Modified h5 influenza hemagglutinin polypeptides and nucleic acids and uses thereof

Abstract

This application relates to modified H5 influenza hemagglutinin polypeptides and messenger ribonucleic acids (mRNAs) encoding the same, as well as compositions and vaccines comprising the same and methods of using the same, such as in the prevention and / or treatment of diseases or conditions caused by influenza A viruses, particularly influenza A virus, subtype H5, such as H5N8 or H5N1.

Description

MODIFIED H5 INFLUENZA HEMAGGLUTININ POLYPEPTIDES AND NUCLEICACIDS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and relies on the filing date of, European Application No. 24306294.0, filed 31 July 2024, the entire disclosure of which is herein incorporated by reference.FIELD

[0002] This application relates to modified H5 influenza hemagglutinin polypeptides and messenger ribonucleic acids (mRNAs) encoding the same, as well as compositions and vaccines comprising the same and methods of using the same, such as in the prevention and / or treatment of diseases or conditions caused by influenza A viruses, particularly influenza A virus subtype H5, such as H5N8 or H5Nl.BACKGROUND

[0003] Influenza A viruses infect a wide variety of birds and mammals, including humans, pigs, ferrets, and chickens. Most influenza A viruses cause mild localized infections of the respiratory and intestinal tract. However, highly pathogenic influenza A strains, such as H5N1, exist that cause systemic infections in poultry in which mortality can reach 100%. These highly pathogenic H5N1 avian influenza (HP Al H5N1) viruses occasionally infect, but typically do not transmit, in mammals. Nonetheless, in the spring of 2024, an unprecedented outbreak of HP Al H5N1 in bovine herds occurred in the US, with virus spread within and between herds, infections in poultry and cats, and spillover into humans, collectively indicating an increased public health risk, including a real epidemic and pandemic threat. The scale of the threat is illustrated by the 1918 influenza pandemic which killed over 50 million people.

[0004] Accordingly, there is an urgent need to develop vaccines, either recombinant vaccines or nucleic acid vaccines, for immunizing against an influenza A virus subtype H5 strain, particularly H5N8 or H5N1.SUMMARY

[0005] Disclosed herein are modified H5 influenza hemagglutinin (HA) polypeptides having characteristics, such as increased immunogenic properties and improved stability in the prefusion conformation, that would enable elicitation of higher neutralization response that can translate into better vaccine efficacy. Accordingly, in one aspect, provided herein is an artificial nucleic acid comprising a nucleotide sequence encoding a modified H5 influenza HA polypeptide, wherein the modified H5 influenza HA polypeptide comprises an amino acid substitution at amino acid position 447 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at amino acid position 447 is M447I. In some embodiments, the modified H5 influenza HA polypeptide is from an influenza A virus subtype H5N8 or an influenza A virus subtype H5N1. In some embodiments, the modified H5 influenza HA polypeptide is from influenza A virus strain A / Astrakhan / 3212 / 2020 or an influenza A virus A / Indonesia / 05 / 2005 strain. In some embodiments, the modified H5 influenza HA polypeptide comprises a polybasic cleavage site at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polybasic cleavage site comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the modified H5 influenza HA polypeptide further comprises one or more mutations in a polybasic cleavage site located at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more mutations comprise removing one or more, optionally all but one, basic amino acids in the polybasic cleavage site. In some embodiments, the one or more mutations converts the polybasic cleavage site to a monobasic cleavage site. In some embodiments, the polybasic cleavage site is converted to the monobasic cleavage site by replacing the polybasic cleavage site with the amino acid sequence TR. In some embodiments, the modified H5 influenza HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified H5 influenza HA polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0006] In some embodiments, the artificial nucleic acid disclosed herein is a messenger RNA (mRNA). In some embodiments, the mRNA comprises a 5'-cap structure and / or a 3'-poly(A) sequence. In some embodiments, the artificial mRNA comprises at least one chemically modified nucleotide and / or a phosphorothioate bond. In some embodiments, the at least one chemically modified nucleotide comprises a pseudouridine, a 2'-fluoro ribonucleotide, or a 2'-methoxyribonucleotide, optionally wherein the pseudouridine is a N1 -methylpseudouridine. In some embodiments, the artificial mRNA comprises a nucleic acid sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the artificial mRNA comprises the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the artificial mRNA comprises or consists of: a 5' cap structure, such as a 5' cap having the following structure

[0007] a 5' untranslated region (5' UTR) having the nucleic acid sequence of SEQ ID NO: 5, a protein coding region having the nucleic acid sequence of SEQ ID NO: 3, a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ ID NO: 6, and a poly A tail.

[0008] Also provided herein, in some embodiments, is a composition comprising the artificial nucleic acid of the present disclosure encapsulated in a lipid nanoparticle (LNP). In some embodiments, the LNP comprises a cationic lipid. In some embodiments, the cationic lipid comprises or is OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4- E10, GL-HEPES-E3-E12-DS-3-E14, (4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyldecanoate) (ALC-0315), or IM-001. In some embodiments, the LNP further comprises a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid. In some embodiments, the PEGylated lipid comprises or is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000). In some embodiments, the cholesterol- based lipid comprises or is cholesterol. In some embodiments, the helper lipid comprises or is dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the cationic lipid is present at a molar ratio between about 35% and about 55%, the PEGylated lipid is present at a molar ratio between about 0.25% and about 2.75%, the cholesterol-based lipid is present at a molar ratio between about 20% and about 45%, and the helper lipid is present at a molar ratio between about 5% and about 35%, wherein all of the molar ratios are relative to the total lipid content of the LNP. In some embodiments, the cationic lipid is present at a molar ratio of about 40%, the PEGylated lipid is present at a molar ratio of about 1.5%, the cholesterol -based lipid is present at a molar ratio ofabout 28.5%, and the helper lipid is present at a molar ratio of about 30%, wherein all of the molar ratios are relative to the total lipid content of the LNP. In some embodiments, the artificial nucleic acid is an artificial mRNA encoding the modified H5 influenza HA polypeptide of SEQ ID NO: 2, and wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, the composition is an immunogenic composition. In some embodiments, in the composition of the present disclosure, the artificial nucleic acid is an artificial mRNA and the artificial mRNA comprises or consists of: a 5' cap structure, such as a 5' cap having the following structure:a 5' untranslated region (5' UTR) having the nucleic acid sequence of SEQ ID NO: 5, a protein coding region having the nucleic acid sequence of SEQ ID NO: 3, a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ ID NO: 6, and a poly A tail.

[0009] In another aspect, provided herein is a modified H5 influenza hemagglutinin (HA) polypeptide comprising an amino acid substitution M447I as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified H5 influenza HA polypeptide is from an influenza A virus subtype H5N8 or an influenza A virus subtype H5N 1. In some embodiments, the modified H5 influenza HA polypeptide is from influenza A virus strain A / Astrakhan / 3212 / 2020 or an influenza A virus A / Indonesia / 05 / 2005 strain. In some embodiments, the modified H5 influenza HA polypeptide comprises a polybasic cleavage site with one or more mutations as described herein elsewhere. In some embodiments, the modified H5 influenza HA polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0010] Also provided is a trimeric influenza A HA polypeptide complex comprising three copies of any of the modified H5 influenza HA polypeptides disclosed herein. In some embodiments, the trimeric influenza A HA polypeptide complex of the present disclosure is more immunogenic as compared to a trimeric influenza A HA polypeptide complex prepared from acontrol H5 influenza HA polypeptide comprising the amino acid of SEQ ID NO: 1. In some embodiments, the immunogenicity is measured using a hemagglutination-inhibition assay.

[0011] Further provided is an artificial nucleic acid (e.g., mRNA) encoding any of the modified H5 influenza HA polypeptides disclosed herein. In some embodiments, the artificial nucleic acid comprises at least one chemically modified nucleotide and / or a phosphorothioate bond. In some embodiments, the disclosure provides a vector comprising the artificial nucleic acid disclosed herein. In some embodiments, the vector is a messenger RNA (mRNA) production vector. In some embodiments, the disclosure provides a host cell comprising the vector.

[0012] In a further aspect, the present disclosure provides a composition comprising any of the modified H5 influenza HA polypeptides, the trimeric influenza A HA polypeptide complexes, the artificial nucleic acids, or the vectors provided herein. In some embodiments, the composition is an immunogenic composition.

[0013] Also provided is a vaccine comprising any of the immunogenic compositions disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the vaccine further comprises an adjuvant.

[0014] Further provided herein is a vaccine comprising any of the artificial nucleic acids disclosed herein encapsulated in a lipid nanoparticle (LNP), wherein the artificial nucleic acid is an artificial mRNA, wherein the vaccine comprises from about 0.1 pg to about 150 pg, such as from about 5 pg to about 120 pg, from about 10 pg to about 60 pg, or about 15 pg to about 45 pg, of the artificial nucleic acid (e.g., artificial mRNA). In some embodiments, the vaccine comprises about 30 pg, about 75 pg, or about 150 pg of the artificial nucleic acid (e.g., artificial mRNA). In some embodiments, the artificial nucleic acid (e.g., artificial mRNA) encodes the modified H5 influenza HA polypeptide of SEQ ID NO: 2 and the LNP comprises a cationic lipid, wherein the cationic lipid comprises GL-HEPES-E3-E12-DS-4-E10.

[0015] In some embodiments, the vaccine of the present disclosure is for use in a method for treatment of a human or animal body. In some embodiments, the vaccine of the present disclosure is for use in a method for immunization of a human or animal body. In some embodiments, the vaccine of the present disclosure is for use in a method for immunizing a subject against influenza A or reducing one or more symptoms of an influenza virus infection. In some embodiments, the vaccine of the present disclosure is for use in a method for immunizing a subject against influenza A or reducing one or more symptoms of an influenza A virus infection.

