1-14 alkyl, C2-14 alkenyl, -R*YR", -YR" and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle; R4 is selected from hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2 and unsubstituted C1-6 alkyl, wherein Q is selected from carbocycle, heterocycle, -OR, -0(CH2)nN(R)2, -C(0)OR, -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 C1-3 alkyl, C2-3 alkenyl and H; each Re is independently selected from C1-3 alkyl, C2-3 alkenyl and H; M and M' are independently selected from -C(0)0-, -OC(O)-, -OC(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)(OR')0-, -S(0)2-, -SS-, aryl groups and heteroaryl groups, wherein M" is a bond, C1-13 alkyl or C2-13 alkenyl; R7 is selected from C1-3 alkyl, C2-3 alkenyl and H; Re is selected from C3-6 carbocycle and heterocycle; R9 is selected from H, CN, NO2, C1-6 alkyl, -OR, -S(0)2R, -S(0)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from C1-3 alkyl, C2-3 alkenyl and H; each R' is independently selected from C1-is alkyl, C2-is alkenyl, -R*YR", -YR" and H;each R" is independently selected from C3-15 alkyl and C3-15 alkenyl; each R* is independently selected from C1-i2 alkyl and C2-i2 alkenyl; each Y is independently C3-6 carbocycle; each X is independently selected from 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) when n is 1, 2, 3, 4 or 5, Q is not -N(R)2, or (ii) when n is 1 or 2, Q is not a 5-, 6- or 7-membered heterocycloalkyl. In some embodiments, the ionizable lipid is:

[0223]

[0224] In some embodiments, the compound has the following structure (I):

[0225]

[0226] 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—. )NRa—, NRaC(═O)NRa—, —OC(═O)NRa— or —NRaC(═O)O— or a direct bond; G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, —C(═O)OR4, —OC(═O)R4 or —NR5C(═O)R4; R4 is C1-C12 alkyl; R5 is H or C1-C6 alkyl; and x is 0, 1 or 2. In a preferred embodiment, the ionizable lipid is:

[0227] The lipid component of lipid nanoparticle composition may include one or more molecules including polyethylene glycol, such as PEG or PEG modified lipids. Such species may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids may be selected from the non-limiting groups including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof. In some embodiments, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipids. As used herein, the term "PEG lipid" refers to a lipid modified by polyethylene glycol (PEG). Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerCl4 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropane-3-amine. Such lipids are also referred to as PEGylated lipids. In some embodiments, the PEG lipid can be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid. In some embodiments, the PEG-modified lipid is a modified form of PEG DMG. In some embodiments, the PEG-modified lipid is a PEG lipid with formula (IV):

[0228]

[0229] wherein R8 and R9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has an average value ranging from 30 to 60.

[0230] L. Preparation

[0231] In one aspect, the present disclosure relates to an immunogenic composition, comprising: (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen, the antigen comprising at least one influenza virus antigen polypeptide or its immunogenic fragment, and (ii) a second RNA polynucleotide having an open reading frame encoding a second antigen, the second antigen comprising at least one influenza virus antigen polypeptide or its immunogenic fragment, wherein the first RNA polynucleotide and the second RNA polynucleotide are formulated in lipid nanoparticles (LNPs). In some embodiments, the first antigen and the second antigen comprise hemagglutinin (HA), or an immunogenic fragment or variant thereof. In some embodiments, the first antigen comprises HA from influenza viruses of different subtypes from the influenza virus antigen polypeptide or its immunogenic fragment of the second antigen. In some embodiments, the composition further comprises (iii) a third antigen, comprising at least one influenza virus antigen polypeptide or its immunogenic fragment, wherein the third antigen is from influenza virus, but from influenza virus strains different from the first antigen and the second antigen. In some embodiments, the first RNA polynucleotide, the second RNA polynucleotide, and the third RNA polynucleotide are formulated in lipid nanoparticles.

[0232] In some embodiments, the composition further comprises (iv) a fourth RNA polynucleotide having an open reading frame encoding a fourth antigen, the antigen comprising at least one influenza virus antigen polypeptide or an immunogenic fragment thereof, wherein the fourth antigen is from an influenza virus, but from an influenza virus strain different from the first antigen, the second antigen, and the third antigen. In some embodiments, the first RNA polynucleotide, the second RNA polynucleotide, the third RNA polynucleotide, and the fourth RNA polynucleotide are formulated in lipid nanoparticles.

[0233] In some embodiments, RNA polynucleotides are mixed in a single container with a desired ratio, and are subsequently formulated into lipid nanoparticles. In some embodiments, the initial input of different RNA polynucleotides with known ratios to be prepared in a single LNP process causes encapsulation of LNPs with different RNA polynucleotides of roughly the same ratio as the input ratio. Such embodiments may be referred to as "premixing" herein. Accordingly, in some embodiments, the first RNA polynucleotide and the second RNA polynucleotide are formulated in a single lipid nanoparticle. In some embodiments, the first RNA polynucleotide, the second RNA polynucleotide, the third RNA polynucleotide and the fourth RNA polynucleotide are formulated in a single LNP. In some embodiments, the first RNA polynucleotide, the second RNA polynucleotide, the third RNA polynucleotide, the fourth RNA polynucleotide and the fifth RNA polynucleotide are formulated in a single LNP. In some embodiments, the first RNA polynucleotide, the second RNA polynucleotide, the third RNA polynucleotide, the fourth RNA polynucleotide, the fifth RNA polynucleotide and the sixth RNA polynucleotide are formulated in a single LNP. In some embodiments, the first RNA polynucleotide, the second RNA polynucleotide, the third RNA polynucleotide, the fourth RNA polynucleotide, the fifth RNA polynucleotide, the sixth RNA polynucleotide, and the seventh RNA polynucleotide are formulated in a single LNP. In some embodiments, the first RNA polynucleotide, the second RNA polynucleotide, the third RNA polynucleotide, the fourth RNA polynucleotide, the fifth RNA polynucleotide, the sixth RNA polynucleotide, the seventh RNA polynucleotide, and the eighth RNA polynucleotide are formulated in a single LNP.

[0234] In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide in the mixture of RNA polynucleotides prior to formulation into LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50: 1. In some embodiments, the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide is greater than 1:1.

[0235] In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide in the mixture of RNA polynucleotides prior to formulation into LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50: 1. In some embodiments, the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide is greater than 1:1.

[0236] In some embodiments, the molar ratio of the first RNA polynucleotide to the fourth RNA polynucleotide in the mixture of RNA polynucleotides prior to formulation into LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50: 1. In some embodiments, the molar ratio of the first RNA polynucleotide to the fourth RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the fifth RNA polynucleotide in the mixture of RNA polynucleotides prior to formulation into LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50: 1. In some embodiments, the molar ratio of the first RNA polynucleotide to the fifth RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the sixth RNA polynucleotide in the mixture of RNA polynucleotides prior to formulation into LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50: 1. In some embodiments, the molar ratio of the first RNA polynucleotide to the sixth RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the seventh RNA polynucleotide in the mixture of RNA polynucleotides prior to formulation into LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50: 1. In some embodiments, the molar ratio of the first RNA polynucleotide to the seventh RNA polynucleotide is greater than 1:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide in the mixture of RNA polynucleotides prior to formulation into LNP is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1, or about 50: 1. In some embodiments, the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide is greater than 1:1.

[0237] In an alternative embodiment, each RNA polynucleotide encoding a specific antigen is formulated in an individual LNP so that each LNP encapsulates an RNA polynucleotide encoding the same antigen. Such embodiments may be referred to herein as "post-mixing". Accordingly, in some embodiments, a first RNA polynucleotide is formulated in a first LNP; a second RNA polynucleotide is formulated in a second LNP; a third RNA polynucleotide is formulated in a third LNP; a fourth RNA polynucleotide is formulated in a fourth LNP; a fifth RNA polynucleotide is formulated in a fifth LNP; a sixth RNA polynucleotide is formulated in a sixth LNP; a seventh RNA polynucleotide is formulated in a seventh LNP; and an eighth RNA polynucleotide is formulated in an eighth LNP.

[0238] In some embodiments, the molar ratio of the first LNP to the second LNP in the mixture of LNPs prior to formulation into the 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.

[0239] In some embodiments, the molar ratio of the first LNP to the third LNP in the mixture of LNPs prior to formulation into the 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.

[0240] In some embodiments, the molar ratio of the first LNP to the fourth LNP in the mixture of LNPs prior to formulation into the 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 mixture of LNPs prior to formulation into the 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 mixture of LNPs prior to formulation into the 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 mixture of LNPs prior to formulation into the 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 mixture of LNPs prior to formulation into the 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.

[0241] In some embodiments, the relative amount of RNA encoding an antigen of influenza B virus may be increased compared to RNA encoding influenza A virus (e.g., an immune response comprising a higher neutralization titer against influenza B virus (e.g., a higher neutralization titer (e.g., as determined by a pseudovirus neutralization assay described herein) compared to a composition comprising equal amounts of RNA encoding an influenza A antigen and RNA encoding an influenza B antigen)). The disclosure also provides exemplary doses of RNA that can generate a strong immune response against both types of influenza virus (e.g., neutralization titers and / or seroconversion rates at clinically relevant levels (e.g., (i) neutralization titers comparable to or better than those previously shown to prevent influenza symptoms, and / or (ii) neutralization titers and / or seroconversion rates comparable to or better than those induced by relevant comparators (e.g., commercially approved influenza vaccines or influenza RNA vaccines)). In some embodiments, a composition comprising a greater amount of RNA encoding an influenza B antigen than RNA encoding an influenza A antigen generates an immune response against each of influenza B virus and influenza A virus, which is comparable to that obtained by a non-RNA influenza vaccine (e.g., an approved vaccine) and / or a composition comprising equal The amount of RNA vaccine induced by RNA encoding influenza A antigen and RNA encoding influenza B antigen is comparable to or better than that induced by RNA vaccine.

[0242] In some embodiments, the RNA concentration in the pharmaceutical RNA preparation is about 0.1-0.2 mg / ml. In some embodiments, the RNA concentration in the pharmaceutical RNA preparation is about 0.1 mg / ml. In some embodiments, the RNA concentration in the pharmaceutical RNA preparation is about 0.12 mg / ml. In some embodiments, the RNA concentration in the pharmaceutical RNA preparation is about 0.14 mg / ml. In some embodiments, the RNA concentration in the pharmaceutical RNA preparation is about 0.16 mg / ml. In some embodiments, the RNA concentration in the pharmaceutical RNA preparation is about 0.18 mg / ml. In some embodiments, about 30 ug of RNA is administered by administering about 200 uL of RNA preparation. In some embodiments, the RNA in the pharmaceutical RNA preparation is diluted (e.g., diluted to a concentration of about 0.05 mg / ml) before administration. In some embodiments, the application volume is between about 200 μl and about 300 μl. In some embodiments, the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer and about 10% sucrose.

[0243] In some embodiments, the pharmaceutical RNA preparation comprises RNA at a concentration of about 0.1 mg / ml, and is prepared in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation comprises RNA at a concentration of about 0.12 mg / ml, and is prepared in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation comprises RNA at a concentration of about 0.14 mg / ml, and is prepared in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation comprises RNA at a concentration of about 0.16 mg / ml, and is prepared in about 10 mM Tris buffer and about 10% sucrose. In some embodiments, the pharmaceutical RNA preparation comprises RNA at a concentration of about 0.18 mg / ml, and is prepared in about 10 mM Tris buffer and about 10% sucrose. Such preparations can be diluted as needed before administration to administer different doses of RNA while keeping the total injection volume relatively constant. For example, an RNA dose of about 10 μg can be administered by diluting such a pharmaceutical RNA formulation about 1:1, and administering about 200 μl of the diluted pharmaceutical RNA formulation.

[0244] In some embodiments, the vaccine is formulated in a vial (e.g., a glass vial). In some embodiments, the glass vial is sealed with a bromobutyl rubber stopper and an aluminum seal with a flip-top plastic cap.

[0245] In some embodiments, the composition comprises RNA encoding an antigen (e.g., HA protein) of an influenza virus, which is recommended by relevant health authorities for inclusion in seasonally adapted vaccines (e.g., cell-based, recombinant or attenuated live viruses). In some embodiments, the composition comprises a variety of RNAs encoding antigens (e.g., HA proteins) of each influenza virus recommended by relevant health authorities for inclusion in seasonally adapted vaccines (e.g., cell-based, recombinant or attenuated live viruses). In some embodiments, the influenza virus is influenza A virus, influenza B virus, or influenza C virus. In some embodiments, influenza A virus is H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N7, or H10N8 virus. In some embodiments, influenza A virus is H1N1, H3N2, H5N1 or H5N8 virus. In some embodiments, influenza A virus is H1N1 virus (e.g., A / Wisconsin / 588 / 2019 or A / Sydney / 5 / 2021). In some embodiments, influenza A virus is H3N2 virus. In some embodiments, H3N2 virus is A / Cambodia / e0826360 / 2020 or A / Darwin / 6 / 2021. In some embodiments, influenza B virus belongs to B / Yamagata or B / Victoria lineage. In some embodiments, B / Victoria lineage influenza virus is B / Washington / 02 / 2019. In some embodiments, B / Victoria lineage virus is B / Austria / 1359417 / 2021. In some embodiments, the B / Yamagata lineage influenza virus is B / Phuket / 3073 / 2013.

[0246] In some embodiments, compositions described herein include polyvalent influenza vaccines. In some embodiments, polyvalent influenza vaccines include 2 to 50 different RNA molecules (e.g., 2 to 40, 2 to 30 or 2 to 20 RNA molecules), in some embodiments, each of the RNA molecules may encode different antigenic polypeptides (or different forms of specific antigenic polypeptides) related to influenza, for example, as described in Arevalo, Claudia P., et al. "A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes." Science 378.6622 (2022): 899-904. In some embodiments, compositions described herein include trivalent influenza vaccines. In some embodiments, trivalent influenza vaccines include RNA encoding antigenic polypeptides related to two A viruses and one B virus that are prevalent in relevant jurisdictions. In some embodiments, compositions described herein include quadrivalent influenza vaccines. In some embodiments, quadrivalent influenza vaccines include RNA encoding antigenic polypeptides related to two A viruses and two B viruses that are prevalent in relevant jurisdictions. In some embodiments, compositions described herein include eight-valent influenza vaccines.In some embodiments, eight-valent influenza vaccines include coding and two antigenic polypeptides (e.g., HA protein and NA protein related to each virus, or its immunogenic fragment) RNA related to two A viruses and two B viruses prevalent in related jurisdictions.In some embodiments, compositions disclosed herein include quadrivalent influenza vaccines, including RNA including nucleotide sequences encoding HA proteins related to H1N1 viruses (e.g., A / Wisconsin / 588 / 2019), RNA including nucleotide sequences encoding HA proteins related to H3N2 viruses (e.g., A / Cambodia / e0826360 / 2020), RNA including nucleotide sequences encoding HA proteins related to B / Victoria lineage influenza viruses (e.g., B / Washington / 02 / 2019), and HA proteins related to B / Yamagata lineage influenza viruses (e.g., B / Phuket / 3073 / 2013).