[0016] In a yet another aspect, the present disclosure provides a method of immunizing a subject, or a method of reducing one or more symptoms of an influenza A virus infection, the method comprising administering to the subject in need thereof any of the immunogenic compositions or vaccines disclosed herein. In some embodiments, the disclosed method prevents an influenza A virus infection in the subject, decreases the subject’s likelihood of getting an influenza A virus infection, or reduces the subject’s likelihood of getting serious illness from an influenza A virus infection. In some embodiments, the disclosed method induces influenza seroprotection in the subject by at least day 43 following the administration of the vaccine to the subject. In some embodiments, the subject is a human, such as one of 6 months of age or older, less than 18 years of age, at least 6 months of age and less than 18 years of age, at least 18 years of age and less than 65 years of age, at least 6 months of age and less than 5 years of age, at least 5 years of age and less than 65 years of age, at least 60 years of age, or at least 65 years of age. In some embodiments, the immunogenic composition or vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally. In some embodiments, the immunogenic composition or vaccine is administered as part of a primeboost vaccination strategy. In some embodiments, the prime-boost vaccination strategy comprises administering a boosting vaccine about 2-3 weeks after administration of a priming vaccine.

[0017] In a further aspect, provided herein is an in vitro method of preparing any of the trimeric H5 influenza HA polypeptide complexes disclosed herein, the method comprising culturing any of the host cells disclosed herein in a cell culture medium, and expressing the trimeric H5 influenza HA polypeptide complex. In some embodiments, the method further comprises a step of purifying the trimeric H5 influenza HA polypeptide complex from the cell culture medium.

[0018] In another further aspect, the present disclosure provides use of any one or any combination of (i) the artificial nucleic acid disclosed herein, (ii) the composition disclosed herein, (iii) the modified H5 influenza HA polypeptide disclosed herein, (iv) the trimeric influenza A HA polypeptide complex disclosed herein, optionally with a pharmaceutically acceptable carrier, and for the manufacture of a medicament for immunizing a subject against influenza A or reducing one or more symptoms of an influenza A virus infection.

[0019] Another aspect is directed to a method of preparing a composition as disclosed herein, comprising the steps of: providing an aqueous buffered solution comprising an artificial nucleic acid molecule as disclosed herein, providing an amphiphilic solution comprising the cationic lipid,the PEGylated lipid, the cholesterol-based lipid, and the helper lipid, and mixing the aqueous buffered solution and the amphiphilic solution at a ratio of 5 : 1 to 3 : 1.BRIEF DESCRIPTION OF THE DRAWING

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods and compositions disclosed herein.

[0021] FIG. 1 depicts the structure of an influenza HA trimer in the prefusion conformation with the M447I mutation described in Example 1. The numbering of the amino acid position is as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0022] FIG. 2A-2B depict the percent total 293FT cells positive for monoclonal antibody binding measured by flow cytometry. The graphs depict the percentage of live cells that bound to each individual monoclonal antibody targeting HA from 30,000 cells. Black bars depict 65C6 (RBD) binding, horizontal stripes depict CR9114 (stem) binding, vertical lines depict FluA-20 (interprotomer interface) binding, and bricks depict secondary antibody binding in the absence of a primary antibody (background). Construct names are listed on the x-axis and percent values are listed on top of the bar graphs. pDNA denotes that constructs were transfected as plasmid DNA and mRNA denotes that constructs were transfected with LNP-encapsulated mRNA. The wildtype HA polypeptide from A / Astrakhan / 3212 / 2020 (H5_Base) was used as the baseline.

[0023] FIG. 3A-3B depict the assessment of surface expression levels of the M447I mutant on 293FT cells measured by flow cytometry. The graphs depict the MFI of live cells that bound to each individual monoclonal antibody targeting HA from 30,000 cells. Black bars depict 65C6 (RBD) binding, horizontal stripes depict CR9114 (stem) binding, vertical lines depict FluA-20 (interprotomer interface) binding, and bricks depict secondary antibody binding in the absence of a primary antibody (background). Construct names are listed on the x-axis. pDNA denotes that constructs were transfected as plasmid DNA and mRNA denotes that constructs were transfected with LNP-encapsulated mRNA.

[0024] FIG. 4 depicts the assessment of the ratio of stem to head binding monoclonal antibodies against the M447I mutant measured by flow cytometry for both pDNA and mRNA transfections. The graph depicts the normalized ratio of stem to head binding antibodies MFI for each construct. Construct names are listed on the x-axis. Data were calculated as (MFI of CR9114binding / MFI of 65C6 binding) per construct. MFI is median fluorescence intensity x percent positive cells for monoclonal antibody binding. H5_Base is the wild-type HA polypeptide from A / Astrakhan / 3212 / 2020. pDNA denotes that constructs were transfected as plasmid DNA and mRNA denotes that constructs were transfected with LNP -encapsulated mRNA.

[0025] FIG. 5 depicts the in vitro expression of the M447I mutant in mRNA-transfected human skeletal muscle cells. The graph on the left shows the HA expression in HSkM cells transfected with mRNA formulated in LNP and the graph on the right shows the expression as normalized to the wild-type control. The M447I mutant is identified as “monobasic M447I (D680- 643)” in the left panel and “D680-643 (M447I LRETR)” in the right panel. The wild-type HA polypeptide from A / Astrakhan / 3212 / 2020 is identified as “WT Polybasic (D680-667)” in the left panel and “D680-667 (WT Polybasic)” in the right panel.

[0026] FIG. 6 depicts the immunogenicity of the M447I mutant (“M447I”) as compared to the wild-type HA polypeptide from A / Astrakhan / 3212 / 2020 (“WT (poly)”), as described in Example 4.

[0027] FIG. 7 depicts a graphic design of the safety and immunogenicity study of a pandemic influenza H5 mRNA vaccine in healthy adults aged 18 years and older as described in Example 5. Ab: antibody; BLV: blood sample visit; D: day; SL: safety labs; TC: telephone call; V: visit; VAC: vaccination.

[0028] FIG. 8 depicts seroprotection rates of a pandemic influenza H5 mRNA vaccine administered to healthy adults 18-64 years old or healthy adults at least 65 years old at different timepoints as described in Example 5.

[0029] FIG. 9A-9B depict seroprotection rates (FIG. 9A) and seroconversion rates (FIG. 9B) of a pandemic influenza H5 mRNA vaccine administered to healthy adults 18-64 years old or healthy adults at least 65 years old at all dose levels as described in Example 5.

[0030] FIG. 10 depicts the geometric mean titers (GMTs) of a pandemic influenza H5 mRNA vaccine administered to healthy adults 18-64 years old or healthy adults 65 years old or older obtained from the immunogenicity analysis as described in Example 5.

[0031] FIG. 11 depicts a summary of solicited reactions following administration of a pandemic influenza H5 mRNA vaccine to healthy adults 18-64 years old or healthy adults aged 65 or older within 7 days after any vaccine injection as described in Example 5.

[0032] FIG. 12A-12B depict a comparison of solicited reactions following administration of a pandemic influenza H5 mRNA vaccine to healthy adults 18-64 years old or healthy adults aged 65 or older by dose (i.e., dose 1 or dose 2) as described in Example 5. FIG. 12A: shows reactions of healthy adults 18-64 years old; FIG. 12B: shows reactions of healthy adults at least 65 years old.

[0033] FIG. 13 depicts a summary of unsolicited adverse event (AE) and unsolicited adverse reaction (AR) following administration of a pandemic influenza H5 mRNA vaccine to healthy adults 18-64 years old or healthy adults aged 65 or older within the follow-up period after any vaccine injection (21 days following the first injection and 28 days following the second injection) as described in Example 5.

[0034] FIG. 14A-14B depict the immunogenicity of the M448I mutant (“Agl”) as compared to the wild-type HA polypeptide from A / Indonesia / 05 / 2005 (“WT (poly)”), as described in Example 6. Functional HIH antibody responses in mice against H5 strain A / Indonesia / 05 / 2005 at D21 post one dose and D35 post two doses of H5 mRNA vaccine formulations. Dose levels of 1 pg (FIG. 14A) or 0.1 pg (FIG. 14B) of the M448I mutant or WT HA H5 mRNA vaccines were injected by the IM route in mice (n = 6). First injection was given at DO, and 2nd injection was given at D21. Functional immunogenicity by HIH titers is reported as the dilution titer. Bars are the geometric mean titer (GMT) of N = 6 mice with circles showing individual responses.DETAILED DESCRIPTION

[0035] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings and discussed in the detailed description that follows. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the disclosure, and should not be interpreted as limiting the scope of the disclosure.

[0036] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology by Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, may provide one of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein.

[0038] Units, prefixes, and symbols are denoted in their International System of Units (SI), unless indicated otherwise.

[0039] The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.Definitions

[0040] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0041] The term “about,” or “approximately,” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%,±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value as such variations are appropriate to perform the disclosed methods and / or to make and use the disclosed compositions. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

[0042] The term “adjacent to” as used herein, in the context of a polypeptide, means that a first polypeptide sequence or domain is positioned N-terminally or C-terminally to a second polypeptide sequence or domain with no other sequences or domains being positioned between the first polypeptide sequence or domain and the second polypeptide sequence or domain.

[0043] The terms “administer,” “administering,” and “administration,” as used herein refer to contacting a subject with a nucleic acid (e.g., mRNA) or a composition or vaccine or to prescribing, instructing, managing or supervising the contacting of a subject with a nucleic acid (e.g., mRNA) or a composition or vaccine.

[0044] An “amino acid” according to the present disclosure can be any of the twenty naturally occurring (or “standard” amino acids) or variants thereof, such as, for example, D-proline (the D- enantiomer of proline), or any variants that are not naturally found in proteins, such as norleucine. The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties, such as cysteine that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that forms a cycle to the polypeptide backbone, and glycine that is more flexible than other amino acids. Table 1 shows the abbreviations and properties of the standard amino acids.Table 1. Standard amino acids, abbreviations, and properties

[0045] The term “analog” as used herein, refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. An analog can be a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. An analog can be a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. An analog can also be generated through performance of a synthetic process different from that used to generate the reference substance.

[0046] The term “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionallyincluding elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0047] As used herein, the term “antibody” refers to an immunoglobulin molecule produced by B lymphoid cells with a specific amino acid sequence. In some embodiments, antibodies are evoked in humans or other animals by a specific antigen (immunogen). Antibodies are characterized by reacting specifically with the antigen in some demonstrable way, antibody and antigen each being defined in terms of the other. The terms “eliciting an antibody response,” “eliciting neutralizing antibody,” “eliciting immunogenic response,” or grammatical equivalents, refer to the ability of an antigen or other molecule to induce the production of antibodies. In some embodiments, the term “antibodies” refers to any recombinant antibodies used in in vitro assays, such as in HA screening assays, including one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Such antibodies may exist as intact immunoglobulins or as fragments of the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Exemplary antibody fragments include, but are not limited to, F(ab)'2, Fab', and single chain Fv (scFv).

[0048] As used herein, the term “antigen” or “immunogen” and variations thereof (e.g., “antigenic” or “immunogenic”) refer to an agent that elicits an immune response; and / or (ii) an agent that is bound by a T cell receptor (e.g., when presented by an MHC molecule) or to a membrane-bound B cell receptor or a soluble antibody (e.g., produced by a B cell) when exposed or administered to an organism. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies) in an organism; alternatively or additionally, in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen) in an organism. It will be appreciated by those skilled in the art that a particular antigen may elicit an immune response in one or several members of a target organism (e.g., mice, ferrets, rabbits, primates, humans), but not in all members of the target organism species. In some embodiments, an antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, including all values and subranges therebetween, of the members of a target organism species. In some embodiments, an antigen binds to an antibody and / or T cell receptor and may or may not induce a particular physiological response in an organism. In some embodiments, for example, an antigen may bind to an antibody and / or to a Tcell receptor in vitro, whether or not such an interaction occurs in vivo. In some embodiments, an antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. Antigens include the modified H5 influenza HA polypeptides described herein.

[0049] As used herein, an “artificial nucleic acid” may typically be understood to be a nucleic acid molecule, e.g., a DNA or an RNA, that does not occur naturally. Thus, an “artificial messenger ribonucleic acid (mRNA)” refers to an mRNA that does not occur naturally. In other words, an artificial nucleic acid or an artificial mRNA may be understood as a non-natural nucleic acid molecule or mRNA molecule. Such nucleic acid or mRNA molecule may be non-natural due to its individual sequence (which does not occur naturally) and / or due to other modifications, e.g., structural modifications of nucleotides that do not occur naturally. An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule, or a hybrid molecule comprising DNA and RNA portions. Typically, artificial nucleic acid molecules may be designed and / or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). Further, the term “artificial nucleic acid” or “artificial mRNA” is not restricted to mean one single molecule but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.

[0050] The phrase “as indexed by reference to the amino acid sequence of SEQ ID NO: 1,” as used herein, refers to a normalized biological sequence alignment that allows the comparison of a query sequence (e.g., a modified HA polypeptide sequence to which one or more of the modifications described herein have been or will be applied) to a subject sequence (e.g., a wildtype influenza HA polypeptide sequence, such as the HA polypeptide sequence of A / Astrakhan / 3212 / 2020 (SEQ ID NO: 1)), thereby identifying amino acid residues in the target sequence that correspond to the same positions in the subject sequence. In general, the target sequence and the query sequence share characteristic portions or features but differ slightly in length and / or sequence identity. For example, the numbering of residues in a specific target sequence or for targeted modification can be identified and described based on the A / Astrakhan / 3212 / 2020 HA amino acid sequence. Sequences are aligned to the full-length HA protein sequence (including signal peptide, transmembrane and cytoplasmic tail domains) of A / Astrakhan / 3212 / 2020 (SEQ ID NO: 1). The N-terminal methionine of the signal peptide isresidue 1. Accordingly, the phrase “amino acid position x as indexed by reference to the amino acid sequence of SEQ ID NO: 1” is used herein to designate the position / identity of an amino acid residue in a polypeptide of interest (e.g., a modified H5 influenza HA polypeptide) by referring to the corresponding amino acid at position x in the HA polypeptide sequence of A / Astrakhan / 3212 / 2020 (SEQ ID NO: 1).

[0051] The term “at least,” “less than,” “more than,” or “up to” prior to a number or series of numbers (e.g., “at least two”) is understood to include the number adjacent to the term “at least,” “less than” or “more than,” and all subsequent numbers or integers that could logically be included, as clear from context. When the term “at least,” “less than,” “more than,” or “up to” is present before a series of numbers or a range, it is understood that “at least,” “less than,” “more than,” or “up to” can modify each of the numbers in the series or range.

[0052] The term “biological activity,” as used herein, refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest. In some embodiments, the biological activity of an HA polypeptide refers to the ability of the HA polypeptide to elicit neutralizing antibody. In these cases, the term “biological activity” is used interchangeably with “immunogenic activity.”

[0053] As used herein, a “codon-optimized” nucleic acid sequence refers to a nucleic acid sequence that has been altered such that expression of the encoded protein is improved and optimized for a particular expression system. A “codon-optimized” nucleic acid sequence encodes the same protein as a non-optimized parental sequence upon which the “codon-optimized” nucleic acid sequence is based. For example, a nucleic acid sequence may be “codon-optimized” for expression in mammalian cells (e.g., CHO cells, human cells, mouse cells etc.), bacterial cells (e.g., E. coli), insect cells, yeast cells or plant cells.

[0054] The terms “comprise,” “include,” or “contain” and grammatical equivalents thereof as used herein are understood to imply the inclusion of stated elements, numbers, or steps, without excluding the presence of additional elements, numbers, or steps. For instance, a composition 'comprising' X may consist solely of X or may include additional elements e.g. X + Y. It isunderstood that wherever aspects are described herein with the language “comprising,” aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0055] As used herein, the term “epitope” includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component in whole or in part. In some embodiments, an epitope is comprised of a plurality of amino acid residues in an antigen. In some embodiments, the amino acid residues are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, the amino acid residues are physically near to or continuous with each other in space when the antigen adopts such a conformation. In some embodiments, at least some of the amino acids are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized; e.g., a non-linear epitope).

[0056] As used herein, the term “hemagglutinin polypeptide” or “HA polypeptide” refers to a polypeptide whose amino acid sequence includes at least one characteristic sequence of an influenza type A or type B HA. A wide variety of HA sequences from influenza isolates are known in the art; indeed, the National Center for Biotechnology Information (NCBI) maintains a database (ncbi.nlm.nih.gov / genomes / FLU / ) that includes more than 40,000 HA sequences (for type A and B viruses). Those of ordinary skill in the art, referring to this database, can readily identify sequences that are characteristic of HA polypeptides generally, and / or of particular HA polypeptides (e.g., influenza type B HAs or influenza type A HAs, such as Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, or H16 polypeptides; or of HAs that mediate infection of particular hosts, e.g., human, avian, seal etc.).

[0057] The term “H5 influenza HA polypeptide” as used here refers to an influenza HA polypeptide from an influenza A virus subtype H5. A “modified H5 influenza HA polypeptide” refers to an influenza HA polypeptide from an influenza A virus subtype H5 with one or more modifications, such as deletions, additions, or substitutions of one or more amino acid residues.

[0058] The term “host” is used herein to refer to a system (e.g., a cell, organism, etc.) in which a polypeptide of interest is present. In some embodiments, a host is a system that is susceptible to infection with a particular infectious agent. In some embodiments, a host is a system that expresses a particular polypeptide of interest.

[0059] As used herein, the term “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. For example, host cells may be used to produce the modified H5 influenza HA polypeptides described herein by standard recombinant techniques.Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell: as used herein. In some embodiments, host cells include any prokaryotic and eukaryotic cells suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence). Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO KI, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2 / 0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

[0060] As used herein, the term “in some embodiments,” “in certain embodiments,” “in other embodiments,” “in some other embodiments,” or the like, refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0061] The term “messenger RNA (mRNA)” as used herein refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. An mRNA can be purified from natural sources, produced using recombinant expression systems (e.g., in vitro transcription) and optionally purified, or chemically synthesized. An mRNA may contain one or more coding and non-coding regions. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.

[0062] The term “mutation” refers to deletion, addition, or substitution of an amino acid residue in the amino acid sequence of a modified protein or polypeptide as compared to the amino acid sequence of a reference protein or polypeptide.

[0063] The term “prevent,” “preventing,” or “prevention,” as used herein, refers to prophylaxis, avoidance of disease manifestation, a delay of onset, and / or reduction in frequency and / or severity of one or more symptoms of a particular disease, disorder or condition (e.g., infection with, for example, a virus, such as influenza virus). In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition.

[0064] As used herein, the term “prophylactically effective amount” means an amount sufficient to avoid disease manifestation, delay onset of and / or reduce in frequency and / or severity one or more symptoms of a particular disease, disorder or condition (e.g., infection with, for example, a virus, such as influenza virus).

[0065] The term “sequence identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Sequence identity” can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., Siam J. Applied Math., 48: 1073 (1988). Typical methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity and similarity are codified in publicly available computer programs. Typical computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410(1990). The well-known Smith Waterman algorithm may also be used to determine identity. In some embodiments, the sequence identity is determined using the BLAST program with the default parameters.

[0066] As used herein, the term “subject” means any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig). In some embodiments, a subject may be a transgenic animal, genetically- engineered animal, and / or a clone. In some embodiments, the subject is an adult, an adolescent or an infant. In some embodiments, the term “individual” or “patient” is used and is intended to be interchangeable with the term “subject.”

[0067] The term “substantially” as used herein refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0068] As used herein, the terms “treat,” “treating,” or “treatment” refer to actions or interventions in a subject which has been diagnosed with a particular disease, disorder, or condition, with the aim to partially or completely alleviate, ameliorate, relieve, inhibit, delay onset of, reduce severity of, and / or reduce incidence of one or more characteristics or symptoms associated with said disease, disorder or condition (e.g., infection with, for example, a virus, such as influenza virus). A vaccine which aims to treat a disease, disorder, or condition may be referred to as a therapeutic vaccine.

[0069] The term “variant” as used herein, with respect to a reference polypeptide, refers to any polypeptides that structurally differ from the reference polypeptide, while retaining at least one of the properties of the reference polypeptide, such as specific antigenic property of the reference polypeptide. Variants of polypeptides may include fragments of polypeptides. Fragments of polypeptides may be N-terminally and / or C-terminally truncated fragments, e.g. C- terminal fragments and N-terminal fragments, as well as deletion fragments, but do not include thenaturally occurring full-length polypeptide (or mature polypeptide). A deletion fragment refers to a polypeptide with one or more internal amino acids deleted from the full-length polypeptide. Variants of polypeptides may also include polypeptides with altered amino acid sequences due to one or more amino acid substitution(s), deletion(s), and / or insertion(s). Variants can be naturally or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Such variations (i.e. truncations and / or amino acid substitutions, deletions, or insertions) may occur on the amino acid level or correspondingly on the nucleic acid level. Variant polypeptides can comprise conservative and / or non-conservative amino acid substitution(s). Conservative substitutions may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. For example, as summarized in Table 1 above, the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups shown above.