[0247] In some embodiments, the composition comprises a quadrivalent influenza vaccine comprising RNA encoding antigenic polypeptides associated with two A viruses and two B viruses predicted to be prevalent in the relevant jurisdiction. In some embodiments, the quadrivalent influenza vaccine comprises RNA encoding antigenic polypeptides associated with H1N1 influenza virus, RNA encoding antigenic polypeptides associated with H3N2 influenza virus, RNA encoding antigenic polypeptides associated with Victoria lineage influenza virus, and RNA encoding antigenic polypeptides associated with Yamagata lineage influenza virus. In some embodiments, the quadrivalent influenza vaccine comprises RNA associated with influenza types predicted to be prevalent in the relevant jurisdiction (e.g., HA polypeptides associated with H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses predicted to be prevalent in the relevant jurisdiction).

[0248] In some embodiments, the composition comprises an octavalent influenza vaccine comprising RNA encoding antigenic polypeptides associated with two A viruses and two B viruses predicted to be prevalent in the relevant jurisdiction. In some embodiments, the octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide derived from HA from influenza A virus, RNA encoding an antigenic polypeptide derived from HA from influenza A virus, RNA encoding an antigenic polypeptide derived from HA from influenza B virus, RNA encoding an antigenic polypeptide derived from HA from influenza B virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from influenza A virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from influenza A virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from influenza A virus, RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from influenza B virus, and RNA encoding an antigenic polypeptide derived from one antigenic polypeptide selected from NA, NP, M1, M2, NS1 and NS2 from influenza B virus. In some embodiments, an octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide derived from HA from influenza A virus, RNA encoding an antigenic polypeptide derived from HA from influenza A virus, RNA encoding an antigenic polypeptide derived from HA from influenza B virus, RNA encoding an antigenic polypeptide derived from HA from influenza B virus, RNA encoding an antigenic polypeptide derived from NA from influenza A virus, RNA encoding an antigenic polypeptide derived from NA from influenza A virus, RNA encoding an antigenic polypeptide derived from NA from influenza B virus, and RNA encoding an antigenic polypeptide derived from NA from influenza B virus. In some embodiments, an octavalent influenza vaccine comprises RNA encoding an antigenic polypeptide associated with H1N1 influenza virus, RNA encoding an antigenic polypeptide associated with H3N2 influenza virus, RNA encoding an antigenic polypeptide associated with Victoria lineage influenza virus, and RNA encoding an antigenic polypeptide associated with Yamagata lineage influenza virus. In some embodiments, the octavalent influenza vaccine comprises RNA associated with influenza types predicted to circulate in the relevant jurisdiction (e.g., HA polypeptides associated with H1N1, H3N2, B / Victoria, and B / Yamagata influenza viruses predicted to circulate in the relevant jurisdiction).

[0249] In some embodiments, each RNA in the compositions disclosed herein encodes an antigenic polypeptide associated with an infectious agent predicted to be prevalent in the relevant jurisdiction.Such compositions can reduce the number of vaccinations required.

[0250] In some embodiments, the particle containing nucleic acid comprises two or more RNA molecules, each of which comprises a nucleotide sequence encoding an antigen (e.g., HA protein) associated with different influenza viruses. In some embodiments, the particle containing nucleic acid comprises three or more RNA molecules, each of which comprises a nucleotide sequence encoding an antigen (e.g., HA protein) associated with different influenza viruses. In some embodiments, the particle containing nucleic acid comprises four or more RNA molecules, each of which comprises a nucleotide sequence encoding an antigen (e.g., HA protein) associated with different influenza viruses. In some embodiments, the particle containing nucleic acid comprises an RNA molecule comprising a nucleotide sequence encoding an antigen polypeptide associated with H1N1 influenza virus, an RNA molecule comprising a nucleotide sequence encoding an antigen polypeptide associated with H3N2 influenza virus, an RNA molecule comprising a nucleotide sequence encoding an antigen polypeptide associated with B / Victoria lineage influenza virus, and an RNA molecule comprising a nucleotide sequence encoding an antigen polypeptide associated with B / Yamagata influenza virus. In some embodiments, each RNA in the composition comprising a nucleotide sequence encoding an antigen polypeptide associated with influenza virus is prepared in the same particle containing nucleic acid. In some embodiments, each RNA in a composition comprising a nucleotide sequence encoding an antigenic polypeptide associated with influenza virus is formulated in a separate nucleic acid-containing particle.

[0251] In some embodiments, a nucleic acid-containing particle (eg, in some embodiments, a LNP as described herein) comprising two or more RNA molecules comprises each RNA molecule in the same amount (ie, in a 1:1 ratio).

[0252] In some embodiments, a nucleic acid-containing particle comprising two or more RNA molecules (eg, in some embodiments, a LNP as described herein) comprises different amounts of each RNA molecule. For example, in some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the first RNA molecule is present in an amount that is 0.01 to 100 times greater than the second RNA molecule (e.g., wherein the amount of the first RNA molecule is 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to 4, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 to 1.5 times greater than the second RNA molecule). In some embodiments, the particles containing nucleic acid include a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 10 times that of the second RNA molecule. In some embodiments, the particles containing nucleic acid include a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 5 times that of the second RNA molecule. In some embodiments, the particles containing nucleic acid include a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 3 times that of the second RNA molecule. In some embodiments, the particles containing nucleic acid include a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 2 times that of the second RNA molecule. In some embodiments, the particles containing nucleic acid include a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 3 times that of the second RNA molecule.

[0253] In some embodiments, a nucleic acid-containing particle (eg, in some embodiments, a LNP as described herein) comprising three RNA molecules comprises each RNA molecule in the same amount (ie, in a 1:1:1 ratio).

[0254] In some embodiments, a nucleic acid-containing particle comprising three RNA molecules (e.g., in some embodiments, an LNP as described herein) comprises different amounts of each RNA molecule. For example, in some embodiments, the ratio of the first RNA molecule: the second RNA molecule: the third RNA molecule is 1:0.01-100:0.01-100 (e.g., 1:0.01-50:0.01-50; 1:0.01-40:0.01-40; 1:0.01-30:0.01-25; 1:0.01-25:0.01-25; 1:0.01-20:0.01-20; 1:0.01-15:0.01-1 In some embodiments, the ratio of the first RNA molecule: the second RNA molecule: the third RNA molecule is 1:1:3. In some embodiments, the ratio of the first RNA molecule: the second RNA molecule: the third RNA molecule is 1:3:3.

[0255] As used herein, the term "dose" generally refers to the "amount of the dose", which relates to the amount of RNA administered per administration, ie, per dosing.

[0256] In some embodiments, administration of an immunogenic composition or vaccine of the present disclosure can be performed by a single administration or boosted by multiple administrations.

[0257] In some embodiments, the regimen described herein includes at least one dose. In some embodiments, the regimen includes a first dose and at least one subsequent dose. In some embodiments, the first dose is the same amount as at least one subsequent dose. In some embodiments, the first dose is the same amount as all subsequent doses. In some embodiments, the first dose is a different amount from at least one subsequent dose. In some embodiments, the first dose is a different amount from all subsequent doses. In some embodiments, the regimen comprises two doses. In some embodiments, the provided regimen consists of two doses. In some embodiments, the regimen comprises three doses.

[0258] In one embodiment, the disclosure contemplates the use of a single dose. In one embodiment, the disclosure contemplates the use of a first immunization dose, followed by one or more booster doses. Booster doses or the first booster dose can be used 7 to 28 days or 14 to 24 days after the first immunization dose is used. In some embodiments, the first booster dose can be used 1 week to 3 months (for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks) after the first immunization dose is used. In some embodiments, subsequent booster doses can be used at least 1 week or longer after the previous booster dose, including, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks or longer. In some embodiments, subsequent booster doses can be used at intervals of about 5-9 weeks or 6-8 weeks. In some embodiments, at least one subsequent booster dose (e.g., after a first booster dose) can be administered at least 3 months or more after the previous dose, including, for example, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months or more.

[0259] In some embodiments, the dosage comprises the following total amount of RNA: 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg, such as about 1 μg, about 2 μg, about 3 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg or about 100 μg. In some embodiments, the dosage comprises up to about 100 μg of RNA (e.g., modRNA) total amount. In some embodiments, the dosage comprises 0.1 μg to 100 μg of one or more first RNAs and 0.1 μg to 100 μg of one or more second RNAs, wherein the one or more first RNAs each comprise a nucleotide sequence encoding an antigenic polypeptide associated with a first infectious agent (e.g., coronavirus), and the one or more second RNAs each comprise a nucleotide sequence encoding an antigenic polypeptide associated with a second infectious agent (e.g., influenza). In some embodiments, the dosage comprises 3 to 60 μg of one or more first RNAs and 3 to 90 μg of one or more second RNAs. In some embodiments, the dosage comprises 3 to 60 μg of one or more first RNAs and 3 to 90 μg of one or more second RNAs, wherein the dosage comprises up to 100 μg of RNA in total. In some embodiments, the dosage comprises 3 to 30 μg of one or more first RNAs and 3 to 60 μg of one or more second RNAs, wherein the dosage comprises up to 100 μg of RNA in total. In some embodiments, the dosage comprises 3 μg of one or more first RNAs and 3 μg of one or more second RNAs. In some embodiments, the dosage comprises 3 μg of one or more first RNAs and 6 μg of one or more second RNAs. In some embodiments, the dosage comprises 10 μg of one or more first RNAs and 10 μg of one or more second RNAs. In some embodiments, the dosage comprises 10 μg of one or more first RNAs and 20 μg of one or more second RNAs. In some embodiments, the dosage comprises 30 μg of one or more first RNAs and 30 μg of one or more second RNAs. In some embodiments, the dosage comprises 30 μg of one or more first RNAs and 60 μg of one or more second RNAs. In some embodiments, the dosage comprises 60 μg of one or more first RNAs and 30 μg of one or more second RNAs.

[0260] In some embodiments, the subsequent dose given to an individual (for example, as part of a primary immunization scheme or a booster scheme) can have the same RNA amount as previously given to an individual. In some embodiments, compared with the amount previously given to an individual, the subsequent dose given to an individual (for example, as part of a primary immunization scheme or a booster scheme) can be different in terms of the amount of RNA. For example, in some embodiments, the subsequent dose can be higher or lower than the previous dose, for example, based on the consideration of various factors, the factors include, for example, the immunogenicity and / or the reactionogenicity induced by the previous dose, the prevalence of the disease, etc. In some embodiments, the subsequent dose can be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or higher than the previous dose. In some embodiments, the subsequent dose can be at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times or higher than the previous dose. In some embodiments, the subsequent dose can be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or higher than the previous dose. In some embodiments, the subsequent dose can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or lower than the previous dose. In some embodiments, 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg, such as about 1 μg, about 2 μg, about 3 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg or about 100 μg of the amount of RNA described herein can be administered per dose (e.g., in a given dose).

[0261] In some embodiments, an amount of 60 μg or less, 55 μg or less, 50 μg or less, 45 μg or less, 40 μg or less, 35 μg or less, 30 μg or less, 25 μg or less, 20 μg or less, 15 μg or less, 10 μg or less, 5 μg or less, 3 μg or less, 2.5 μg or less, or 1 μg or less of an RNA described herein can be administered per dose (e.g., in a given dose).

[0262] In some embodiments, at least 0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 10 μg, at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 40 μg, at least 50 μg or at least 60 μg of RNA described herein can be administered per dose (e.g., in a given dose). In some embodiments, at least 3 ug of RNA described herein can be administered in at least one given dose. In some embodiments, at least 10 ug of RNA described herein can be administered in at least one given dose. In some embodiments, at least 15 ug of RNA described herein can be administered in at least one given dose. In some embodiments, at least 20 ug of RNA described herein can be administered in at least one given dose. In some embodiments, at least 25 ug of RNA described herein can be administered in at least one given dose. In some embodiments, at least 30 ug of RNA described herein can be administered in at least one given dose. In some embodiments, the amount of RNA described herein of at least 50ug can be administered in at least one given dose. In some embodiments, the amount of RNA described herein of at least 60ug can be administered in at least one given dose. In some embodiments, the combination of the above amounts can be administered in a scheme including two or more doses (e.g., the previous dose and subsequent dose can be different amounts as described herein). In some embodiments, the combination of the above amounts can be administered in a primary immunization scheme and a booster scheme (e.g., different doses can be given in a primary immunization scheme and a booster scheme).

[0263] In some embodiments, 0.25 μg to 60 μg, 0.5 μg to 55 μg, 1 μg to 50 μg, 5 μg to 40 μg, or 10 μg to 30 μg of RNA described herein can be administered per dose. In some embodiments, 3 μg to 30 μg of RNA described herein can be administered in at least one given dose. In some embodiments, 3 μg to 20 μg of RNA described herein can be administered in at least one given dose. In some embodiments, 3 μg to 15 μg of RNA described herein can be administered in at least one given dose. In some embodiments, 3 μg to 10 μg of RNA described herein can be administered in at least one given dose. In some embodiments, 10 μg to 30 μg of RNA described herein can be administered in at least one given dose.

[0264] In some embodiments, the regimen applied to the subject may include multiple doses (e.g., at least two doses, at least three doses or more). In some embodiments, the regimen applied to the subject may include a first dose and a second dose, which are given at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart or longer. In some embodiments, such doses may be given at least 1 month apart, at least 2 months apart, at least 3 months apart, at least 4 months apart, at least 5 months apart, at least 6 months apart, at least 7 months apart, at least 8 months apart, at least 9 months apart, at least 10 months apart, at least 11 months apart, at least 12 months apart or longer. In some embodiments, the doses can be administered at intervals of several days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 days or more. In some embodiments, the doses can be administered at intervals of about 1 to about 3 weeks, or at intervals of about 1 to about 4 weeks, or at intervals of about 1 to about 5 weeks, or at intervals of about 1 to about 6 weeks, or at intervals of about 1 to more than 6 weeks. In some embodiments, the doses may be separated by a period of about 7 to about 60 days, such as about 14 to about 48 days, etc. In some embodiments, the minimum number of days between doses may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more. In some embodiments, the maximum number of days between doses may be about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or less. In some embodiments, the doses may be separated by about 21 to about 28 days. In some embodiments, the doses may be spaced from about 19 to about 42 days apart. In some embodiments, the doses may be spaced from about 7 to about 28 days apart. In some embodiments, the doses may be spaced from about 14 to about 24 days apart. In some embodiments, the doses may be spaced from about 21 to about 42 days apart.

[0265] In some embodiments, the vaccination regimen comprises a first dose and a second dose. In some embodiments, the first dose and the second dose are administered at least 21 days apart. In some embodiments, the first dose and the second dose are administered at least 28 days apart.

[0266] In some embodiments, the vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is the same as the amount of RNA administered in the second dose. In some embodiments, the vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is different from the amount of RNA administered in the second dose.

[0267] In some embodiments, the vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is less than the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10%-90% of the second dose. In some embodiments, the amount of RNA administered in the first dose is 10%-50% of the second dose. In some embodiments, the amount of RNA administered in the first dose is 10%-20% of the second dose. In some embodiments, the first dose and the second dose are administered at least 2 weeks apart, including at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, at least 6 weeks apart, or longer. In some embodiments, the first dose and the second dose are administered at least 3 weeks apart.

[0268] In some embodiments, the first dose comprises less than about 30ug of RNA, and the second dose comprises at least about 30ug of RNA. In some embodiments, the first dose comprises about 1 to less than about 30ug of RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25 or less than about 30ug of RNA), and the second dose comprises about 30 to about 100ug of RNA (e.g., about 30, about 40, about 50 or about 60ug of RNA). In some embodiments, the first dose comprises about 1 to about 20ug of RNA, about 1 to about 10ug of RNA, or about 1 to about 5ug of RNA, and the second dose comprises about 30 to about 60ug of RNA.

[0269] In some embodiments, the first dose comprises about 1 to about 10 ug of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 ug of RNA), and the second dose comprises about 30 to about 60 ug of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 ug of RNA).

[0270] In some embodiments, the first dose comprises about 1 ug of RNA and the second dose comprises about 30 ug of RNA. In some embodiments, the first dose comprises about 3 ug of RNA and the second dose comprises about 30 ug of RNA. In some embodiments, the first dose comprises about 5 ug of RNA and the second dose comprises about 30 ug of RNA. In some embodiments, the first dose comprises about 10 ug of RNA and the second dose comprises about 30 ug of RNA. In some embodiments, the first dose comprises about 15 ug of RNA and the second dose comprises about 30 ug of RNA.

[0271] In some embodiments, the first dose comprises about 1ug of RNA, and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA, and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 5ug of RNA, and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 6ug of RNA, and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 10ug of RNA, and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 15ug of RNA, and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 20ug of RNA, and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 25ug of RNA, and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA, and the second dose comprises about 60ug of RNA.

[0272] In some embodiments, the first dose comprises less than about 10ug of RNA, and the second dose comprises at least about 10ug of RNA. In some embodiments, the first dose comprises about 0.1 to less than about 10ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8 or less than about 10ug of RNA), and the second dose comprises about 10 to about 30ug of RNA (e.g., about 10, about 15, about 20, about 25 or about 30ug of RNA). In some embodiments, the first dose comprises about 0.1 to about 10ug of RNA, about 1 to about 5ug of RNA, or about 0.1 to about 3ug of RNA, and the second dose comprises about 10 to about 30ug of RNA.

[0273] In some embodiments, the first dose comprises about 0.1 to about 5 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5 ug of RNA), and the second dose comprises about 10 to about 20 ug of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20 ug of RNA).

[0274] In some embodiments, the first dose comprises about 0.1 ug of RNA and the second dose comprises about 10 ug of RNA. In some embodiments, the first dose comprises about 0.3 ug of RNA and the second dose comprises about 10 ug of RNA. In some embodiments, the first dose comprises about 1 ug of RNA and the second dose comprises about 10 ug of RNA. In some embodiments, the first dose comprises about 3 ug of RNA and the second dose comprises about 10 ug of RNA.

[0275] In some embodiments, the first dose comprises less than about 3ug of RNA, and the second dose comprises at least about 3ug of RNA. In some embodiments, the first dose comprises about 0.1 to less than about 3ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5ug of RNA), and the second dose comprises about 3 to about 10ug of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10ug of RNA). In some embodiments, the first dose comprises about 0.1 to about 3ug of RNA, about 0.1 to about 1ug of RNA, or about 0.1 to about 0.5ug of RNA, and the second dose comprises about 3 to about 10ug of RNA.

[0276] In some embodiments, the first dose comprises about 0.1 to about 1.0 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 ug of RNA), and the second dose comprises about 1 to about 3 ug of RNA (e.g., about 1.0, about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA).

[0277] In some embodiments, the first dose comprises about 0.1 ug of RNA and the second dose comprises about 3 ug of RNA. In some embodiments, the first dose comprises about 0.3 ug of RNA and the second dose comprises about 3 ug of RNA. In some embodiments, the first dose comprises about 0.5 ug of RNA and the second dose comprises about 3 ug of RNA. In some embodiments, the first dose comprises about 1 ug of RNA and the second dose comprises about 3 ug of RNA.

[0278] In some embodiments, the vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is greater than the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the second dose is 10%-90% of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10%-50% of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10%-20% of the first dose. In some embodiments, the first dose and the second dose are administered at least 2 weeks apart, including at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, at least 6 weeks apart, or more. In some embodiments, the first dose and the second dose are administered at least 3 weeks apart.

[0279] In some embodiments, the first dose comprises at least about 30ug of RNA, and the second dose comprises less than about 30ug of RNA. In some embodiments, the first dose comprises about 30 to about 100ug of RNA (e.g., about 30, about 40, about 50, or about 60ug of RNA), and the second dose comprises about 1 to about 30ug of RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or about 30ug of RNA). In some embodiments, the second dose comprises about 1 to about 20ug of RNA, about 1 to about 10ug of RNA, or about 1 to 5ug of RNA. In some embodiments, the first dose comprises about 30 to about 60ug of RNA, and the second dose comprises about 1 to about 20ug of RNA, about 1 to about 10ug of RNA, or about 0.1 to about 3ug of RNA.

[0280] In some embodiments, the first dose comprises about 30 to about 60 ug of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 ug of RNA), and the second dose comprises about 1 to about 10 ug of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 ug of RNA).

[0281] In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 1ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 3ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 5ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 10ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 15ug of RNA.

[0282] In some embodiments, the first dose comprises about 60ug of RNA, and the second dose comprises about 1ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA, and the second dose comprises about 3ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA, and the second dose comprises about 5ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA, and the second dose comprises about 6ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA, and the second dose comprises about 10ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA, and the second dose comprises about 15ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA, and the second dose comprises about 20ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA, and the second dose comprises about 25ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA, and the second dose comprises about 30ug of RNA.

[0283] In some embodiments, the first dose comprises at least about 10ug of RNA, and the second dose comprises less than about 10ug of RNA. In some embodiments, the first dose comprises about 10 to about 30ug of RNA (e.g., about 10, about 15, about 20, about 25, or about 30ug of RNA), and the second dose comprises about 0.1 to less than about 10ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about 10ug of RNA). In some embodiments, the first dose comprises about 10 to about 30ug of RNA, or about 0.1 to about 3ug of RNA, and the second dose comprises about 1 to about 10ug of RNA, or about 1 to about 5ug of RNA.

[0284] In some embodiments, the first dose comprises about 10 to about 20 ug of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20 ug of RNA), and the second dose comprises about 0.1 to about 5 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, or about 5 ug of RNA).

[0285] In some embodiments, the first dose comprises about 10 ug of RNA and the second dose comprises about 0.1 ug of RNA. In some embodiments, the first dose comprises about 10 ug of RNA and the second dose comprises about 0.3 ug of RNA. In some embodiments, the first dose comprises about 10 ug of RNA and the second dose comprises about 1 ug of RNA. In some embodiments, the first dose comprises about 10 ug of RNA and the second dose comprises about 3 ug of RNA.

[0286] In some embodiments, the first dose comprises at least about 3ug of RNA, and the second dose comprises less than about 3ug of RNA. In some embodiments, the first dose comprises about 3 to about 10ug of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10ug of RNA), and the second dose comprises 0.1 to less than about 3ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5ug of RNA). In some embodiments, the first dose comprises about 3 to about 10ug of RNA, and the second dose comprises about 0.1 to about 3ug of RNA, about 0.1 to about 1ug of RNA, or about 0.1 to about 0.5ug of RNA.

[0287] In some embodiments, the first dose comprises about 1 to about 3 ug of RNA (e.g., about 1, about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA), and the second dose comprises about 0.1 to 0.3 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 ug of RNA).

[0288] In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 0.1ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 0.3ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 0.6ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 1ug of RNA.

[0289] In some embodiments, the vaccination regimen comprises at least two doses, including, for example, at least three doses, at least four doses or more doses. In some embodiments, the vaccination regimen comprises three doses. In some embodiments, the time interval between the first dose and the second dose can be the same as the time interval between the second dose and the third dose. In some embodiments, the time interval between the first dose and the second dose can be longer than the time interval between the second dose and the third dose, for example, a few days or weeks (including, for example, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks or longer). In some embodiments, the time interval between the first dose and the second dose can be shorter than the time interval between the second dose and the third dose, for example, a few days or weeks (including, for example, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks or longer). In some embodiments, the time interval between the first dose and the second dose can be shorter than the time interval between the second dose and the third dose, for example, at least 1 month shorter (including, for example, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months or longer).

[0290] In some embodiments, the last dose of the priming regimen and the first dose of the boosting regimen are given at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months or longer apart. In some embodiments, the priming regimen may comprise two doses. In some embodiments, the priming regimen may comprise three doses.

[0291] In some embodiments, the first dose and the second dose (and / or other subsequent doses) may be administered by intramuscular injection. In some embodiments, the first dose and the second dose (and / or other subsequent doses) may be administered in the deltoid muscle. In some embodiments, the first dose and the second dose (and / or other subsequent doses) may be administered in the same arm.

[0292] In some embodiments, the mRNA compositions described herein are administered (e.g., by intramuscular injection) as a series of two doses (e.g., each 0.3 mL) spaced 21 days apart. In some embodiments, the mRNA compositions described herein are administered (e.g., by intramuscular injection) as a series of two doses (e.g., each 0.2 mL) spaced 21 days apart. In some embodiments, the mRNA compositions described herein are administered (e.g., by intramuscular injection) as a series of three doses (e.g., 0.3 mL or lower, including, for example, 0.2 mL), wherein the dose interval is at least 3 weeks. In some embodiments, the first dose and the second dose can be administered at intervals of 3 weeks, and the second dose and the third dose can be administered at intervals longer than the time interval between the first dose and the second dose, for example, at intervals of at least 4 weeks or longer (including at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks or longer). In some embodiments, each dose is about 60 ug. In some embodiments, each dose is about 50 ug. In some embodiments, each dose is about 30 ug. In some embodiments, each dose is about 25 ug. In some embodiments, each dose is about 20ug. In some embodiments, each dose is about 15ug. In some embodiments, each dose is about 10ug. In some embodiments, each dose is about 3ug.

[0293] In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 60ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 50ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 30ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 25ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 20ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 15ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 10ug. In some embodiments, at least one dose administered in a vaccination regimen (eg, a priming vaccination regimen and / or a booster vaccination regimen) is about 3 ug.

[0294] In one embodiment, each dose is administered about 60 μg of RNA described herein. In one embodiment, each dose is administered about 50 μg of RNA described herein. In one embodiment, each dose is administered about 30 μg of RNA described herein. In one embodiment, each dose is administered about 25 μg of RNA described herein. In one embodiment, each dose is administered about 20 μg of RNA described herein. In one embodiment, each dose is administered about 15 μg of RNA described herein. In one embodiment, each dose is administered about 10 μg of RNA described herein. In one embodiment, each dose is administered about 5 μg of RNA described herein. In one embodiment, each dose is administered about 3 μg of RNA described herein. In one embodiment, at least two such doses are administered. For example, the second dose may be administered about 21 days after the first dose is administered.

[0295] In some embodiments, the efficacy of the RNA vaccines described herein (e.g., administered in two doses, wherein the second dose may be administered about 21 days after the first dose, and, for example, administered in an amount of about 30 μg per dose) is at least 70%, at least 80%, at least 90%, or at least 95% starting 7 days after the second dose (e.g., if the second dose is administered 21 days after the first dose, then 28 days after the first dose). In some embodiments, such efficacy is observed in a population of at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, or older. In some embodiments, the efficacy of the RNA vaccine described herein (e.g., administered in two doses, wherein the second dose may be administered about 21 days after the first dose, and, for example, administered in an amount of about 30 μg per dose) is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% in a population of at least 65 years old, such as 65 to 80 years old, 65 to 75 years old, or 65 to 70 years old. Such efficacy can be observed for a period of up to 1 month, 2 months, 3 months, 6 months, or even longer.

[0296] In one embodiment, vaccine efficacy is defined as the percentage reduction in the number of subjects with evidence of infection (vaccinated subjects relative to unvaccinated subjects).

[0297] In one embodiment, the method and medicament described herein are applied to a pediatric colony. In various embodiments, the pediatric colony includes or is composed of: a subject under 18 years old, for example, 5 to less than 18 years old, 12 to less than 18 years old, 16 to less than 18 years old, 12 to less than 16 years old, 5 to less than 12 years old, or 6 months to less than 12 years old. In various embodiments, the pediatric colony includes or is composed of: a subject under 5 years old, for example, 2 to less than 5 years old, 12 to less than 24 months, 7 to less than 12 months or less than 6 months. In some such embodiments, the mRNA composition described herein is applied to a subject less than 2 years old, for example, 6 months to less than 2 years old. In some such embodiments, the mRNA composition described herein is applied to a subject less than 6 months, for example, 1 month to less than 4 months. In some embodiments, the dosing regimen (e.g., dosage and / or dosing schedule) for a pediatric colony may be different for age groups. For example, in some embodiments, subjects from 6 months to 4 years old can be administered according to a primary immunization scheme comprising at least three doses, wherein the initial two doses are administered at least 3 weeks apart (including, for example, at least 4 weeks, at least 5 weeks, at least 6 weeks or longer), followed by a third dose administered at least 8 weeks after the second dose (including, for example, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks or longer). In some such embodiments, at least one dose administered is 3ug RNA described herein. In some embodiments, subjects 5 years old and above can be administered according to a primary immunization scheme comprising at least two doses, wherein the two doses are administered at least 3 weeks apart (including, for example, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks or longer). In some such embodiments, at least one dose administered is 10ug RNA described herein. In some embodiments, immunocompromised subjects 5 years of age and older (e.g., in some embodiments, subjects who have undergone a solid organ transplant or have been diagnosed with an equivalent condition considered to be immunocompromised) can be administered according to a priming regimen comprising at least three doses, wherein the initial two doses are administered at least 3 weeks (including, for example, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or more) apart, followed by a third dose administered at least 4 weeks (including, for example, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or more) after the second dose.