[0070] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0071] As used herein, the term “wild-type” generally refers to a normal form of a protein or nucleic acid, as is found in nature. For example, wild-type HA polypeptides are found in natural isolates of influenza virus. A variety of different wild-type HA sequences can be found in the NCBI influenza virus sequence database (ncbi.nlm.nih.gov / genomes / FLU / ).Structure of Influenza HA Polypeptides

[0072] Influenza HA is a trimeric glycoprotein that is anchored to the viral coat and has two main functions. During the entry process, HA first mediates attachment of the virus to the surface of target cells through interactions with sialic acid receptors and then, after endocytosis of the virus, it triggers the fusion of the viral and endosomal membranes to release its genome into the cytoplasm of the target cell. Influenza HA comprises a large ectodomain of about 500 amino acids that is cleaved by host-derived enzymes to generate 2 polypeptides that remain linked by a disulfide bond. The majority of the N-terminal fragment (about 320-330 amino acids), known as HA1, forms a membrane-distal globular domain, also called the head region, that contains the receptor-binding domain (RBD; receptor-binding site (RBS)) and most determinants recognized by virus-neutralizing antibodies. The smaller C-terminal portion (about 180 amino acids), known as HA2, consisting of a fusion peptide, a N-terminal refolding region 1 (RR1), a central helix, and a C-terminal refolding region 2 (RR2), forms a stem-like structure, also called the stem region, that anchors the head region to the cellular or viral membrane. Three monomeric HA subunits combine to form the trimeric biological HA molecule through symmetry operations. A schematic structure of an influenza HA trimer is shown in FIG. 1.

[0073] Like other class I fusion proteins, influenza HA transforms from a high-energy, metastable prefusion state to a postfusion conformation, a transition triggered by low pH. Without intending to be bound by any theory, the pH-based triggering of HA conformational changes is generally thought to be due to the protonation of amino acids that act as pH sensors. However, the residue or combination of residues acting as pH sensors has not yet been fully elucidated. Since the membrane fusion event occurs in the pH range of about 5-6, the most likely residues to function as pH sensors are histidines, aspartates and / or glutamates, which possess a pKain the appropriate pH range.

[0074] Most HA neutralizing antibodies bind to the loops that surround the RBS and interfere with receptor binding and attachment. Since these loops are highly variable, most antibodies targeting these regions are strain specific. Functional and structural analysis of recently developed fully human monoclonal antibodies against influenza A HA with broad cross-neutralizing potency revealed that, rather than targeting the receptor binding and attachment, these antibodies interfere with the membrane fusion process and are directed against highly conserved epitopes in the stem domain of the influenza A HA protein. See, Throsby et al., PLoS One, 2008, 3(12):e3942, Ekiert et al., Science, 2009, 324(5924):246-251, WO 2008 / 028946, W02010 / 130636, and WO 2013 / 007770, all incorporated herein by reference in their entireties.

[0075] The amino acid sequences of a large number of influenza HA polypeptides from different influenza A viruses, including subtype H5, as well as nucleic acid sequences encoding such polypeptides, are known in the art and readily available in, for instance, the Influenza Virus Database maintained by the National Center for Biotechnology Information (NCBI) (ncbi.nlm.nih.gov / genomes / FLU / ). By way of example, the full-length amino acid sequences of the wild-type HA polypeptide from a representative influenza A virus subtype H5, i.e., influenza A virus A / Astrakhan / 3212 / 2020 strain, is set forth in SEQ ID NO: 1 (EPI_ISL_1038924) and is 567 amino acids in length:MENIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEK THNGKLCDLNGVKPLILKDCSVAGWLLGNPMCDEFIRVPEWSYIVERANP ANDLCYPGSLNDYEELKHLLSRINHFEKILIIPKSSWPNHETSLGVSAACPY QGAPSFFRNVVWLII<I<NDAYPTII<ISYNNTNREDLLILWGIHHSNNAEEQT NLYKNPTTYISVGTSTLNQRLVPKIATRSQVNGQRGRMDFFWTILKPDDA IHFESNGNFIAPEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSM PFHNIHPLTIGECPKYVKSNKLVLATGLRNSPLREKRRKRGLFGAIAGFIEG GWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKM NTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTL DFHDSNVI<NLYDI<VRLQLRDNAI<ELGNGCFEFYHI<CDNECMESVRNGT YDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTAASSLALAIMMAGLSL WMCSNGSLQCRICI (SEQ ID NO: 1).

[0076] For the purposes of the present disclosure (unless context indicates otherwise), the amino acid positions in the modified H5 influenza HA polypeptides are given with reference tothe amino acid sequence of the full length wild-type HA polypeptide of influenza A virus A / Astrakhan / 3212 / 2020 strain set forth in SEQ ID NO: 1. However, it should be noted, and one of skill in the art will understand, that different H5 influenza HA sequences may have different numbering systems, for example, if there are additional amino acid residues added or removed as compared to SEQ ID NO: 1. As such, it is to be understood that when specific amino acid residues are referred to by their number, the description is not limited to only amino acids located at precisely that numbered position when counting from the beginning of a given amino acid sequence, but rather that the equivalent / corresponding amino acid residue in any and all H5 influenza HA polypeptide sequences is intended even if that residue is not at the same precise numbered position, for example if a given H5 influenza HA polypeptide sequence is shorter or longer than SEQ ID NO: 1, or has insertions or deletions as compared to SEQ ID NO: 1.

[0077] For example, the full-length amino acid sequence of the wild-type HA polypeptide from the H5N1 influenza A virus A / Indonesia / 5 / 2005 strain, is set forth in SEQ ID NO: 13 (GenBank Accession No. ABP51969.1) and is 568 amino acids in length:MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEK THNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAN PTNDLC YPGSFNDYEELKHLLSRINHFEKIQIIPKS SWSDHEAS SGVS S ACP YLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAE QTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPND AINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSS MPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRESRRKKRGLFGAIAGF IEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIID I<MNTQFEAVGREFNNLERRIENLNI<I<MEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRN GTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMAGL SLWMCSNGSLQCRICI (SEQ ID NO: 13)

[0078] Thus, the full-length wild-type HA polypeptide from the H5N1 influenza A virus A / Indonesia / 5 / 2005 strain is one amino acid longer than the full-length wild-type HA polypeptide from the H5N8 influenza A virus A / Astrakhan / 3212 / 2020 strain (568 amino acids versus 567 amino acids). By aligning the HA polypeptide sequences, it is apparent that the M447 amino acidin the A / Astrakhan / 3212 / 2020 strain corresponds to the M448 amino acid in the A / Indonesia / 5 / 2005 strain.Modified H5 Influenza HA Polypeptides

[0079] The present disclosure provides modified H5 influenza HA polypeptides in which one or more mutations have been introduced in the amino acid sequence relative to the amino acid sequence of the corresponding wild-type H5 influenza HA polypeptide. In some embodiments, the modified H5 influenza HA polypeptides disclosed herein possess certain beneficial characteristics, such as increased immunogenic properties and / or improved stability in the prefusion conformation, as compared to the corresponding wild-type H5 influenza HA polypeptide. In some embodiments, the modified H5 influenza HA polypeptides disclosed herein present epitopes for recognition by broadly protecting antibodies and thus, are useful for creating a universal epitope-based vaccine for inducing protection against a broad range of influenza A virus strains, particularly those of subtype H5, such as H5N1, H5N3, or H5N8. Also provided are artificial nucleic acid molecules, such as messenger RNAs (mRNAs), that encode the modified H5 influenza HA polypeptides disclosed herein.

[0080] The present disclosure is based, in part, on the surprising discovery that a single amino acid substitution in the HA polypeptide of influenza A virus subtype H5 strains, A / Astrakhan / 3212 / 2020 strain and A / Indonesia / 5 / 2005, led to an increase in immunogenicity as compared to the wild-type HA polypeptide. Accordingly, in some embodiments, the modified H5 influenza HA polypeptides of the present disclosure comprise an amino acid substitution at amino acid position 447 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, which is the amino acid sequence of the wild-type HA polypeptide of influenza A virus A / Astrakhan / 3212 / 2020 strain (e.g., amino acids position 448 of the wild-type HA polypeptide of the A / Indonesia / 5 / 2005 strain). The amino acid substitutions may be conservative substitutions or non-conservative substitutions.

[0081] In some embodiments, the amino acid substitutions may be substitutions of non- classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine P-alanine, GABA and 5- Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, P-alanine, fluoro-amino acids, designer amino acids such as P methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general). In some embodiments, the amino acid substitutions are chosen based on factors which include, but are not limited to, potential for steric hindrance, charge attraction, charge repulsion, common properties of the amino acid side chain, secondary and / or tertiary structure considerations, and / or frequency of use in respective host cells. A person skilled in the art would understand which factors to consider when designing amino acid substitutions for the modified H5 influenza HA polypeptides of the present disclosure.

[0082] In some embodiments, the amino acid substitutions are conservative substitutions. The amino acid residue located at amino acid position 447 of SEQ ID NO: 1 or amino acid position 448 of SEQ ID NO: 13 is a Met and thus, in such embodiments, it may be substituted with Ala, Vai, Leu, or He. Accordingly, in some embodiments, the amino acid substitution at amino acid position 447 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 is M447I. In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447A or M448A, respectively. In some embodiments, the amino acid substitution at amino acid position 447 is of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447V or M448V, respectively. In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447L or M448L, respectively. In embodiments where the modified HA polypeptide is based on a corresponding wild-type H5 influenza HA polypeptide other than that of A / Astrakhan / 3212 / 2020, the amino acid residue in the position corresponding to amino acid position 447 of SEQ ID NO: 1 may not be a Met. Accordingly, references herein to a substitution of Met are intended to apply equally to circumstances where the amino acid residue of the wild-type H5 influenza HA polypeptide at the position corresponding to amino acid position 447 of SEQ ID NO: 1 is not Met. For example, any amino acid at this amino acid position can receive a conservative substitution, and can be substituted with Ala, Vai, Leu, or He, typically, with He.