[0298] In some embodiments, the mRNA compositions described herein are applied to subjects aged 12 years or more, and each dose is about 30ug. In some embodiments, the mRNA compositions described herein are applied to subjects aged 12 years or more (including, for example, 18 years or more), and each dose is higher than 30ug, including, for example, 35ug, 40ug, 45ug, 50ug, 55ug, 60ug, 65ug, 70ug or higher. In some such embodiments, the mRNA compositions described herein are applied to subjects aged 12 years or more, and each dose is about 60ug. In some such embodiments, the mRNA compositions described herein are applied to subjects aged 12 years or more, and each dose is about 50ug. In one embodiment, the pediatric colony includes or is composed of the following: 12 to less than 18 years of subjects, including 16 to less than 18 years of subjects and / or 12 to less than 16 years of subjects. In this embodiment, treatment may include 2 vaccinations at intervals of 21 days, wherein in one embodiment, the vaccine is administered, for example, by intramuscular administration with an amount of 30 μg RNA per dose. In some embodiments, higher doses are administered to older pediatric patients and adults, e.g., patients 12 years of age or older, compared to younger children or infants, e.g., 2 to less than 5 years of age, 6 months to less than 2 years of age, or less than 6 months of age. In some embodiments, higher doses are administered to children 2 to less than 5 years of age, compared to toddlers and / or infants, e.g., 6 months to less than 2 years of age, or less than 6 months of age.

[0299] In one embodiment, the pediatric population comprises or consists of: 5 to less than 18 years of age, including 12 to less than 18 years of age and / or 5 to less than 12 years of age. In this embodiment, treatment may include 2 vaccinations 21 days apart, wherein in various embodiments, the vaccine is administered, for example, by intramuscular administration in an amount of 10 μg, 20 μg or 30 μg RNA per dose. In some such embodiments, the mRNA compositions described herein are administered to subjects aged 5 to 11 years, and each dose is about 10 ug.

[0300] In one embodiment, the pediatric colony comprises the following or is composed of the following: less than 5 years old subjects, including 2 to less than 5 years old subjects, 12 to less than 24 months of subjects, 7 to less than 12 months of subjects, 6 to less than 12 months of subjects and / or less than 6 months of subjects. In this embodiment, treatment may include, for example, 21 to 42 days at intervals, for example, 2 vaccinations at intervals of 21 days, wherein in each embodiment, the vaccine is, for example, administered by intramuscular administration with the amount of 3 μg, 10 μg, 20 μg or 30 μg RNA per dosage. In some such embodiments, mRNA compositions described herein are applied to 2 to less than 5 years old subjects, and each dosage is about 3ug. In some such embodiments, mRNA compositions described herein are applied to about 6 months to less than about 5 years old subjects, and each dosage is about 3ug.

[0301] In some embodiments, the mRNA compositions described herein are applied to subjects aged 12 years or more, and at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 60ug. In some embodiments, the mRNA compositions described herein are applied to subjects aged 12 years or more, and at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 30ug. In some embodiments, the mRNA compositions described herein are applied to subjects aged 12 years or more, and at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 15ug. In some embodiments, the mRNA compositions described herein are applied to subjects aged 5 to less than 12 years, and at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 10ug. In some embodiments, the mRNA compositions described herein are applied to subjects aged 2 to less than 5 years, and at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3ug. In some embodiments, the mRNA compositions described herein are administered to subjects aged 6 months to less than 2 years, and at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3 ug or less, including, for example, 2 ug, 1 ug or less). In some embodiments, the mRNA compositions described herein are administered to infants less than 6 months old, and at least one dose administered in a vaccination regimen (e.g., a primary vaccination regimen and / or a booster vaccination regimen) is about 3 ug or less, including, for example, 2 ug, 1 ug, 0.5 ug or less).

[0302] In some embodiments, a dose administered to a subject in need thereof may comprise administration of a single mRNA composition described herein.

[0303] In some embodiments, the dosage administered to a subject in need thereof may include administration of at least two or more (including, for example, at least three or more) different pharmaceutical products / formulations. For example, in some embodiments, at least two or more different pharmaceutical products / formulations may include at least two different mRNA compositions described herein (e.g., in some embodiments, each comprising a different RNA construct).

[0304] In some embodiments, two or more RNAs are administered to a subject (e.g., as part of a primary immunization regimen or a booster regimen), wherein the two or more RNAs are administered on the same day or at the same visit. In some embodiments, the two or more RNAs are administered in separate compositions, e.g., by administering each RNA to separate portions of the subject (e.g., by intramuscular administration to different arms of the subject or different parts of the same arm of the subject). In some embodiments, the two or more RNAs are mixed before administration (e.g., for example, by administering a practitioner to mix just before administration). In some embodiments, the two or more RNAs are formulated together (e.g., by (a) mixing separate populations of LNPs, each population comprising different RNAs; or (b) by mixing two or more RNAs before LNP formulation, so that each LNP comprises two or more RNAs).

[0305] In some embodiments, the composition administered to a subject comprises one or more first RNAs and one or more second RNAs, each RNA in the same amount (ie, in a 1:1 ratio).

[0306] In some embodiments, a composition is administered to a subject or comprises one or more first RNAs and one or more second RNAs, each in different amounts. For example, in some embodiments, the composition is administered to a subject or comprises one or more first RNAs in an amount that is 0.01 to 100 times the amount of the one or more second RNAs (e.g., wherein the amount of the one or more first RNAs is 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to 4, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 to 1.5 times the amount of the one or more second RNAs). In some embodiments, the subject is administered or the composition comprises one or more first RNA and one or more second RNA, wherein the concentration of the one or more first RNA is 1 to 10 times of the one or more second RNA. In some embodiments, the subject is administered or the composition comprises one or more first RNA and one or more second RNA, wherein the amount of the one or more first RNA is 1 to 5 times of the one or more second RNA. In some embodiments, the subject is administered or the composition comprises one or more first RNA and one or more second RNA, wherein the concentration of the one or more first RNA is 1 to 3 times of the one or more second RNA. In some embodiments, the subject is administered or the composition comprises one or more first RNA and one or more second RNA, wherein the amount of the one or more first RNA is 2 times of the one or more second RNA. In some embodiments, the subject is administered or the composition comprises one or more first RNA and one or more second RNA, wherein the concentration of the one or more first RNA is 3 times of the one or more second RNA.

[0307] In some embodiments, the subject is administered or the composition comprises two first RNAs, each encoding an antigen derived from an influenza strain or variant, wherein the amount of each RNA is different. For example, in some embodiments, the ratio of the two first RNAs is 1:0.01-100 (e.g., 1:0.01-50; 1:0.01-40; 1:0.01-30; 1:0.01-25; 1:0.01-20; 1:0.01-15; 1:0.01-10; 1:0.01-9; 1:0.01-8; 1:0.01-7; 1:0.01-6; 1:0.01-5; 1:0.01-4; 1:0.01-3; 1:0.01-2; 1:0.01-1.5, 1:0.1-10, 1:0.1-5, 1:0.1-3, 1:2-10, 1:2-5, or 1:2-3). In some embodiments, the composition is administered to a subject or comprises two first RNAs in a ratio of 1:3. In some embodiments, the subject is administered or the composition comprises two first RNAs in a ratio of 1:2.

[0308] For example, in some embodiments, the ratio of the three first RNAs is 1:0.01-100:0.01-100 (e.g., 1:0.01-50:0.01-50; 1:0.01-40:0.01-40; 1:0.01-30:0.01-30; 1:0.01-25:0.01-25; 1:0.01-20:0.01-20; 1:0.01-15:0.01-15; 1:0.01-10:0.01-10; 1:0.01-9:0.01-9; 1:0.01-8:0. 01-8; 1:0.01-7:0.01-7; 1:0.01-6:0.01-6; 1:0.01-5:0.01-5; 1:0.01-4:0.01-4; 1:0.01-3:0.01-3; 1:0.01-2:0.01-2; 1:0.01-1.5:0.01-1.5; 1:0.1-10:0.1-10, 1:0.1-5:0.1-5, 1:0.1-3:0.1-3, 1:2-10:2-10, 1:2-5:2-5, or 1:2-3:2-3). In some embodiments, the subject is administered or the composition comprises the three first RNAs in a ratio of 1:1:3. In some embodiments, the subject is administered or the composition comprises the three first RNAs in a ratio of 1:3:3.

[0309] In some embodiments, the subject is administered or the composition comprises two or more second RNAs, one or more of which encode the HA protein of influenza A virus, and one or more of which encode the HA protein of influenza B virus. In some embodiments, one or more second RNAs encoding the HA protein of influenza A virus and one or more second RNAs encoding the HA protein of influenza B virus are present or administered in the same amount (i.e., at a ratio of 1: 1). In some embodiments, one or more second RNAs encoding the HA protein of influenza A virus and one or more second RNAs encoding the HA protein of influenza B virus are administered in different amounts (e.g., at a ratio of 1: 10 to 10: 1, or at a ratio of 1: 2, 1: 3, 1: 4, 1: 5, 2: 1, 3: 1, 4: 1 or 5: 1 (total RNA encoding A antigen: total RNA encoding B antigen).

[0310] In some embodiments, the subject is administered or the composition comprises two second RNAs, each of which encodes an HA protein of a different influenza virus type (e.g., a second RNA encoding an HA protein of influenza A virus and a second RNA encoding an HA protein of influenza B virus). In some embodiments, the second RNA is administered or present in the same amount (i.e., at a ratio of 1:1). In some embodiments, the second RNA is administered or present in different amounts (e.g., at a ratio of 1:10 to 10:1, or at a ratio of 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1 (A:B)).

[0311] In some embodiments, the subject is administered or the composition comprises three second RNAs, each of which encodes an HA protein of a different influenza virus subtype (e.g., an HA protein of A / Wisconsin (H1N1) virus, A / Darwin (H3N2) virus, and B / Austria (Victoria) virus). In some embodiments, the subject is administered or the composition comprises each of the three second RNAs in the same amount (i.e., in a 1:1:1 ratio). In some embodiments, the subject is administered or the composition comprises different amounts of one or more of the three second RNAs (e.g., at a ratio of 1:1:2 to 1:1:10 (e.g., at a ratio of 1:1:2, 1:1:3, 1:1:4, or 1:1:5), or at a ratio of 2:2:1 to 2:2:10 (e.g., at a ratio of 2:2:1, 3:3:1, 4:4:1, or 5:5:1). In some embodiments, the subject is administered or the composition comprises three second RNAs, two of which encode HA proteins of different influenza A viruses, and one of which encodes an HA protein of influenza B virus. In some such embodiments, the second RNA encoding the HA protein of influenza B virus is present or administered in a higher amount than any second RNA encoding the HA protein from influenza A virus (e.g., in some embodiments, the two second RNAs encoding the HA proteins from influenza A virus are present or administered relative to the second RNAs encoding the HA proteins from influenza B virus). In some embodiments, the ratio of the second RNA encoding the HA protein of influenza A virus to the second RNA encoding the HA protein of influenza B virus is 1:1:1-10, 1:1:2, 1:1:3, 1:1:4, or 1:1:5 (A:A:B). In some embodiments, the subject is administered or the composition comprises three second RNAs, two encoding HA proteins of influenza A virus and one encoding HA protein of influenza B virus, wherein the ratio of the three second RNAs is 1:1:4 (A:A:B). In some embodiments, the two second RNAs encoding the HA protein of influenza A virus are each present in a higher amount or are each administered in a higher amount compared to the second RNA encoding the HA protein from influenza B virus (for example, in some embodiments, the ratio of the two second RNAs encoding the HA protein from influenza A virus to the second RNA encoding the HA protein from influenza B virus is 1-10:1-10:1, 2:2:1, 3:3:1, 4:4:1, or 5:5:1 (A:A:B)).

[0312] In some embodiments, the subject is administered or the composition comprises four second RNAs, each of which encodes the HA protein of different influenza virus subtypes. In some such embodiments, the four second RNAs comprise two second RNAs encoding the HA protein of different influenza A viruses and two second RNAs encoding the HA protein of different influenza B viruses (e.g., HA protein of H1N1 virus, HA protein of H3N2 virus, HA protein of B / Victoria lineage virus, and HA protein of B / Yamagata lineage virus). In some embodiments, each of the two second RNAs encoding the HA protein of influenza A virus and the two second RNAs encoding the HA protein of influenza B virus are present in the same amount (i.e., the ratio of the four second RNAs is 1:1:1:1). In some embodiments, the two second RNAs encoding the HA protein of influenza B virus are each administered in a higher amount or are each present in a higher amount than any second RNA encoding the HA protein from influenza A virus (e.g., in some embodiments, the ratio of the two second RNAs encoding the HA protein from influenza A virus relative to the two second RNAs encoding the HA protein from influenza B virus is 1:1:2-10:2-10, 1:1:2-5:2-5, 1:1:2:2, 1:1:3:3, 1:1:4:4, 1:1:5:5, 1:1:6:6, 1:1:7:7, 1:1:8:8, 1:1:9:9, 1:1:10:10 (A:A:B:B)). In some embodiments, the subject is administered or the composition comprises four second RNAs, two encoding the HA protein of influenza A virus and two encoding the HA protein of influenza B virus, wherein the ratio of the four second RNAs is 1:1:5:5 (A:A:B:B). In some embodiments, the two second RNAs encoding HA proteins from influenza A virus are each administered in a higher amount or are each present in a higher amount than any second RNA encoding HA proteins from influenza B virus (e.g., in some embodiments, the ratio of the two second RNAs encoding HA proteins from influenza A virus relative to the two second RNAs encoding HA proteins from influenza B virus is 2-10:2-10:1:1, 2-5:2-5:1:1, 2:2:1:1, 3:3:1:1, 4:4:1:1, 5:5:1:1, 6:6:1:1, 7:7:1:1, 8:8:1:1, 9:9:1:1, 10:10:1:1 (A:A:B:B)).

[0313] In some embodiments, the composition comprises or administers to a subject four second RNAs, comprising three second RNAs encoding HA proteins of different influenza A viruses and one second RNA encoding HA proteins of influenza B viruses (e.g., A / Wisconsin (H1N1), A / Darwin (H3N2), A / Cambodia (H3N2), and B / Austria (Victoria)). In some such embodiments, each of the four second RNAs is administered or present in the same amount (i.e., in a ratio of 1:1:1:1). In some embodiments, the amount of the second RNA encoding the HA protein of influenza B virus is higher than any of the second RNAs encoding the HA protein of influenza A virus (e.g., in some embodiments, the ratio of the second RNAs is 1:1:1:1-10, 1:1:1:1-5, 1:1:1:2, 1:1:1:3, 1:1:1:4, or 1:1:1:5 (A:A:A:B)). In some embodiments, the ratio of the second RNA administered or in the composition is 1:1:1:5 (A:A:A:B). In some embodiments, the amount of each second RNA encoding the HA protein of influenza A virus is higher than the amount of the second RNA encoding the HA protein of influenza B virus (e.g., in some embodiments, the ratio of the second RNA is 1-10:1-10:1-10:1, 1-5:1-5:1-5:1, 2:2:2:1, 3:3:3:1, 4:4:4:1, or 5:5:5:1 (A:A:A:B)).

[0314] In some embodiments, the subject is administered or the composition comprises one or more second RNAs encoding the HA protein of influenza virus (e.g., two second RNAs, three second RNAs, or four second RNAs, each encoding a different HA protein of influenza virus), in a total amount of 0.1 to 100 μg (e.g., 1 to 90 μg, 3 to 90 μg, 1 to 60 μg, 3 to 60 μg, 5 to 60 μg, 10 to 60 μg, 30 to 60 μg, 3 to 30 μg). In some embodiments, the subject is administered or the composition comprises one or more second RNAs encoding the HA protein of influenza virus, in a total amount of 3 μg, 5 μg, 6 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 45 μg, 60 μg, 75 μg, or 90 μg.

[0315] In some embodiments, the subject is administered or the composition comprises three or four second RNAs, each encoding an HA antigen of a different influenza strain, in one of the amounts listed in Table C below (each "influenza component" corresponds to a second RNA encoding an HA antigen (e.g., a second RNA as described herein).

[0316] In some embodiments, a composition described herein is characterized in that it produces an influenza neutralizing antibody titer that is at least twice the neutralizing antibody titer produced by a reference vaccine for each influenza virus for which it encodes an antigen (e.g., where the reference vaccine is a quadrivalent influenza RNA vaccine or an approved (non-RNA) influenza vaccine administered alone).

[0317] In some embodiments, the influenza vaccine is an alpha influenza virus vaccine, a beta influenza virus vaccine, a gamma influenza virus vaccine, or a delta influenza virus vaccine. In some embodiments, the vaccine is an influenza virus vaccine, an influenza virus vaccine, an influenza virus vaccine, or an influenza virus vaccine. In some embodiments, the influenza virus vaccine comprises a hemagglutinin selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18, or an immunogenic fragment or variant thereof, or a nucleic acid encoding any one thereof (e.g., RNA). In some embodiments, the influenza vaccine comprises or encodes a neuraminidase (NA) selected from N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11, or an immunogenic fragment or variant thereof, or a nucleic acid encoding any one thereof (e.g., RNA). In some embodiments, the influenza vaccine comprises at least one influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2 and / or polymerase basic protein 2 (PB2), or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any of them. Example

[0318] Example 1: Description of the manufacturing process

[0319] A description of the manufacturing process and process control of influenza saRNA vaccine APIs is provided in this section. The manufacturing process includes RNA synthesis via an in vitro transcription (IVT) step and a purification step by ultrafiltration / diafiltration (UFDF-1). RNA is then enzymatically capped and purified by chromatography and final UFDF-2, followed by final filtration and distribution.

[0320] The process has been scaled up to a 1.5L starting IVT volume for manufacturing clinical material. Other than the changes required to scale up the process to 1.5L, there were no significant changes from the nonclinical toxicology / development process.

[0321] RT-ddPCR (identification of encoded RNA sequences)

[0322] If after performing a one-step reverse transcription (RT)-ddPCR assay of the RNA in the sample, the test sample is positive for the replicase sequence (confirming the self-amplification of the RNA construct) and the target sequence (confirming the encoded influenza sequence), the identity of the influenza saRNA is confirmed. Digital droplet polymerase chain reaction (ddPCR) technology is a digital form of polymerase chain reaction (PCR) that uses a water-oil emulsion system to quantify target nucleic acids. The RNA sample is diluted to a final theoretical input concentration that is within the linear range of the ddPCR assay. The reaction mixture containing reverse transcriptase, DNA polymerase, and sequence-specific primers and probes is divided into droplets, and the PCR reaction is performed individually in each partition. The results are calculated by counting the amplified target sequence (positive droplets, as measured by fluorescence amplitude above background) and the number of partitions in which there is no amplification (negative droplets). After the positive and negative controls have been checked and determined to be valid and acceptable, if the positive droplet count is above the established threshold, the identity is confirmed.

[0323] Reversed-phase HPLC (presence of pseudouridine)

[0324] After complete digestion of mRNA, the presence of pseudouridine is determined by reverse phase high performance liquid chromatography (RP-HPLC). The individual nucleosides obtained have a characteristic elution pattern, including the resolution of uridine and pseudouridine. The presence of pseudouridine is confirmed by comparison with uridine and pseudouridine references and limit standards.

[0325] Capillary gel electrophoresis (RNA integrity)

[0326] RNA integrity is determined by capillary gel electrophoresis (CGE) based on the differential migration of RNA of different molecular weights in an applied electric field. RNA is subjected to a denaturant, which causes RNA to unfold and dissociate non-covalent complexes. When subjected to an electric field, denatured RNA species migrate toward the anode through a gel matrix according to length and size. Intercalating dyes bind to RNA and related fragments during migration, allowing fluorescent detection. Intact RNA is separated from any fragmented species, allowing quantitative RNA integrity to be determined by determining the relative percentage time-corrected area about the intact (main) peak.

[0327] qPCR (residual DNA template)

[0328] The level of residual DNA template is determined by quantitative polymerase chain reaction (qPCR) using fluorescence technology. The qPCR master mix comprising target-specific primers and fluorescent qPCR quantitative reagents is added to all sample wells. The sample is prepared with a series of dilutions and analyzed in real time by qPCR. The measured fluorescence signal is directly proportional to the amount of PCR product. The quantification of DNA is performed under the cycle threshold (Ct) during the exponential phase of the reaction, wherein the target sequence amplification above the signal threshold established is detected for the first time. The Ct point depends on the amount of DNA initially present in the sample. Taking into account the dilution factor, the DNA concentration in the test sample is calculated according to the linear regression of the standard curve. The results are reported in ng DNA / mg RNA.

[0329] Batch details and batch analytical summary data for 1 batch of regulatory toxicology material and 1 GMP batch of drug substance to be used in clinical trials are provided in Table 1.

[0330] Table 1

[0331] S.4.4.6-2. Batch results for influenza saRNA vaccine TC83-delkozak-HA-SGP-NA-80A API

[0332]

[0333] a. Identification via reverse transcription and quantitative polymerase chain reaction

[0334] Specifications For clinical supply only Abbreviations: NTU = nephelometric turbidity units; NT = not tested; TBP = to be provided in IND amendment; ddPCR = digital droplet polymerase chain reaction; qPCR = quantitative polymerase chain reaction; LAL = horseshoe crab amebocyte lysate; NMT = not more than; EU = endotoxin units; CFU = colony forming units

[0335] Example 2: S.4.1 Description and composition of drug product

[0336] The PF-07867246 (Construct 6 (TC83-delkozak-HA-SGP-NA-80A) (SEQ ID NO: 1)) drug product is a preservative-free, sterile dispersion of liquid nanoparticles (LNPs) in an aqueous cryoprotectant buffer for intramuscular administration. The drug product is formulated at 0.06 mg / mL RNA in 10 mM Tris buffer, 10% sucrose, and optionally 20 mM glutamate, pH 7.4. The drug product is supplied in 2 mL glass vials sealed with chlorobutyl rubber stoppers and aluminum seals with flip-top plastic caps (nominal capacity of 0.5 mL). The composition of the drug product is given in Table 2, including the unit formula, amount / vial, function, and quality standards applicable to each component.

[0337] Table 2

[0338]

[0339] Table 3

[0340] 3.6.TC83-delkozak-HA-SGP-NA-80A (A / Wisconsin / 588 / 2019) -- VV00050 (Construct 6) Nucleotide sequence 5'-->3'

[0341] Sequence length: 10936 nucleotides; 3113 A; 2761 C; 2796 G; 2266 UA = adenine; C = cytosine; G = guanine; U = uridine

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348] Example 3: S.4.1. Description and composition of drug product

[0349] The PF07871987 (Construct 7TC83-HA-40A 50U-50pU (SEQ ID NO: 2)) drug product is a preservative-free, sterile dispersion of liquid nanoparticles (LNPs) in an aqueous cryoprotectant buffer for intramuscular administration. The drug product is formulated at 0.06 mg / mL RNA in 10 mM Tris buffer, 10% sucrose, and optionally 20 mM glutamate, pH 7.4.

[0350] The drug product is supplied in 2 mL glass vials sealed with chlorobutyl rubber stoppers and aluminum seals with flip-top plastic caps (nominal capacity of 0.5 mL).

[0351] The composition of the drug product is given in Table 4, including the unit formula, amount / vial, function, and the specification applicable to each component.

[0352] Table 4:

[0353]

[0354] Table 5 S.4.4.6-4. Batch results for influenza saRNA vaccine TC83-HA-40A 50U-50pU API

[0355]

[0356] a. Identification via reverse transcription and quantitative polymerase chain reaction

[0357] b. Nonclinical toxicology reports the approximate percentage of pseudoU. Its presence can be inferred from the results reported as being “confirmed”.

[0358] Specifications For clinical supply only Abbreviations: NTU = nephelometric turbidity units; NT = not tested; TBP = to be provided in IND amendment; ddPCR = digital droplet polymerase chain reaction; qPCR = quantitative polymerase chain reaction; LAL = horseshoe crab amebocyte lysate; NMT = not more than; EU = endotoxin units; CFU = colony forming units

[0359]

[0360]

[0361]

[0362]

[0363]

[0364] Example 4: S.4.4.1 Construct 7: TC83-HA-40A 50U-50PU (PF-07871987)

[0365] Batch details and batch analytical summary data for 1 batch of regulatory toxicology material and 1 GMP batch of drug substance to be used in clinical trials are provided in Table 7.

[0366] Table 7

[0367] S.4.4.6-7. Batch results for influenza saRNA vaccine TC83-HA-40A 50U-50pU API

[0368]

[0369] a. Identification via reverse transcription and quantitative polymerase chain reaction

[0370] b. Nonclinical toxicology reports the approximate percentage of pseudoU. Its presence can be inferred from the results reported as being “confirmed”.

[0371] Specifications apply only to clinical supply

[0372] Abbreviations: NTU = nephelometric turbidity units; NT = not tested; TBP = to be provided in IND amendment; ddPCR = digital droplet polymerase chain reaction; qPCR = quantitative polymerase chain reaction; LAL = horseshoe crab amebocyte lysate; NMT = not more than; EU = endotoxin unit; CFU = colony forming unit

[0373] Example 5: Determination

[0374] Hemagglutination inhibition assay

[0375] The main serological assay for measuring vaccine-induced immune responses to influenza is the hemagglutinin inhibition assay (HAI). HAI quantitatively measures functional antibodies in serum, which prevents HA-mediated hemagglutination of red blood cells in the reaction of serum samples, influenza viruses and red blood cells derived from turkeys or guinea pigs containing receptor-destroying enzyme pretreatment. The HAI titer is the reciprocal of the highest serum dilution that causes HA activity loss, and when the microtiter plate is tilted, it appears as a teardrop shape. The titer report from multiple determinations / samples is the geometric mean titer (GMT). ≥1:40 HAI titers are generally recognized as protective in people.

[0376] Influenza microneutralization assay

[0377] The influenza virus microneutralization assay (MNT) quantitatively measures functional antibodies in serum that neutralize influenza virus activity, preventing productive infection of host cell monolayers. When influenza virus is incubated with a serum sample, a neutralization reaction occurs; the reaction mixture is then applied to a monolayer of Madin-Darby Canine Kidney (MDCK) cells to measure the degree of neutralization. MNT titers are reported as the reciprocal of the dilution that results in a 50% or 90% reduction in infection when compared to a serum-free control. The 1-day MNT measures anti-HA neutralizing antibodies, while the 3-day MNT measures both anti-HA neutralizing antibodies and anti-NA neutralizing antibodies.

[0378] Neuraminidase inhibition assay

[0379] The neuraminidase inhibition assay (NAI) quantitatively measures functional antibodies in serum that prevent NA-mediated sialic acid cleavage in an enzyme-linked lectin assay. Briefly, serum containing antibodies is incubated with influenza virus and the mixture is then transferred to a plate coated with fetuin lectin. The cleavage of sialic acid from fetuin is monitored by binding of horseradish peroxidase-conjugated peanut agglutinin to exposed galactose moieties and a colorimetric reaction following substrate addition. The NAI titer is the reciprocal of the highest serum dilution that results in a 50% reduction in NA activity compared to a serum-free control. Titers from multiple assays / samples are reported as geometric mean titers (GMTs).

[0380] Example 6: Evaluation of influenza bicistronic HA-NA saRNA vaccine design in mice

[0381] This study was conducted to compare the immunogenicity of bicistronic saRNA vaccine candidates encoding influenza hemagglutinin (HA) and neuraminidase (NA) to determine the optimal bicistronic HA-NA saRNA vaccine design. The saRNA vectors used in this study were all based on the TC-83 backbone, however the study was designed to evaluate the immunogenicity with or without an exogenous kozak sequence upstream of the first gene of interest and the effect of the poly A tail length (40A or 80A).

[0382] The key bicistronic design elements evaluated in this study include regulatory elements (subgenomic promoter (SGP) relative to internal ribosome entry site (IRES)) for driving expression of the second gene of interest and the antigen placement order (HA-NA or NA-HA) on the vector.

[0383] Intramuscular immunization of Balb / c mice with LNP-formulated saRNA vaccines encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA antigens induced functional and neutralizing antibody responses.

[0384] Overall, similar titers were elicited with all bicistronic saRNA vaccines tested, and these titers were also similar to saRNA vaccines composed of individually formulated saRNA-HA+saRNA-NA. These results confirm that the bicistronic saRNA approach elicits titers and is feasible.

[0385] The saRNA vectors used in this study are all based on the TC-83 backbone, but the study is designed to evaluate the immunogenicity of the first target gene with or without an exogenous kozak sequence and the effect of the poly A tail length (40A or 80A). The key bicistronic design elements evaluated in this study include the regulatory elements required to drive the expression of the second target gene and the order of antigen placement on the vector (HA-NA or NA-HA). The regulatory elements selected for comparison are the natural VEEV subgenomic promoter (SGP; 61 nucleotides) and the internal ribosome entry site (IRES, 587 nucleotides) derived from encephalomyocarditis virus. As a comparator, modRNA vaccines encoding influenza HA or NA are also included in the study.