[0083] In some embodiments, the amino acid substitutions are non-conservative substitutions. In such embodiments, the amino acid residue Met at amino acid position 447 of SEQ ID NO: 1 or the amino acid position 448 of SEQ ID NO: 13 (or the corresponding amino acid residue of anyH5 influenza HA polypeptide whether or not it is Met) may be substituted with an amino acid that is not Ala, Vai, Leu, or He. Accordingly, in some embodiments, the amino acid substitution at amino acid position 447 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 is M447C, M447S, M447T, M447N, M447Q, M447D, M447E, M447H, M447K, M447R, M447G, M447P, M447W, M447Y, or M447F. In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447S or M448S, respectively. In some embodiments, the amino acid substitution at amino acid position 447of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447T or M448T, respectively. In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447N or M448N, respectively. In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447Q or M448Q, respectively. In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447D or M448D, respectively. In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447E or M448E, respectively. In some embodiments, the amino acid substitution at amino acid position 447of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447H or M448H, respectively. In some embodiments, the amino acid substitution at amino acid position 447of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447K or M448K, respectively. In some embodiments, the amino acid substitution at amino acid position 447of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447R or M448R, respectively. In some embodiments, the amino acid substitution at amino acid position 447of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447G or M448G, respectively. In some embodiments, the amino acid substitution at amino acid position 447of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447P or M448P, respectively. In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447W or M448W, respectively. In some embodiments, the amino acid substitution at amino acid position 447of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447Y or M448Y, respectively. In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or at amino acid position 448 of SEQ ID NO: 13 is M447F or M448F, respectively.

[0084] In some embodiments, the amino acid substitution at amino acid position 447 of SEQ ID NO: 1 or amino acid position 448 of SEQ ID NO: 13 creates a cavity filling effect in the stem region of the resultant modified H5 influenza HA polypeptide. As shown in FIG. 1, the M447I mutation in the modified H5 influenza HA polypeptide of SEQ ID NO: 2 creates such an effect which leads to an HA trimer that is more stable in the prefusion conformation. A mutation leading to a cavity filling effect generally refers to a mutation that results in a substitution of an amino acid residue in a parental polypeptide, such as a wild-type H5 influenza A HA polypeptide (e.g., HA polypeptide of influenza A virus strain A / Astrakhan / 3212 / 2020) by an amino acid that is expected to fill an internal cavity (i.e., a void space) that exists in the folded structure of the parental polypeptide. Without wishing to be bound by any theory, such cavity filling mutations can contribute to stabilizing the pH-sensitive interfaces between the head and stem regions and thus, stabilize the prefusion conformation of the modified H5 influenza A HA polypeptide.

[0085] The modified H5 influenza HA polypeptides of the present disclosure can be generated from any wild-type HA polypeptides of any influenza A viruses subtype H5 known in the art or discovered in the future, including but not limited to, subtype H5N1, H5N3, or H5N8. In some embodiments, the modified H5 influenza HA polypeptides of the present disclosure are generated from a wild-type HA polypeptide of an influenza A virus subtype H5N1. In some embodiments, the modified H5 influenza HA polypeptides of the present disclosure are generated from a wildtype HA polypeptide of the influenza A virus A / Indonesia / 05 / 2005 strain. In some embodiments, the modified H5 influenza HA polypeptides of the present disclosure are generated from a wildtype HA polypeptide of an influenza A virus subtype H5N8. In some embodiments, the modified H5 influenza HA polypeptides of the present disclosure are generated from a wild-type HA polypeptide of the influenza A virus A / Astrakhan / 3212 / 2020 strain. In some embodiments, the modified H5 influenza HA polypeptides of the present disclosure are generated from a wild-type HA polypeptide of an influenza A virus subtype H5N3. It should be understood that any H5 influenza A strain not specifically mentioned herein can be the source of the HA polypeptide for generation of the modified H5 influenza HA polypeptides according to the present disclosure.

[0086] A key determinant of influenza virus pathogenesis can be one or more mutations in the proteolytic cleavage site of the HA polypeptide. The presence of a polybasic cleavage site is important for the systemic spread and increased virulence associated with highly pathogenic avian influenza (HP Al) viruses, such as H5N1 or H5N8, whereas low-pathogenicity forms of influenzavirus generally contain a monobasic cleavage site. For instance, the wild-type HA polypeptide of influenza A virus strain A / Astrakhan / 3212 / 2020 (SEQ ID NO: 1) contains a polybasic cleavage site at amino acid positions 341-345 (KRRKR; SEQ ID NO: 8), which makes this influenza strain highly pathogenic. To work with zoonotic HP Al strains, the polybasic cleavage site of the wildtype HA polypeptide generally needs to be mutated to create low pathogenic variants. For example, the polybasic cleavage site can be converted to a monobasic cleavage site like the HA polypeptides of seasonal influenza viruses. A polybasic cleavage site can be mutated to a monobasic cleavage site by removing one or more basic amino acids. A polybasic cleavage site can also be mutated to a monobasic cleavage site by replacing it with a monobasic cleavage site found in any low-pathogenicity forms of influenza virus, such as a seasonal influenza virus. A polybasic cleavage site can also be mutated to a monobasic cleavage site by replacing it with an amino acid sequence representing a monobasic cleavage site.

[0087] Accordingly, in some embodiments, the modified H5 influenza HA polypeptides of the present disclosure contain a polybasic cleavage site at amino acid positions 341-345 (e.g., KRRKR; SEQ ID NO: 8), as indexed by reference to the amino acid sequence of SEQ ID NO: 1. For example, the wild-type polypeptide of the influenza A virus A / Indonesia / 05 / 2005 strain comprises the polybasic cleavage site or RRKKR (SEQ ID NO: 15) at amino acid positions 342- 346 of SEQ ID NO: 13. In some embodiments, the polybasic cleavage site is mutated to a monobasic cleavage site by removing one or more basic amino acid residues. In some embodiments, the polybasic cleavage site is mutated to a monobasic cleavage site by removing all but one basic amino acid. In some embodiments, the polybasic cleavage site is mutated to a monobasic cleavage site by replacing the polybasic cleavage site with a monobasic cleavage site found in a seasonal influenza virus. In some embodiments, the polybasic cleavage site of SEQ ID NO: 8 or SEQ ID NO: 15 is replaced by an amino acid sequence representing a monobasic cleavage site, such as the amino acid sequence TR.

[0088] Accordingly, in some embodiments, the modified H5 influenza HA polypeptide of the present disclosure further comprises one or more mutations in a polybasic cleavage site located at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 such that the polybasic cleavage site is converted to a monobasic cleavage site. In some embodiments, the polybasic cleavage site comprises the amino acid sequence KRRKR (SEQ ID NO: 8) or RRKKR (SEQ ID NO: 15) and the polybasic cleavage site is converted to the monobasiccleavage site by removing one or more, optionally all but one, basic amino acids. In some embodiments, the polybasic cleavage site is converted to the monobasic cleavage site by replacing the polybasic cleavage site with the amino acid sequence TR.

[0089] In some embodiments, the modified H5 influenza HA polypeptides of the present disclosure comprise a monobasic cleavage site and an amino acid substitution at amino acid position 447 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified H5 influenza HA polypeptides of the present disclosure comprise a monobasic cleavage site having the amino acid sequence of TR at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 and amino acid substitution M447I as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the modified H5 influenza HA polypeptides of the present disclosure comprise a monobasic cleavage site having the amino acid sequence of TR that replaces amino acids 341-346 of SEQ ID NO: 13.

[0090] A first representative modified H5 influenza HA polypeptide according to the present disclosure may have the amino acid sequence set forth in SEQ ID NO: 2 (the M447I mutation (bold, underlined, and italic) with a monobasic cleavage site (bold and underlined)):MENIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEK THNGKLCDLNGVKPLILKDCSVAGWLLGNPMCDEFIRVPEWSYIVERANP ANDLCYPGSLNDYEELKHLLSRINHFEKILIIPKSSWPNHETSLGVSAACPY QGAPSFFRNVVWLII<I<NDAYPTII<ISYNNTNREDLLILWGIHHSNNAEEQT NLYKNPTTYISVGTSTLNQRLVPKIATRSQVNGQRGRMDFFWTILKPDDA IHFESNGNFIAPEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSM PFHNIHPLTIGECPKYVKSNKLVLATGLRNSPLRETRGLFGAIAGFIEGGW QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVL / ENERTLDFHD SNVI<NLYDI<VRLQLRDNAI<ELGNGCFEFYHI<CDNECMESVRNGTYDYP QYSEEARLKREEISGVKLESIGTYQILSIYSTAASSLALAIMMAGLSLWMC SNGSLQCRICI (SEQ ID NO: 2).

[0091] A second representative modified H5 influenza HA polypeptide according to the present disclosure may have the amino acid sequence set forth in SEQ ID NO: 14 (the M448I mutation (bold, underlined, and italic) with a monobasic cleavage site (bold and underlined)):MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEK THNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKAN PTNDLC YPGSFNDYEELKHLLSRINHFEKIQIIPKS SWSDHEAS SGVS S ACP YLGSPSFFRNVVWLIKKSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQ TRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAI NFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSM PFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRETRGLFGAIAGFIEGGW QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVL7ENERTLDFHD SNVI<NLYDI<VRLQLRDNAI<ELGNGCFEFYHI<CDNECMESIRNGTYNYP QYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWMC SNGSLQCRICI (SEQ ID NO: 14)

[0092] Accordingly, in some embodiments, the modified H5 influenza HA polypeptides of the present disclosure may comprise an amino acid sequence having at least about 80%, such as at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, including all values and subranges therebetween, sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14. In some embodiments, the modified H5 influenza HA polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified H5 influenza HA polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0093] Similar to wild-type H5 influenza HA polypeptides, the modified H5 influenza HA polypeptides disclosed herein are capable of forming a trimeric HA complex through symmetry operations. Thus, in some embodiments, provided herein is a trimeric H5 influenza HA polypeptide complex comprising three copies of any of the modified H5 influenza HA polypeptides according to the present disclosure. In certain embodiments, the trimeric H5 influenza HA polypeptide complex of the present disclosure has improved stability in the prefusion conformation as compared to a trimeric H5 influenza HA polypeptide complex prepared from the corresponding wild-type H5 influenza HA polypeptide without the one or more modifications. Conformation stability can be measured using any methods known in the art. In some embodiments, stability in the prefusion conformation is measured by an increased binding of the trimeric H5 influenza HA polypeptide complex of the present disclosure to a stem region-specific antibody (e.g., CR9114) as compared to a trimeric H5 influenza HA polypeptide complex preparedfrom the corresponding wild-type H5 influenza HA polypeptide without the one or more modifications (e.g., substitutions). In some embodiments, stability in the prefusion conformation is measured by an increased binding ratio of the stem region-specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) as compared to a trimeric H5 influenza HA polypeptide complex prepared from the corresponding wild-type H5 influenza HA polypeptide without the one or more modifications.