[0386] Mice were immunized with saRNA or modRNA LNP formulations on days 0 and 28, and sera were collected 21 days after the prime and 14 days after the boost. Neutralizing and functional antibodies were measured on days 21 and 42 to determine immunogenicity.

[0387] The study was designed to have 15 groups as shown in Table 8, each containing a total of 10 female mice (mouse strain: BALB / c). Evaluation of mRNA drug product at 0.05 mL dose volume

[0388] Table 8. Study design

[0389]

[0390] Table 9. Study schedule

[0391] sky program 0 Vax #1 21 bloodletting 28 Vax #2 42 Final bloodletting

[0392] Table 10. Test articles and diluents used in the study (provided for both priming and boosting)

[0393]

[0394]

[0395] M. Analytical test results of test items

[0396] Table 11. Drug product materials and analytical results

[0397]

[0398] 2. Results and Discussion

[0399] Intramuscular immunization of Balb / c mice with LNP-formulated saRNA vaccines encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA antigens induced functional and neutralizing antibody responses as measured by HAI, 1-day MNT, 3-day MNT, and NAI (1.0 and 1.0, respectively). Figure 1 , Figure 2 , Figure 3 and Figure 4 ), with a clear boosting effect two weeks after the second immunization. The 3-day MNT results at day 42 showed that NA contributed the least to neutralization compared to HA in this assay. Overall, similar titers were achieved with all bicistronic saRNA vaccine designs evaluated, and these titers were also similar to saRNA vaccines composed of individually formulated HA and NA monocistronic saRNAs (HA / NA post-mixing) and modRNA. The titers of saRNA vaccines expressing two antigens were similar or slightly lower than those of saRNA-HA or -NA controls alone. These data confirm that the bicistronic saRNA approach is feasible.

[0400] The absence of the Kozak sequence, the length of the poly(A) tail, the regulatory element used to drive the second gene of interest (IRES versus SGP), and the order of antigen placement (HA-NA or NA-HA) had no clear effect on the titers elicited.

[0401] about Figure 1 Female Balb / c mice were immunized IM with different LNP-formulated influenza saRNA vaccine constructs and influenza modRNA comparators encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA on days 0 and 28. The HA / NA post-mix formulation consisted of a 1:1 mixture of individually formulated saRNA-HA plus saRNA-NA. Functional antibody responses to A / Wisconsin / 588 / 2019 were measured by HAI on days 21 (3 weeks after the primary immunization) and 42 (2 weeks after the boost).

[0402] about Figure 2Female Balb / c mice were immunized IM with different LNP-formulated influenza saRNA vaccine constructs and influenza modRNA comparators encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA on days 0 and 28. The HA / NA post-mix formulation consisted of a 1:1 mixture of individually formulated saRNA-HA plus saRNA-NA. Functional antibody responses to A / Wisconsin / 588 / 2019 were measured by a 1-day MNT assay on days 21 (3 weeks after the primary immunization) and 42 (2 weeks after the boost). A neutralization titer of 50% was reported.

[0403] about Figure 3 Female Balb / c mice were immunized IM with different LNP-formulated influenza saRNA vaccine constructs and influenza modRNA comparators encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA on days 0 and 28. The HA / NA post-mix formulation consisted of a 1:1 mixture of individually formulated saRNA-HA plus saRNA-NA. At day 42 (2 weeks after boost), functional antibody responses against A / Wisconsin / 588 / 2019 were measured by a 3-day MNT assay. A neutralization titer of 50% was reported.

[0404] about Figure 4 Female Balb / c mice were immunized IM with different LNP-formulated influenza saRNA vaccine constructs and influenza modRNA comparators encoding A / Wisconsin / 588 / 2019 (H1N1) HA and / or NA on days 0 and 28. The HA / NA post-mix formulation consisted of a 1:1 mixture of individually formulated saRNA-HA plus saRNA-NA. Functional antibody responses to A / Wisconsin / 588 / 2019 were measured by NAI on days 21 (3 weeks after the primary immunization) and 42 (2 weeks after the boost).

[0405] 3. Conclusion

[0406] Seasonal influenza saRNA vaccines are designed to express 4 different HA proteins and 4 different NA proteins to match the dominant influenza strains of each season. This can potentially be achieved using 8 individual saRNA components or 4 bicistronic saRNA components. For this reason, the feasibility of the bicistronic saRNA method was evaluated in mouse immunogenicity studies. Bicistronic saRNA vaccine candidates (TC83-delkozak-HA-SGP-NA-80A) encoding both HA and NA antigens were compared with monocistronic saRNA-HA or saRNA-NA controls (TC83-HA-40A or TC83-NA-40A) encoding a single antigen, and were also compared with a 1: 1 mixture of individually formulated saRNA-HA+saRNA-NA components. ModRNA vaccine candidates encoding the same A / Wisconsin / 588 / 2019 (H1N1) HA or NA antigens were also included as additional comparators. Balb / c mice were immunized IM on day 0 with 20 ng of bicistronic and monocistronic saRNA vaccine formulations, a total of 40 ng (20 ng each) of a 1:1 mixture of saRNA-HA + saRNA-NA, and 200 ng of modRNA comparator. All saRNA LNPs were in a 10 mM Tris / 10% sucrose + 20 mM glutamate, pH 7.4 matrix selected for clinical use. At day 21 (3 weeks after primary immunization), anti-HA antibody responses were induced as measured by HAI and MNT ( Figure 1 and Figure 2 ), and also induced anti-NA antibody responses as measured by NAI ( Figure 4 ). The bicistronic saRNA vaccine candidate achieved similar titers to saRNA vaccines consisting of a 1:1 mixture of individually formulated monocistronic saRNA HA + saRNA-NA. The titers for saRNA vaccine formulations expressing two antigens were similar to or slightly lower than those produced by saRNA controls expressing only a single antigen. These results confirm that the bicistronic saRNA approach is feasible and indicate that saRNA vaccines provide dose savings compared to modRNA in Balb / c mice based on antibody titers after a single immunization.

[0407] Overall, the functional and neutralizing antibody titers produced by the evaluated bicistronic saRNA constructs were similar to the levels induced by saRNA vaccines, which consisted of individually formulated HA and NA monocistronic saRNAs. The titers of saRNA vaccines expressing two antigens were similar or slightly lower than those of saRNA-HA or -NA controls alone. These preliminary results confirm that the bicistronic saRNA approach is feasible, regardless of the regulatory elements used to drive the second gene of interest (IRES relative to SGP), the order of antigen placement (HA-NA or NA-HA), the absence of kozak sequences, and the length of the poly A tail (40A relative to 80A).

[0408] Example 7: Immunogenicity of saRNA influenza vaccines containing modified nucleosides

[0409] To determine whether the incorporation of modified bases can produce more tolerable and potent saRNA vaccines, saRNA formulations expressing influenza HA were generated by replacing uridine with different amounts of N1-methylpseudouridine from 0% to 100%. The effect of increasing the percentage of modified bases on in vitro antigen expression depends on the cell type. In particular, in immunocompetent human cell lines such as HeLa, saRNA containing 25-75% N1-methylpseudouridine resulted in in vitro antigen expression higher than that of unmodified (0%) saRNA controls. However, regardless of the cell type, saRNA with 100% base modification always produced low levels of antigen, most likely due to impaired replicase function. Increasing the percentage of modified nucleosides incorporated into saRNA is also associated with reduced activation levels of different PRRs or RNA sensors such as TLR3, TLR7, and RIG-1 in reporter cell lines (data not shown).

[0410] To evaluate immunogenicity, Balb / c mice were immunized IM with 200 ng of saRNA vaccine formulations containing varying amounts of N1-methylpseudouridine on day 0. Incorporation of higher amounts of modified nucleosides was associated with less innate immune activation as measured by cytokine and chemokine secretion in serum on day 1 post-vaccination ( Figure 5 ), which may improve the tolerability of the vaccine. However, a higher amount of modified nucleosides in saRNA was also associated with a decrease in neutralizing antibody titers at 3 weeks after vaccination (Figure 6 ), which could potentially reflect an effect on replicase activity. These results were confirmed in a different C57BL6 / J mouse species ( Figure 7 and Figure 8). Based on mouse data, saRNA constructs with 50% incorporation of modified nucleosides substantially reduced cytokine and chemokine secretion compared to unmodified controls, with antibody titers more modestly reduced to levels similar to or higher than the modRNA-HA benchmark. Overall, the data suggest that partial incorporation of modified bases can be tolerated by saRNA to reduce early innate immune stimulation while still eliciting a strong adaptive humoral response.

[0411] The usage of 50% modified bases may partially affect replicase function, but the enhanced tolerance can potentially lead to improved saRNA vaccines in humans.

[0412] about Figure 5 Female Balb / c mice were immunized IM on day 0 with 200ng LNP-formulated influenza saRNA vaccine formulations containing varying amounts (0% to 100%) of N1-methylpseudouridine or influenza modRNA comparators encoding A / Wisconsin / 588 / 2019 (H1N1) HA. Serum cytokines and chemokines were measured 24 hours after the first immunization using the mouse antiviral response panel LEGENDplex assay. Data are reported as medians with interquartile ranges.

[0413] about Figure 6 Female Balb / c mice were immunized IM on day 0 with 200 LNP-formulated influenza saRNA vaccine formulations containing varying amounts (0% to 100%) of N1-methylpseudouridine or a modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA. Antibody responses against A / Wisconsin / 588 / 2019 were measured by HAI or 1-day MNT assays on day 21 (3 weeks post-immunization). HAI and 50% neutralization titers (geometric means with geometric SD) are reported.

[0414] about Figure 7 Female C57BL6 / J mice were immunized IM on day 0 with 200ng LNP-formulated influenza saRNA vaccine formulations containing different amounts (0% or 50%) of N1-methylpseudouridine or influenza modRNA comparators encoding A / Wisconsin / 588 / 2019 (H1N1) HA. Serum cytokines and chemokines were measured 24 hours after the first immunization using the mouse antiviral response panel LEGENDplex assay. Data are reported as medians with interquartile ranges.

[0415] about Figure 8Female C57BL6 / J mice were immunized IM with 200LNP-formulated influenza saRNA vaccine formulations containing different amounts (0% or 50%) of N1-methylpseudouridine or a modRNA comparator encoding A / Wisconsin / 588 / 2019 (H1N1) HA on day 0. Antibody responses against A / Wisconsin / 588 / 2019 were measured by HAI or 1-day MNT assay on day 21 (3 weeks after immunization). HAI and 50% neutralization titers (geometric means with geometric SD) are reported.

[0416] Example 8: Immunogenicity of a tetravalent bicistronic saRNA influenza vaccine encoding HA and NA from four seasonal influenza strains

[0417] The main pharmacology of influenza saRNA vaccines was evaluated in in vitro and in vivo non-clinical studies. In vitro and in vivo studies have confirmed that influenza saRNA vaccines encode influenza HA and / or NA proteins, which induce strong functional and neutralizing antibody responses and stable CD4+ and CD8+ T cell responses, and allow significantly smaller doses compared to modRNA in mice. Replication of saRNA also leads to innate immune activation, which may enhance adaptive immune responses to expressed antigens. Effective in vitro expression of HA and NA glycoproteins from influenza saRNA vaccines was confirmed in cultured cells. Immunogenicity studies in mice, rats, and ferrets have confirmed that different influenza saRNA vaccine preparations induce strong functional and neutralizing antibody responses and T cell responses. Innate immune activation is also confirmed in mice and rats as measured by serum cytokine and chemokine release 24 hours after immunization. Immunogenicity studies in mice based on influenza modRNA vaccines also support the use of bicistronic influenza saRNA constructs, which express two separate influenza antigens (HA and NA) from the same saRNA vector. Immunogenicity studies in mice also showed that saRNA can tolerate partial incorporation of modified nucleosides to reduce early innate immune stimulation while still eliciting a strong adaptive humoral response. Finally, immunogenicity studies in mice also supported the combination of four bicistronic influenza saRNA constructs, each encoding different HA and NA, to target four seasonal influenza strains.

[0418] The influenza saRNA vaccine candidates selected for initial POC testing contain full-length, codon-optimized coding sequences for either the HA or NA glycoprotein from the cell-based A / Wisconsin / 588 / 2019 (H1N1) strain recommended for use in the 20212022 and 2022-2023 Northern Hemisphere and 2022 Southern Hemisphere influenza seasons.

[0419] Table 12. saRNA formulations

[0420]

[0421] The use of four bicistronic saRNA components can achieve a seasonal influenza saRNA vaccine that expresses four different HA and four different NA proteins to match the dominant influenza strains of each season. The feasibility of the quadrivalent bicistronic saRNA approach was evaluated in a mouse immunogenicity study based on the 2021-22 licensed adjuvanted seasonal influenza vaccine (QIV; FluAd) in the northern hemisphere. BALB / c mice were immunized IM with a total dose of 0.8 μg of the quadrivalent saRNA vaccine (0.2 μg / component) or 2.4 μg of the licensed QIV comparator on days 0 and 28. At day 42 (2 weeks after the second immunization), anti-HA antibody responses to each of the four components were induced as measured by HAI and MNT ( Fig. 9 ), and also induced anti-NA antibody responses as measured by NAI ( Fig.10 ). The tetravalent bicistronic saRNA vaccine candidate achieved similar or higher HA and NA titers than the QIV comparator.

[0422] about Fig. 9 Female Balb / c mice were immunized IM with 20ng LNP-formulated tetravalent saRNA (composed of 4 bicistronic constructs encoding HA and NA from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage)) or 2.4μg of a licensed adjuvanted tetravalent inactivated vaccine (QIV; FluAd) on day 0. Antibody responses to each vaccine component were measured by HAI or 1-day MNT assays on day 42 (2 weeks after the second dose). HAI and 50% neutralization titers (geometric means with geometric SD) are reported.

[0423] about Fig.10Female Balb / c mice were immunized IM with 20ng LNP-formulated tetravalent saRNA (composed of 4 bicistronic constructs encoding HA and NA from A / Wisconsin / 588 / 2019 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), B / Washington / 2 / 2019 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage)) or 2.4μg of a licensed adjuvanted tetravalent inactivated vaccine (QIV; FluAd) on day 0. Antibody responses to each vaccine component were measured by NAI at day 42 (2 weeks after the second dose). NAI titers (geometric means with geometric SD) are reported for 3 of the 4 strains. Due to technical issues using the NAI assay for this strain, H3N2 NAI titers could not be reported for both saRNA and QIV.

[0424] Example 9: Effects in humans

[0425] C4861001 is an ongoing Phase 1 FIH study that evaluates the safety, tolerability and immunogenicity of PF-07845104. As of the data cutoff date of November 15, 2022, 253 participants were randomly assigned, and 248 participants received vaccination. A total of 5 participants were not vaccinated (1 participant from vaccine formulation 4 in the 2.5 μg group, 2 participants from vaccine formulation 5 in the 2.5 μg group, 1 participant from vaccine formulation 5 in the 10 μg group, and 1 participant from vaccine formulation 6 in the 2.5 μg group). Licensed QIV is used as a comparator.

[0426] Safety and Efficacy - Study C4861001

[0427] C4861001 is an ongoing FIH Phase 1, randomized, placebo-controlled, observer-blinded, sponsor-unblinded, dose-finding, and vaccine composition / formulation selection study in healthy adults. This study evaluates the safety, tolerability, and immunogenicity of a single dose of different monocistronic and ultimately bicistronic saRNA vaccine formulations against influenza. Participants aged 18 to 49 years were randomized 4:1 to receive either saRNA vaccine formulation or placebo. An additional group of participants was independently enrolled to receive a licensed QIV as a control.

[0428] Table 12 provides details of the saRNA vaccine formulations.

[0429] Table 13. Vaccine formulation details

[0430]

[0431] Abbreviations: 50pU = 50% N1-methylpseudouridine; 50U = 50% uridine; HA = hemagglutinin; NA = neuraminidase; poly A = polyadenylation; SGP = subgenomic promoter; TRD = Trinidad donkey strain.

[0432] NOTE: This list is not exhaustive and preparations may be added or omitted.

[0433] Vaccine formulations 1, 2, and control groups - randomized participants

[0434] A total of 36 participants were randomly assigned to each of the vaccine formulation 1 and vaccine formulation 2 groups, 63 participants were randomly assigned to the placebo group, and 33 participants were randomly assigned to the control group.

[0435] Vaccine preparations 1

[0436] Within this group, 11 participants received the 1 μg vaccination, 13 participants received the 2.5 μg vaccination, and 12 participants received the 10 μg vaccination. One participant was randomized to 2.5 μg but was mistakenly dosed with 1 μg and has therefore been included in the 1 μg group for the purpose of safety analysis.

[0437] Five participants (1 participant from the 1 μg group and 2 participants each from the 2.5 μg group and the 10 μg group) withdrew from the study after vaccination

[0438] Vaccine preparation 2

[0439] Within this group, 12 participants each received 1 μg, 2.5 μg, and 10 μg of the vaccine. One participant from the 1 μg group was lost to follow-up after vaccination.

[0440] Vaccine formulations 3, 4, 7, and control group—randomly assigned participants

[0441] A total of 36 participants were randomly assigned to each of the vaccine formulation 3, vaccine formulation 4, and vaccine formulation 7 groups, and 63 participants were randomly assigned to placebo. One participant from the vaccine formulation 42.5 μg group did not receive the vaccine.

[0442] Vaccine preparation 3

[0443] Within this group, 12 participants each received 1 μg, 2.5 μg, and 10 μg of the vaccine.

[0444] Two participants from the 1 μg group withdrew from the study after vaccination.

[0445] Vaccine preparations4

[0446] Within this group, 12 participants received the 1 μg and 10 μg doses each, while 11 participants received the 2.5 μg dose.

[0447] Two participants (1 participant each from the 1 μg and 10 μg groups) withdrew from the study after vaccination.

[0448] Vaccine preparations7

[0449] Within this group, 12 participants each received 1 μg, 2.5 μg, and 10 μg of the vaccine.

[0450] Vaccine formulations 5, 6, and control group - randomized participants

[0451] A total of 38 participants were randomly assigned to vaccine formulation 5, 35 to vaccine formulation 6, 63 to placebo, and 33 to the control group. Three participants from vaccine formulation 5 (2 participants from the 2.5-μg group and 1 participant from the 10-μg group) and 1 participant from vaccine formulation 6 2.5-μg were not vaccinated.

[0452] Vaccine preparations5

[0453] Within this group, 12 participants each received the 1 μg and 2.5 μg vaccines, while 11 received the 10 μg vaccine. Two participants from the 2.5 μg group and one participant from the 10 μg group did not receive the vaccine.

[0454] Two participants from the 1 μg group withdrew from the study after vaccination.

[0455] Vaccine preparations6

[0456] Within this group, 11 participants each received the 1 μg and 2.5 μg vaccines, while 12 participants received the 10 μg vaccine. One participant from the 2.5 μg group did not receive the vaccine.

[0457] Five participants (1 participant from the 1 μg group and 4 participants from the 2.5 μg group) withdrew from the study after vaccination.

[0458] Immunogenicity

[0459] A total of 253 participants were randomized to receive vaccination, of whom 196 were evaluable for immunogenicity.

[0460] A dose-dependent increase in HAI GMT was observed after 4 weeks after vaccine administration. HAI GMT at day 1 (before vaccination) and 1, 2 and 4 weeks after vaccine administration are shown in Fig.11 , Fig.12 and Fig.13 middle.

[0461] Vaccine formulations 1 and 2

[0462] The proportion of participants who achieved seroconversion 4 weeks after 10 μg was higher than that after 1 μg and 2.5 μg (Table 14).

[0463] Vaccine formulations 3, 4 and 7

[0464] In vaccine formulation 3, the proportion of participants who achieved seroconversion 4 weeks after 10 μg was higher than the proportion of participants who achieved seroconversion after 1 μg and 2.5 μg (Table 15).

[0465] Table 14. Proportion of Participants Achieving HAI Seroconversion to A / Wisconsin / 588 / 2019 (H1N1) Strain Following Vaccination - Vaccine Formulations 1, 2, and Control Group - Immunogenicity Evaluable Population

[0466]

[0467] a. Include participants in studies receiving licensed QIV whose VRD data are tested concurrently with the VRD data of the Test C1 group.

[0468] b. Include participants in studies receiving licensed QIV whose VRD data were tested concurrently with the VRD data for groups C2-C5 and C7.

[0469] c. Included participants in studies who received a placebo at randomization.

[0470] d. Protocol-specified times for blood sample collection.

[0471] eN = number of participants with valid and conclusive assay results for the specified assay both prior to vaccination and at the specified sampling time point.

[0472] fn = number of participants with valid and conclusive assay results for the specified assay who achieved HAI seroconversion at the specified sampling time point.

[0473] g. Exact two-sided CI based on Clopper and Pearson's method.

[0474] NOTE: HAI refers to the assay name for FLU_HAI_H1N1_WIS19 tested at baseline, 1 week, and 2 weeks after vaccination, and FLU_HAI_H1N1_II_WIS19 tested at baseline and 4 weeks after vaccination.

[0475] Note: Seroconversion was defined as an HAI titer <1:10 before vaccination and ≥1:40 at the time point of interest, or an HAI titer ≥1:10 before vaccination with a 4-fold rise at the time point of interest.

[0476] Table 15. Proportion of Participants Achieving HAI Seroconversion to A / Wisconsin / 588 / 2019 (H1N1) Strain Following Vaccination - Vaccine Formulations 3, 4, 7, and Control Group - Immunogenicity Evaluable Population

[0477]

[0478] a. Include participants in studies receiving licensed QIV whose VRD data were tested concurrently with the VRD data for groups C2-C5 and C7.

[0479] b. Included participants in studies who received a placebo at randomization.

[0480] c. Protocol-specified times for blood sample collection.

[0481] dN = number of participants with valid and conclusive assay results for a given assay both prior to vaccination and at the given sampling time point.

[0482] en = number of participants with valid and conclusive assay results for the specified assay who achieved HAI seroconversion at the specified sampling time point.

[0483] f. Exact two-sided CI based on Clopper and Pearson's method.

[0484] NOTE: HAI refers to the assay name for FLU_HAI_H1N1_WIS19 tested at baseline, 1 week, and 2 weeks after vaccination, and FLU_HAI_H1N1_II_WIS19 tested at baseline and 4 weeks after vaccination.

[0485] Note: Seroconversion was defined as an HAI titer <1:10 before vaccination and ≥1:40 at the time point of interest, or an HAI titer ≥1:10 before vaccination with a 4-fold rise at the time point of interest.

[0486] Table 16. Proportion of Participants Achieving HAI Seroconversion to A / Wisconsin / 588 / 2019 (H1N1) Strain Following Vaccination - Vaccine Formulations 5, 6, and Control Group - Immunogenicity Evaluable Population

[0487]

[0488] a. Include participants in studies receiving licensed QIV whose VRD data were tested concurrently with the VRD data for groups C2-C5 and C7.

[0489] b. Include participants in studies receiving licensed QIV whose VRD data were tested concurrently with the VRD data for group C6.

[0490] c. Included participants in studies who received a placebo at randomization.

[0491] d. Protocol-specified times for blood sample collection.

[0492] eN = number of participants with valid and conclusive assay results for the specified assay both prior to vaccination and at the specified sampling time point.

[0493] fn = number of participants with valid and conclusive assay results for the specified assay who achieved HAI seroconversion at the specified sampling time point.

[0494] g. Exact two-sided CI based on Clopper and Pearson's method.

[0495] NOTE: HAI refers to the assay name for FLU_HAI_H1N1_WIS19 tested at baseline, 1 week, and 2 weeks after vaccination, and FLU_HAI_H1N1_II_WIS19 tested at baseline and 4 weeks after vaccination.

[0496] Note: Seroconversion was defined as an HAI titer <1:10 before vaccination and ≥1:40 at the time point of interest, or an HAI titer ≥1:10 before vaccination with a 4-fold rise at the time point of interest.

[0497] In vaccine formulation 4, the proportion of subjects who achieved seroconversion 4 weeks after 1 μg was higher than the proportion of subjects who achieved seroconversion after 2.5 μg and 10 μg (Table 15).

[0498] In vaccine formulation 7, the proportion of subjects who achieved seroconversion 4 weeks after 1 μg was equal to the proportion of subjects who achieved seroconversion after 2.5 μg (Table 15).

[0499] Vaccine formulations 5 and 6

[0500] In vaccine formulation 5, there were no participants who achieved seroconversion after 4 weeks. In vaccine formulation 6, the proportion of subjects who achieved seroconversion after 4 weeks was higher after 10 μg than after 1 μg and 2.5 μg (Table 16).

[0501] Example 10: Immunogenicity of influenza octavalent HA / NA saRNA vaccine in mice

[0502] This study was performed to test the immunogenicity of an eight-valent saRNA-LNP vaccine encoding HA and NA antigens from four influenza virus strains in a mouse model. The saRNA constructs used in this study encode hemagglutinin (HA) and / or neuraminidase (NA) proteins from influenza virus strains A / Wisconsin / 588 / 2019, A / Cambodia / e0926360 / 2020, B / Phuket / 3073 / 2013, or B / Washington / 02 / 2019. The eight-valent saRNA-LNP vaccine elicited comparable or higher levels of functional anti-HA antibodies, anti-NA antibodies, and virus neutralizing antibodies compared to the licensed comparator FluAd.

[0503] Octavalent vaccine formulations composed of monocistronic or bicistronic saRNA constructs had comparable immunogenicity in mice after two doses. Multivalent saRNA vaccines mixed before ("premix") or after ("postmix") formulation with LNPs had comparable immunogenicity in mice. The octavalent saRNA-LNP vaccine did result in modest interference with immunogenicity for some strains, particularly influenza B strains, compared to mice vaccinated with monocistronic single antigen controls. Overall, these data provide support for the continued evaluation of multivalent saRNA vaccines encoding influenza virus antigens.

[0504] The primary objective of this study was to evaluate the immunogenicity of eight-valent saRNA vaccines encoding hemagglutinin (HA) or neuraminidase (NA) proteins from influenza virus in mice compared with monocistronic and bicistronic saRNA vaccines. The eight-valent vaccine consists of eight monocistronic HA or NA saRNA or four bicistronic HA-NA saRNA constructs. The eight-valent saRNA vaccine was also compared with a licensed quadrivalent inactivated influenza virus vaccine comparator (containing 8 HA / NA antigens from 4 strains) and a quadrivalent modified RNA (modRNA)-LNP vaccine encoding 4 HA proteins. The secondary objective of this study was to compare the immunogenicity of the eight-valent vaccine constructs combined with lipid nanoparticles (LNPs) before or after formulation in a mouse model.

[0505] This study tested saRNA (TC83-delkozak-80A) constructs encoding HA or NA proteins (monocistrons) or both HA and NA proteins (bicistrons) from H1N1 A / Wisconsin / 588 / 2019, H3N2 A / Cambodia / e0926360 / 2020, B / Yam B / Phuket / 3073 / 2013, or B / Vic B / Washington / 02 / 2019. Eight-valent formulations formulated in LNPs were tested in mice, containing 8 monocistronic (TC83-delkozak-HA-80A or TC83-delkozak-NA-80A) or 4 bicistronic (TC83-delkozak-HA-SGP-NA-80A) saRNAs. The eight-valent saRNA vaccines were either mixed before formulation in LNP (premix) or after formulation of each saRNA construct in LNP (postmix). Two alternative quadrivalent vaccine comparators were included in this study: a nucleoside-modified RNA (modRNA)-LNP vaccine encoding four HA proteins, and FluAd, a licensed comparator consisting of four inactivated influenza viruses. Monocistronic and bicistronic saRNA-LNP vaccines for each strain were also included as controls to assess any potential interference with the antibody response observed in the eight-valent vaccine formulation.

[0506] The study was designed to have 20 groups each containing a total of 10 female mice (mouse strain: Balb / c) as shown in Table 17. The study schedule assays used in the study are documented below.

[0507] Table 17 Study design

[0508]

[0509]

[0510] Assays used in the study:

[0511] HAI (groups 1, 3, 4, 6, 8, 10-20);

[0512] NAI (groups 1, 2, 5, 7, 9, 10-17, 19, 20);

[0513] 1D neutral

[0514] D21, 42

[0515] A 0.3 mL syringe was filled to 0.05 mL and the vaccine was administered via the intramuscular route for each animal. This procedure was repeated on day 28 for booster vaccination.

[0516] Table 18 Test articles and diluents used in the study

[0517]

[0518]

[0519]

[0520]

[0521] Table 19 Drug product materials and analytical results

[0522]

[0523]

[0524]

[0525]

[0526] Intramuscular injection of mice with one dose of an octavalent saRNA-LNP vaccine encoding HA and NA antigens from four influenza virus strains (i.e., “4x saRNA bicistronic”) elicited robust functional anti-HA antibodies ( Fig.14 ), anti-NA antibody ( Fig.15 ) and virus neutralizing antibodies ( Fig.16 In general, after a single dose, HAI antibody levels elicited against IAV strains were higher than those elicited against IBV strains ( Fig.14 ). The eight-valent saRNA-LNP vaccine elicited HAI and neutralization titers comparable to or slightly higher than the four-valent modRNA vaccine or FluAd (at 12ug or 2.4ug doses). Neuraminidase inhibition (NAI) antibody levels elicited by the eight-valent saRNA-LNP vaccine on day 21 tended to be slightly lower than the single antigen control, but tended to be higher than the NAI titers elicited by FluAd (at 12ug or 2.4ug doses) ( Fig.15 In general, virus neutralization titers elicited after one dose of the octavalent saRNA vaccine were comparable or better than a single dose of the quadrivalent modRNA vaccine or FluAd (at either 12ug or 2.4ug doses). Fig.16 ).