[0094] In some embodiments, the trimeric H5 influenza HA polypeptide complex of the disclosure is more immunogenic as compared to a trimeric H5 influenza HA polypeptide complex prepared from the corresponding wild-type H5 influenza HA polypeptide without the one or more modifications. Immunogenicity can be measured using any methods known in the art. In some embodiments, immunogenicity is measured using hemagglutination inhibition assay (HAI).Nucleic Acid Construction and Expression

[0095] The present disclosure further provides artificial nucleic acid molecules encoding the disclosed modified H5 influenza HA polypeptides. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. The modified H5 influenza HA polypeptides provided herein can be synthesized as DNA sequences by standard methods known in the art and subsequently cloned and expressed in a recombinant host system using a suitable vector. The modified H5 influenza HA polypeptides provided herein can also be synthesized as RNA, such as messenger RNA (mRNA), sequences. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence and encompasses an RNA molecule (e.g., mRNA) with the specified sequence in which U, or a derivative thereof, such as pseudouridine, is substituted for T, unless context requires otherwise. Other nucleotide derivatives or modified nucleotides can be incorporated into the artificial nucleic acid molecules encoding the disclosed modified H5 influenza HA polypeptide. The synthesized DNA or mRNA sequences encoding the modified H5 influenza HA polypeptides of the present disclosure can be codon- optimized so that expression of the encoded protein is improved and optimized for a particular expression system. Any codon optimization algorithms known in the art can be used to generate codon-optimized nucleic acid sequences.

[0096] Accordingly, in some embodiments, provided herein is an artificial nucleic acid encoding the modified H5 influenza HA polypeptides of the present disclosure. In someembodiments, the artificial nucleic acid is a DNA. In some embodiments, the artificial nucleic acid is an RNA. In some embodiments, the artificial nucleic acid is a mRNA.

[0097] A representative codon-optimized mRNA sequence encoding the modified H5 influenza HA polypeptides of SEQ ID NO: 2 is set forth in SEQ ID NO: 3:AUGGAGAACAUCGUGCUGCUGCUGGCCAUCGUGUCUCUGGUGAAGUCCGACCAGAUUUGUAUCGGCUACCACGCUAACAACAGCACCGAACAGGUGGACACCAUCAUGGAGAAGAACGUGACCGUGACCCACGCCCAGGACAUCCUGGAAAAGACCCACAAUGGCAAGCUGUGCGAUCUGAAUGGCGUGAAGCCCCUGAUCCUGAAGGACUGCUCCGUGGCCGGCUGGCUGCUGGGCAAUCCAAUGUGCGAUGAGUUUAUCCGGGUGCCCGAGUGGAGCUACAUCGUGGAGAGAGCCAACCCCGCCAACGACCUGUGCUACCCCGGCAGCCUGAAUGACUACGAGGAGCUGAAGCAUCUGCUGUCCCGGAUCAAUCACUUCGAGAAGAUCCUGAUCAUUCCCAAGAGCUCCUGGCCUAACCACGAGACAUCCCUGGGCGUGAGCGCCGCCUGUCCUUACCAGGGGGCCCCCUCCUUUUUCAGGAAUGUGGUGUGGCUGAUCAAGAAGAACGACGCCUACCCCACCAUCAAGAUCAGCUACAACAACACCAACCGGGAGGACCUGCUGAUCCUGUGGGGCAUCCACCACAGCAACAACGCCGAGGAGCAGACCAACCUGUAUAAGAAUCCCACCACCUACAUCUCCGU GGGGACCAGCACCCUGAACCAGAGGCUGGUGCCCAAGAUCGCCACCC GGUCCCAGGUGAACGGCCAGAGGGGCAGAAUGGAUUUCUUCUGGAC CAUCCUGAAGCCCGACGAUGCCAUCCACUUUGAGAGCAACGGCAAC UUCAUCGCCCCCGAGUACGCCUACAAGAUCGUGAAGAAGGGCGACU CUACCAUCAUGAAGUCCGGGGUGGAGUACGGCCACUGCAACACCAA GUGCCAGACCCCCGUGGGCGCCAUCAAUAGCAGCAUGCCUUUCCACA AUAUCCACCCCCUGACCAUCGGAGAGUGCCCCAAGUACGUGAAAAG CAACAAGCUGGUGCUGGCCACCGGCCUGAGGAACAGCCCUCUGAGA GAGACUCGGGGCCUGUUCGGCGCCAUCGCCGGCUUCAUCGAGGGCG GCUGGCAGGGGAUGGUGGACGGCUGGUACGGCUACCACCACAGCAA CGAGCAGGGCUCCGGCUACGCCGCCGACAAGGAGUCCACCCAGAAG GCCAUCGACGGCGUGACCAACAAGGUGAACUCUAUCAUCGACAAGA UGAACACCCAGUUCGAGGCCGUGGGCCGGGAGUUCAACAACCUGGAAAGGAGGAUCGAGAACCUGAAUAAAAAGAUGGAGGACGGCUUCCUG GACGUGUGGACAUACAACGCCGAGCUGCUGGUGCUGAUCGAGAAUG AGAGAACCCUGGAUUUCCACGACUCCAACGUGAAGAACCUGUACGA CAAGGUGCGGCUGCAGCUGAGGGACAACGCCAAGGAGCUGGGCAAU GGCUGCUUCGAGUUCUACCACAAGUGCGACAACGAAUGCAUGGAGU CCGUGAGGAACGGCACCUACGAUUAUCCCCAGUACAGCGAGGAGGC CAGACUGAAGCGCGAGGAGAUCAGCGGGGUGAAGCUGGAGUCCAUU GGCACCUACCAGAUCCUGAGCAUCUAUAGCACCGCCGCCUCCAGCCU GGCCCUGGCCAUCAUGAUGGCCGGCCUGUCUCUGUGGAUGUGUAGC AACGGCAGCCUGCAGUGCAGGAUCUGCAUCUAAUAA (SEQ ID NO: 3).

[0098] In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the modified H5 influenza HA polypeptides disclosed herein comprise a nucleic acid sequence having at least about 80%, such as at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, including all values and subranges therebetween, sequence identity to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the modified H5 influenza HA polypeptides disclosed herein comprise or consist of the nucleic acid sequence of SEQ ID NO: 3.

[0099] To express the modified H5 influenza HA polypeptides of the present disclosure, suitable recombinant host cells include, but are not limited to, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a clonal isolate derived from the parental Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include, but are not limited to, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells, typically transformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, but are not limited to, for example, chicken embryonic stem cells (e.g., EBx® cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts, and duck cells. Suitable insect cell expression systems, such as baculovirus-vectored systems, are known to those of skill in the art and described in, for instance, Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, for example, Invitrogen (SanDiego, CA). Avian cell expression systems are also known to those of skill in the art and described in, for example, U.S. Pat. Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668. Similarly, bacterial and mammalian cell expression systems are also known in the art and described in, for example, Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

[0100] In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell). In some embodiments, the host cell is a SF9 cell of Spodoptera frugiperda. See U.S. Patent No. 6,103,526, which is hereby incorporated by reference in its entirety. In some embodiments, the host cell is a SF9 cell of Spodoptera frugiperda which has been infected with a baculovirus vector (e.g., Autographa californica nuclear polyhedrosis virus). In some embodiments, the host cell is a CHO cell.

[0101] A number of suitable vectors for expression of recombinant proteins in insect or mammalian cells are well-known and conventional in the art. Suitable vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, a terminator), and / or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., of mammalian origin or from a heterologous mammalian or nonmammalian species). For example, for expression in insect cells, a suitable baculovirus expression vector, such as pFastBac (Invitrogen), is used to produce recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express recombinant protein. For expression in mammalian cells, a vector that will drive expression of the construct in the desired mammalian host cell (e.g., CHO cells) can be used.

[0102] The modified H5 influenza HA polypeptides of the present disclosure can be purified using any suitable methods. For example, methods for purifying recombinant influenza HA polypeptides are known in the art. See e.g., Wang et al., Vaccine, 2006, 24(12):2176-2185. Suitable methods for purifying desired proteins including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating and size exclusion are well-known in the art. Suitable purification schemes can be created using two or more of these or other suitable methods. If desired, the modified H5 influenza HA polypeptides can include a “tag” that facilitates purification, such as an epitope tag or a histidine (HIS) tag.Such tagged polypeptides can conveniently be purified, for example from conditioned media, by chelating chromatography or affinity chromatography.

[0103] Purified polypeptides can be analyzed by spectroscopic methods known in the art, such as circular dichroism spectroscopy, Fourier-transform infrared spectroscopy, NMR spectroscopy, or X-ray crystallography, to investigate the presence of desired structures like helices and beta sheets. ELISA, Octet and FACS and the like can be used to investigate binding of the modified H5 influenza HA polypeptides of the present disclosure to the broadly neutralizing antibodies known in the art, such as CR9114 (stem-specific), CR8071 (VE-specific), and R95-1D05 (RBS- specific) (Dreyfus et al., Science, 2012, 337(6100): 1343-1348). Thus, modified H5 influenza HA polypeptides according to the present disclosure having a desired conformation (e.g., stabilized prefusion conformation) can be verified and selected.

[0104] Accordingly, in some embodiments, provided herein are artificial nucleic acids encoding any of the modified H5 influenza HA polypeptides described herein. The artificial nucleic acids of the disclosure can be in form of a DNA or a RNA, such as a messenger RNA (mRNA). In some embodiments, the artificial nucleic acids of the disclosure are DNA molecules. In some embodiments, the artificial nucleic acids of the disclosure are RNA molecules. In certain embodiments, the artificial nucleic acids of the disclosure are mRNA molecules.

[0105] Also provided herein are vectors comprising the artificial nucleic acid molecules (e.g., mRNAs) disclosed herein. The RNA sequences encoding a protein of interest (e.g., mRNA encoding an influenza HA protein) can be cloned into a number of types of vectors. For example, the nucleic acids can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest can include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0106] In certain embodiments, the vector can be used to express mRNA in a host cell. In various embodiments, the vector can be used as a template for in vitro transcription (IVT). The construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0107] In some embodiments, the vectors disclosed herein can comprise at least the following, from 5' to 3': an RNA polymerase promoter; a polynucleotide sequence encoding a 5' UTR; apolynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3' UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein may comprise a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal.

[0108] A variety of RNA polymerase promoters are known. In some embodiments, the promoter can be a T7 RNA polymerase promoter. Other useful promoters can include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known.

[0109] Also disclosed herein are host cells (e.g., mammalian cells, e.g., human cells) comprising the vectors or RNA compositions disclosed herein.

[0110] Polynucleotides can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. (2001). Hum Gene Ther. 12(8):861-70, or the TransIT-RNA transfection Kit (Minis, Madison, WI).[OHl] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0112] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the mRNA sequence in the host cell a variety of assays may be performed.Immunogenic Compositions and Vaccines

[0113] The present disclosure also relates to immunogenic compositions that comprise any of the modified H5 influenza HA polypeptides disclosed herein or artificial nucleic acids (e.g., mRNA) or vectors encoding such modified H5 influenza HA polypeptides. As used herein, the term “immunogenic composition” refers to a composition that generates an immune response thatmay or may not be a protective immune response or protective immunity. The term “immune response” refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen, immunogen, or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and / or adaptive immune response. Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and / or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like. An antibody response or humoral response is an immune response in which antibodies are produced. A “cellular immune response” is one mediated by T cells and / or other white blood cells.

[0114] Also provided herein is a vaccine comprising the immunogenic composition of the present disclosure and a pharmaceutically acceptable carrier. As used herein, the term “vaccine” refers to a composition that generates a protective immune response or protective immunity in a subject. A “protective immune response” or “protective immunity” refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection) or reduces the symptoms of infection (for instance, an infection by an influenza virus). Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous, intraperitoneal, intradermal, intranasal, by inhalation, or intramuscular.

[0115] The term “pharmaceutically acceptable” means that the carrier, at the dosages and concentrations employed, will not cause unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see e.g., Remington’s Pharmaceutical Sciences, 19thed., Mack Publishing Co., Easton, PA, 1995; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis, 2000; and Handbook of Pharmaceutical Excipients, 3rded., A. Kibbe, Ed., Pharmaceutical Press, 2000). The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can, e.g., be employed as liquid carriers, particularly for injectablesolutions. The exact formulation should suit the mode of administration. The modified H5 influenza HA polypeptides and / or nucleic acid (e.g., mRNA) molecules preferably are formulated and administered as a sterile solution. Sterile solutions are prepared by sterile filtration or by other methods known in the art. The solutions can then be lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, such as pH 5.0 to 7.5.

[0116] Accordingly, in some embodiments, provided herein is a composition comprising any of the modified H5 influenza HA polypeptides disclosed herein, a trimeric H5 influenza HA polypeptide complex comprising three copies of any of the modified H5 influenza HA polypeptides disclosed herein, an artificial nucleic acid encoding any of the modified H5 influenza HA polypeptides disclosed herein, or a vector comprising such an artificial nucleic acid. In some embodiments, provided herein is a composition comprising one or more mRNA molecules encapsulated in a lipid nanoparticle (LNP), wherein the one or more mRNA molecules encode any of the modified H5 influenza HA polypeptides disclosed herein. In some embodiments, such a composition is an immunogenic composition.

[0117] In some embodiments, also provided herein is an immunogenic composition or vaccine comprising any of the modified H5 influenza HA polypeptides disclosed herein. In some embodiments, provided herein is an immunogenic composition or vaccine comprising a trimeric H5 influenza HA polypeptide complex comprising three copies of any of the modified H5 influenza HA polypeptides disclosed herein. In some embodiments, provided herein is an immunogenic composition or vaccine comprising an artificial nucleic acid molecule, or a vector comprising such an artificial nucleic acid molecule, that encodes any of the modified H5 influenza HA polypeptides disclosed herein. In some embodiments, provided herein is an immunogenic composition or vaccine comprising one or more messenger RNA (mRNA) molecules encoding any of the modified H5 influenza HA polypeptides disclosed herein. In certain embodiments, the one or more mRNA molecules in the immunogenic composition or vaccine of the disclosure are encapsulated in a lipid nanoparticle (LNP).

[0118] Each ribonucleic acid molecule may be present in the compositions disclosed herein in an amount effective to induce an immune response in a subject to which the composition is administered. In certain embodiments, each ribonucleic acid molecule may be present in the vaccine or immunogenic compositions disclosed herein in an amount ranging, for example, fromabout 0.1 pg to about 150 pg, such as from about 1 jug to about 130 jug, from about 30 jug to about 150 jig, from about 5 jug to about 120 jug, from about 10 jug to about 75pg, from about 10 jug to about 60 jig, from about 10 jug to about 30 jug, from about 15 jug to about 45 jug, from about 30 jug to about 75 jug, from about 75 jug to about 150 jug, from about 25 jug to about 35 jug, from about 70 jig to about 80 jug, or from about 145 jug to about 150 jug, including all values and subranges therebetween. In certain embodiments, each ribonucleic acid molecule is present in the vaccine or immunogenic compositions disclosed herein in an amount of about 0.1 pg, about 1 pg, about 5 pg, about 10 pg, about 30 pg, about 50 pg, about 75 pg, about 100 pg, about 125 pg, or about 150 pg, including all values and subranges therebetween. In certain embodiments, each ribonucleic acid molecule is present in the vaccine or immunogenic composition in an amount sufficient to encode, for example, from about 5 pg to about 120 pg, such as from about 10 pg to about 60 pg, or about 15 pg to about 45 pg, including all values and subranges therebetween, of the modified H5 influenza HA polypeptide.

[0119] Accordingly, in some embodiments, provided herein is a vaccine or immunogenic composition comprising any artificial mRNA disclosed herein encapsulated in a LNP, wherein the vaccine comprises from about 0.1 pg to about 150 pg, from about 1 pg to about 130 pg, from about 30 pg to about 150 pg, from about 5 pg to about 120 pg, from about 10 pg to about 75 pg, from about 10 pg to about 60 pg, from about 10 pg to about 30 pg, from about 15 pg to about 45 pg, from about 30 pg to about 75 pg, from about 75 pg to about 150 pg, from about 25 pg to about 35 pg, from about 70 pg to about 80 pg, or from about 145 pg to about 150 pg, including all values and subranges therebetween, of the artificial mRNA. In some embodiments, the vaccine or immunogenic composition comprises about 0.1 pg, about 1 pg, about 5 pg, about 10 pg, about 30 pg, about 50 pg, about 75 pg, about 100 pg, about 125 pg, or about 150 pg, including all values and subranges therebetween, of the artificial mRNA. In some embodiments, the vaccine or immunogenic composition comprises about 30 pg of the artificial mRNA. In some embodiments, the vaccine or immunogenic composition comprises about 75 pg of the artificial mRNA. In some embodiments, the vaccine or immunogenic composition comprises about 150 pg of the artificial mRNA.

[0120] The LNP compositions of the present disclosure may be provided as a frozen liquid form or a lyophilized form. A variety of cryoprotectants may be used, including, without limitations, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant mayconstitute 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition comprises trehalose, e.g., at 5-30% (e.g., 10%) (w / v). Once formulated with the cryoprotectant, the LNP compositions may be frozen (or lyophilized and cryopreserved) at -20°C to -80°C. The LNP compositions may be provided to a patient in an aqueous buffered solution - thawed if previously frozen, or if previously lyophilized, reconstituted in an aqueous buffered solution at bedside. The buffered solution preferably is isotonic and suitable for e.g., intramuscular or intradermal injection. In some embodiments, the buffered solution is a phosphate-buffered saline (PBS).

[0121] In some embodiments, the composition of the disclosure is an immunogenic composition capable of eliciting an immune response against influenza A viruses subtype H5, such as H5N8 or H5N1, in a subject.

[0122] In some embodiments, the immunogenic compositions of the disclosure may further comprise one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients to stabilize the modified H5 influenza HA polypeptides comprised therein, or mRNA molecules encoding the same and / or LNP encapsulating such mRNA molecules, or to facilitate administration of the immunogenic composition. Examples of such excipients include, but are not limited to, parabens, thimerosal, thiomersal, chlorobutanol, bezalkonium chloride, and chelators (e.g., ethylenediaminetetraacetic acid, or EDTA).I. RNA

[0123] In certain embodiments, the vaccine or immunogenic compositions disclosed herein may comprise one or more self-amplifying ribonucleic acids, such as one or more self-amplifying RNA encoding a modified H5 influenza HA polypeptide as disclosed herein. Antigen expression from traditional mRNA is proportional to the number of mRNA molecules successfully delivered to a subject from a vaccine or immunogenic composition. Self-amplifying RNA, however, comprise genetically-engineered replicons derived from self-replicating viruses, and therefore may be added to a vaccine or immunogenic composition in lower dosages than traditional mRNA while achieving comparable results.

[0124] In certain embodiments, the RNAs are messenger RNAs (mRNAs) comprising an open reading frame (ORF) encoding a modified H5 influenza HA polypeptide as disclosed herein. Incertain embodiments, the RNAs (e.g., mRNAs) further comprises at least one 5' UTR, 3' UTR, poly(A) tail, and / or 5' cap.A. 5' Cap

[0125] An mRNA 5' cap can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Several types of 5' caps are known. A 7-m ethylguanosine cap (also referred to as “m7G” or “Cap-0”) comprises a guanosine that is linked through a 5'-5'- triphosphate bond to the first transcribed nucleotide.