[0527] All groups received a second dose of the vaccine on day 28, and blood was collected 14 days later on day 42. After the second dose, functional anti-HA antibodies ( Fig.17 ), anti-NA antibody ( Fig.18 ) and virus neutralizing antibodies ( Fig.19) were boosted in all vaccine groups. HAI and virus neutralization titers elicited against A / Cambodia / e0926360 / 2020 were minimally affected by the multivalent formulation, while the octavalent and single antigen vaccines elicited similar titers against this virus ( Fig.17 , Fig.19 After two doses of the vaccine, NAI titers elicited by the eight-valent saRNA-LNP vaccine were comparable or better than those elicited by 12ug or 2.4ug doses of FluAd ( Fig.18 In addition to the virus neutralization titers elicited against A / Cambodia / e0926360 / 2020, the octavalent saRNA-LNP vaccine elicited comparable or better neutralization titers than the quadrivalent HA-encoding modRNA-LNP vaccine or FluAd (12ug or 2.4ug doses) after two doses of the vaccine ( Fig.19 ).

[0528] To evaluate whether the octavalent vaccine was better prepared by first premixing all saRNA constructs and then formulating with LNPs (premixing) or by mixing the saRNA-LNP vaccine after formulation (postmixing), the immunogenicity of each method was compared. Premixing and postmixing formulations were evaluated for an octavalent vaccine composed of 8 monocistronic HA or NA saRNAs and an octavalent vaccine composed of 4 bicistronic saRNAs (HA-SGP-NA). Both preparation methods had a significant effect on the immunogenicity of the vaccine at one dose ( Fig.14 , Fig.15 , Fig.16 ) or two doses ( Fig.17 , Fig.18 , Fig.19 ) elicited similar levels of functional anti-HA antibodies ( Fig.14 , Fig.17 ), anti-NA antibody ( Fig.15 , Fig.18 ) and virus neutralizing antibodies ( Fig.16 , Fig.19 ). These data suggest that co-formulated multivalent saRNA vaccines and those combined after formulation have comparable immunogenicity.

[0529] The immunogenicity of an octavalent saRNA-LNP vaccine composed of eight monocistronic saRNA constructs or four bicistronic (HA-SGP-NA) saRNA constructs was also evaluated in this study. No differences in HAI titers against IBV strains were observed at this time point for either the monocistronic or bicistronic octavalent vaccines. By day 42 (2 weeks after dose 2), HAI titers against both IAV and IBV strains were comparable in mice administered the octavalent vaccine with either the monocistronic or bicistronic saRNA constructs ( Fig.17 Three weeks after dose 1, neutralizing antibody levels elicited by either the monocistronic or bicistronic octavalent vaccine were generally comparable ( Fig.16 ), except for the titers elicited against A / Cambodia / e0926360 / 2020, where the monocistronic octavalent vaccine formulation elicited titers that were ~10-fold higher than the bicistronic octavalent vaccine. After the second dose, neutralizing antibody levels against all 4 viruses were comparable in mice receiving either the monocistronic or bicistronic octavalent saRNA vaccine ( Fig.19 ).

[0530] The purpose of this study is to evaluate the immunogenicity of eight-valent saRNA-LNP vaccines encoding HA and NA antigens from four influenza virus strains in a mouse model. Functional anti-HA antibodies measured by HAI assay, functional anti-NA antibodies measured by NAI assay, and virus neutralizing antibodies measured by 1-day MNT were all induced after two doses of the eight-valent vaccine formulation. When comparing eight-valent vaccines consisting of eight monocistronic or four bicistronic saRNA constructs, the monocistronic eight-valent vaccine was moderately better after a single dose, but after two doses of the vaccine, the monocistronic and bicistronic eight-valent vaccines had comparable immunogenicity. Similar immunogenicity was observed between the co-formulated eight-valent saRNA vaccines (premix) and those eight-valent saRNA vaccines (postmix) combined after formulation. Compared with the modRNA-LNP vaccines and FluAd encoding HA in tetravalent, the eight-valent saRNA vaccines are generally able to induce comparable or better immunogenicity than these comparators.

[0531] Fig.14 On day 0, mice were vaccinated intramuscularly with saRNA encoding HA or HA / NA formulated in LNP, tetravalent modRNA-LNP vaccines encoding HA, or licensed comparator FluAd. Serum was collected 3 weeks after vaccination (day 21, 3 weeks PD1). Functional anti-HA antibody titers for A / Wisconsin / 588 / 2019 (upper left), A / Cambodia / e0926360 / 2020 (upper right), B / Phuket / 3073 / 2013 (lower left) or B / Washington / 02 / 2019 (lower right) were measured in serum collected from mice (n=5) by hemagglutination inhibition (HAI). The geometric mean titer (GMT) at each time point is shown at the top of each figure and indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by a dotted line.

[0532] Fig.15On day 0, mice were vaccinated intramuscularly with saRNA encoding HA or HA / NA formulated in LNP or a licensed comparator FluAd. Serum was collected 3 weeks after vaccination (day 21, 3 weeks PD1). Functional anti-NA antibody titers for A / Wisconsin / 588 / 2019 (upper left), B / Phuket / 3073 / 2013 (lower left) or B / Washington / 02 / 2019 (lower right) were measured in serum collected from mice (n=5) by enzyme-linked lectin assay (ELLA). Due to technical difficulties for the virus, the NAI titer for A / Cambodia / e0926360 / 2020 was not determined. The geometric mean titer (GMT) at each time point is shown at the top of each figure and indicated by a horizontal line. Negative titers are plotted at the LOD indicated by the dotted line.

[0533] Fig.16 On day 0, mice were vaccinated intramuscularly with saRNA encoding HA or HA / NA formulated in LNP, tetravalent modRNA-LNP vaccines encoding HA, or licensed comparator FluAd. Serum was collected 3 weeks after vaccination (day 21, 3 weeks PD1). Virus neutralizing antibody titers for A / Wisconsin / 588 / 2019 (upper left), A / Cambodia / e0926360 / 2020 (upper right), B / Phuket / 3073 / 2013 (lower left) or B / Washington / 02 / 2019 (lower right) were measured in serum collected from mice (n=5) by 1-day microneutralization test (MNT). The geometric mean titer (GMT) at each time point is shown at the top of each figure and indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by the dotted line.

[0534] Fig.17At day 0 and day 28, mice were vaccinated intramuscularly with saRNA encoding HA or HA / NA formulated in LNP, tetravalent modRNA-LNP vaccines encoding HA, or licensed comparator FluAd. Serum was collected 2 weeks after the second vaccination (day 42, 2 weeks PD2). Functional anti-HA antibody titers for A / Wisconsin / 588 / 2019 (upper left), A / Cambodia / e0926360 / 2020 (upper right), B / Phuket / 3073 / 2013 (lower left) or B / Washington / 02 / 2019 (lower right) were measured in serum collected from mice (n=5) by hemagglutination inhibition (HAI). The geometric mean titer (GMT) at each time point is shown at the top of each figure and indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by a dotted line.

[0535] Fig.18 On day 0 and day 28, mice were vaccinated intramuscularly with saRNA encoding HA or HA / NA formulated in LNP or a licensed comparator FluAd. Serum was collected 2 weeks after the second vaccination (day 42, 2 weeks PD2). Functional anti-NA antibody titers for A / Wisconsin / 588 / 2019 (upper left), B / Phuket / 3073 / 2013 (lower left) or B / Washington / 02 / 2019 (lower right) were measured in serum collected from mice (n=5) by enzyme-linked lectin assay (ELLA). Due to technical difficulties for the virus, the NAI titer for A / Cambodia / e0926360 / 2020 was not determined. 50% inhibition titers are plotted, and the geometric mean titer (GMT) at each time point is displayed at the top of each figure and indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by the dotted line.

[0536] Fig.19On the 0th and 28th days, mice were vaccinated intramuscularly with saRNA encoding HA or HA / NA formulated in LNP, tetravalent modRNA-LNP vaccines encoding HA, or licensed comparator FluAd. Serum was collected 2 weeks after the second vaccination (day 42, 2 weeks PD2). Virus neutralizing antibody titers for A / Wisconsin / 588 / 2019 (upper left), A / Cambodia / e0926360 / 2020 (upper right), B / Phuket / 3073 / 2013 (lower left) or B / Washington / 02 / 2019 (lower right) were measured in serum collected from mice (n=5) by 1-day microneutralization test (MNT). 50% neutralization titers are plotted, and the geometric mean titer (GMT) at each time point is displayed at the top of each figure and indicated by a horizontal line. Negative titers are plotted at the LOD of the assay indicated by the dotted line.

[0537] Exemplary embodiments

[0538] 1. A composition comprising a self-amplifying RNA molecule, the self-amplifying RNA molecule comprising: a 5' cap; a 5' untranslated region; a coding region for a non-structural protein derived from an alphavirus; a subgenomic promoter derived from an alphavirus; an open reading frame encoding a gene of interest; a 3' untranslated region; and a 3' poly A sequence; wherein at least 5% of the total population of specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0539] 2. The composition of technical solution 1, wherein the 5' cap is represented by formula I:

[0540]

[0541] Where R 1 and R 2 are each independently H or Me; and

[0542] B 1 and B 2 Each is independently guanine, adenine or uracil.

[0543] 3. The composition of technical solution 1 or 2, wherein B 1 and B 2 A naturally occurring base.

[0544] 4. The composition of any one of technical solutions 1 to 3, wherein R 1 is methyl and R 2 For hydrogen.

[0545] 5. The composition of any one of technical solutions 1 to 4, wherein B 1 For guanine.

[0546] 6. The composition of any one of technical solutions 1 to 5, wherein B 1 Adenine.

[0547] 7. The composition of any one of technical solutions 1 to 5, wherein B 2 Adenine.

[0548] 8. The composition of any one of technical solutions 1 to 7, wherein B 2 For uracil.

[0549] 9. The composition of any one of technical solutions 1 to 8, wherein the nucleotide immediately downstream (5' to 3') of the 5' cap comprises guanine.

[0550] 10. The composition of any one of technical solutions 1 to 9, wherein B 1 Adenine and B 2 For uracil.

[0551] 11. The composition of any one of technical solutions 1 to 10, wherein B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 For hydrogen.

[0552] 12. The composition of any one of technical solutions 1 to 11, wherein the nucleotide immediately downstream (5' to 3') of the 5' cap comprises guanine, B 1 Adenine, B 2 is uracil, R 1 is methyl, and R 2 For hydrogen.

[0553] 13. The composition of any one of technical solutions 1 to 12, wherein at least 10% of the total population of specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0554] 14. The composition of any one of technical solutions 1 to 13, wherein at least 25% of the total population of specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0555] 15. The composition of any one of technical solutions 1 to 14, wherein at least 50% of the total population of specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0556] 16. The composition of any one of technical solutions 1 to 15, wherein at least 75% of the total population of specific nucleotides in the molecule has been replaced by one or more modified or non-natural nucleotides.

[0557] 17. The composition of any one of technical solutions 1 to 16, wherein substantially all of a specific population of nucleotides in the molecule have been replaced by one or more modified or non-natural nucleotides.

[0558] 18. The composition of any one of technical solutions 1 to 17, wherein the one or more modified or non-natural replace...

Claims

1. A composition comprising a self-amplifying RNA (saRNA), the self-amplifying RNA (saRNA) comprising: a 5' cap; a 5' untranslated region (5'UTR); a coding region for a nonstructural protein derived from an alphavirus; a first subgenomic promoter derived from an alphavirus; a first open reading frame encoding a first gene of interest derived from influenza virus hemagglutinin (HA); a second subgenomic promoter derived from an alphavirus; a second open reading frame encoding a second gene of interest derived from influenza virus; a 3' untranslated region (3'UTR); and a 3' poly A sequence.

2. The composition of claim 1, wherein the 5'UTR sequence has at least 70% sequence identity with SEQ ID NO: 12; wherein the coding region of the nonstructural protein comprises a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO: 13, a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO: 14, a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO: 15, and a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO: 16; wherein the first subgenomic promoter has a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO: 17; wherein the polynucleotide sequence encoding HA has at least 70% sequence identity with SEQ ID NO: 18; wherein the second subgenomic promoter comprises a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO: 19; wherein the 3'UTR comprises a polynucleotide sequence having at least 70% sequence identity with SEQ ID NO: 21; and the poly A tail comprises at least 20 consecutive adenines.

3. The composition according to any one of claims 1-2, wherein the second target gene derived from influenza virus encodes any one polypeptide selected from HA, NA, NP, M1, M2, NS1 and NS2.

4. The composition according to any one of claims 1 to 3, wherein the 5' cap is represented by Formula II:

5. The composition of any one of claims 1-4, wherein at least 10% of the total nucleotides in the saRNA have been replaced with modified or non-natural nucleotides selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deazapseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

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

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

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

9. The composition of any one of claims 1 to 8, wherein the saRNA is complexed or associated with lipid nanoparticles (LNPs) comprising ionizable lipids, neutral lipids, steroids, and polymer-conjugated lipids.

10. The composition of any one of claims 1 to 9, wherein the saRNA is complexed or associated with a lipid nanoparticle (LNP) comprising: (i) Based on at least one cationic lipid; (ii) at least one neutral lipid comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroidal compound comprising cholesterol; and (iv) Based on at least one PEG-lipid, wherein n has an average value ranging from 30 to 60.

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

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

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

14. The composition of claim 13, wherein the 5' cap is represented by Formula II:

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

16. The composition of any one of claims 13 to 15, wherein the saRNA molecule is encapsulated in, bound to or adsorbed to: a liposome, a lipid nanoparticle, a polyplex, a lipid roll, a virosome, an immunostimulatory complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsion body, a polycationic peptide, a cationic nanoemulsion, or a combination thereof.

17. The composition of any one of claims 13 to 16, wherein at least 50% of the total saRNA molecules are full-length.

18. The composition of any one of claims 13 to 17, wherein at least 80% of the total saRNA molecules are full-length.

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

20. The use according to claim 19, wherein the composition elicits an immune response comprising a T cell response.

21. The composition of any one of claims 1-18, wherein the composition comprises a plurality of saRNAs encapsulated in lipid nanoparticles.

22. The composition of any one of claims 1-18, wherein the composition comprises four saRNAs encapsulated in lipid nanoparticles.

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