[0126] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Publication No. US 2016 / 0032356 and U.S. Publication No. US 2018 / 0125989, which are incorporated herein by reference.

[0127] 5' -capping of polynucleotides may be completed concomitantly during the in vitro- transcription reaction using the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5')G (the ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5’)G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies). 5'-capping of modified RNA may be completed post-transcriptionally using a vaccinia virus capping enzyme to generate the Cap 0 structure: m7G(5')ppp(5')G. Cap 1 structure may be generated using both vaccinia virus capping enzyme and a 2'-0 methyltransferase to generate: m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'- O methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-O-m ethylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase.

[0128] In certain embodiments, the mRNA of the disclosure comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (the ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.

[0129] In certain embodiments, the mRNA of the disclosure comprises a 5' cap of:B. Untranslated Region (UTR)

[0130] In some embodiments, the mRNA of the disclosure includes a 5' and / or 3' untranslated region (UTR). In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon. The 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal.

[0131] In some embodiments, the mRNA disclosed herein may comprise a 5' UTR that includes one or more elements that affect an mRNA’s stability or translation. In some embodiments, a 5' UTR may be about 10 to 5,000 nucleotides in length. In some embodiments, a 5' UTR may be about 50 to 500 nucleotides in length. In some embodiments, the 5' UTR is at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or about 5,000 nucleotides in length.

[0132] In some embodiments, the mRNA disclosed herein may comprise a 3' UTR comprising one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' UTR may be 50 to 5,000 nucleotides in length or longer. In some embodiments, a 3' UTR may be 50 to 1,000 nucleotides in length or longer. In some embodiments, the 3' UTR is at least about 50nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1,000 nucleotides in length, about 1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000 nucleotides in length, about 3,500 nucleotides in length, about 4,000 nucleotides in length, about 4,500 nucleotides in length, or about 5,000 nucleotides in length.

[0133] In some embodiments, the mRNA disclosed herein may comprise a 5' or 3' UTR that is derived from a gene distinct from the one encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0134] In certain embodiments, the 5' and / or 3' UTR sequences can be derived from mRNA which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the mRNA. For example, a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof, to improve the nuclease resistance and / or improve the half-life of the mRNA. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof, to the 3' end or untranslated region of the mRNA. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA relative to their unmodified counterparts, and include, for example, modifications made to improve such mRNA resistance to in vivo nuclease digestion.

[0135] Exemplary 5' UTRs include a sequence derived from a CMV immediate-early 1 (IE1) gene (U.S. Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference), or the sequence GGGAUCCUACC (SEQ ID NO: 4) (U.S. Publication No. 2016 / 0151409, incorporated herein by reference).

[0136] In various embodiments, the 5' UTR may be derived from the 5' UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-associated translational regulation. However, TOP genes with a tissue specific translational regulation are also known. Incertain embodiments, the 5' UTR derived from the 5' UTR of a TOP gene lacks the 5' TOP motif (the oligopyrimidine tract) (e.g., U.S. Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0137] In certain embodiments, the 5' UTR is derived from a ribosomal protein Large 32 (L32) gene (U.S. Publication No. 2017 / 0029847, supra).

[0138] In certain embodiments, the 5' UTR is derived from the 5' UTR of a hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No. 2016 / 0166710, supra).

[0139] In certain embodiments, the 5' UTR is derived from the 5' UTR of an ATP5A1 gene (U.S. Publication No. 2016 / 0166710, supra).

[0140] In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.

[0141] In some embodiments, the 5' UTR comprises a nucleic acid sequence of GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG UGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 5).

[0142] In some embodiments, the 3' UTR comprises a nucleic acid sequence of CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCC ACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 6).

[0143] The 5' UTR and 3' UTR are described in further detail in International Pub. No. WO 2012 / 075040, incorporated herein by reference.C. Polyadenylated Tail

[0144] As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to a sequence of adenosine nucleotides at the 3' end of the mRNA molecule. The poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation. The poly(A) tail may enhance translation. In some embodiments, the poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have essentially a length of 100 nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at least one nucleotide different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide, or a stretch ofnucleotides, that are different from an adenosine nucleotide). In certain embodiments, the poly(A) tail comprises the sequence:AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA (SEQ ID NO: 7).

[0145] The “poly(A) tail,” as used herein, typically relates to RNA. However, in the context of the disclosure, the term likewise relates to corresponding sequences in a DNA molecule (e.g., a “poly(T) sequence”).

[0146] The poly(A) tail may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0147] In some embodiments where the nucleic acid is an RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during RNA in vitro transcription. In certain embodiments, the poly(A) tail is obtained in vitro by common methods of chemical synthesis without being transcribed from a DNA template. In various embodiments, poly(A) tails are generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols, or alternatively, by using immobilized poly(A) polymerases, e.g., using methods and means as described in International Pub. No. WO 2016 / 174271.

[0148] The nucleic acid may comprise a poly(A) tail obtained by enzymatic polyadenylation, wherein the majority of nucleic acid molecules comprise about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0149] In some embodiments, the nucleic acid may comprise a poly(A) tail derived from a template DNA and may additionally comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in International Pub. No. WO 2016 / 091391.

[0150] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal.

[0151] In various embodiments, the nucleic acid may comprise at least one poly(C) sequence.

[0152] The term “poly(C) sequence,” as used herein, is intended to be a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides,about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides.D. Chemical Modification

[0153] The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may comprise at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA may be synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines, such as, e.g., 1-methyl-adenine, 2-methyl- adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2- thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1- methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1 -methylinosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5- carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5- bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N- uracil-5-oxy acetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2- thio-uracil, 5 ’-methoxy carbonylmethyl -uracil, 5-methoxy -uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, P-D-mannosyl-queosine, phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7- deazaguanosine, 5-methylcytosine, and inosine.

[0154] In some embodiments, the disclosed mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-m ethyl- 1-deaza-pseudouri dine, 2-thio-l-m ethylpseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl- pseudouridine, 4-thio-pseudouridine, 5 -aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methyluridine, 5-methoxyuridine, and 2'-O-methyl uridine.

[0155] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof.

[0156] In some embodiments, the chemical modification comprises N1 -methylpseudouridine.

[0157] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0158] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0159] The preparation of such analogs is described, e.g., in U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642.E. mRNA Synthesis

[0160] The mRNAs disclosed herein may be synthesized according to any of a variety of methods. For example, mRNAs according to the present disclosure may be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, e.g., in Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530: 101- 14. Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions may vary according to the specific application. The presence of these reagents is generally undesirable in a final mRNA product and these reagents can be considered impurities or contaminants which can be purified or removed to provide a clean and / or homogeneous mRNA that is suitable for therapeutic use. While mRNA provided from in vitro transcription reactions may be desirable in some embodiments, other sources of mRNA can be used according to the instant disclosure including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.II. Lipid Nanoparticle

[0161] The term “lipid nanoparticle” or “LNP” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids, for example a cationic lipid and / or non-cationic lipid, and one or more excipients selected from neutral lipids, anionic lipids, zwitterionic lipids, ionizable lipids, steroids, and polymer conjugated lipids (e.g., a pegylated lipid). Examples of suitable lipids include, but are not limited to, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). RNA-encapsulated LNP compositions are known in the art, such as those described in PCT Publication Nos. WO 2021 / 237084 and WO 2022 / 099003, the entire contents of which are incorporated by reference herein.

[0162] Any known LNP formulations may be used in the embodiments disclosed herein. In some embodiments, the LNPs comprise four categories of lipids: (i) an ionizable lipid (e.g., a cationic lipid); (ii) a PEGylated lipid; (iii) a cholesterol-based lipid, and (iv) a helper lipid.A. Ionizable Lipid

[0163] An ionizable lipid facilitates mRNA encapsulation and may be a cationic lipid. A cationic lipid affords a positively charged environment at low pH to facilitate efficient encapsulation of the negatively charged mRNA drug substance. Exemplary cationic lipids are shown below in Table 2.Table 2. Cationic lipids

[0164] The cationic lipid may be selected from the group comprising [ckkE10] / [OF-02], [(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl]4-(dimethylamino)butanoate (D-Lin- MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA); 1,2-di linoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); di((Z)-non-2-en-l-yl)9-((4-(dimethyl amino)butanoyl)oxy)heptadecanedioate (L319); 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl] amino] octanoate (SM-102); [(4-hydroxybutyl)azanediyl]di(hexane-6, 1-diyl) bis(2-hexyldecanoate) (ALC-0315); [3-(dimethylamino)-2-[(Z)-octadec-9-enoyl] oxypropyl](Z)- octadec-9-enoate (DODAP); 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxo ethyl]pentanamide (DOGS); [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methyl heptan-2-yl]-2,3,4,7,8,9,l l,12,14,15,16,17-dodecahydro-lH-cyclopenta[a] phenanthren-3-yl]N- [2-(dimethylamino)ethyl]carbamate (DC-Chol); tetrakis(8-methylnonyl) 3,3',3",3"'- (((methylazanediyl)bis(propane-3,ldiyl))bis(azanetriyl))tetrapropionate (3060il0); decyl(2- (dioctylammonio)ethyl)phosphate (9A1P9); ethyl 5,5-di((Z)-heptadec-8-en-l-yl)-l-(3- (pyrrolidin-l-yl)propyl)-2,5-dihydro-lH-imidazole-2-carboxylate (A2-Iso5-2DC18); bis(2- (dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl) azanediyl)dipropionate (B AME-016B); 1 , 1 '-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200); 3 , 6-bi s(4-(bi s(2-hy droxy dodecyl)amino)butyl)piperazine-2, 5 -di one (cKK-E 12); hexa(octan-3 -yl) 9,9',9'',9"',9'''',9"'''-((((benzene-l,3,5-tricarbonyl)yris(azanediyl))tris(propane-3,l-diyl))tris (azanetriyl))hexanonanoate (FTT5); (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,l-diyl))bis (azanetriyl))tetraki s(ethane-2, 1 -diyl)(9Z, 9 'Z, 9 "Z, 9"'Z, 12Z, 12 'Z, 12 "Z, 12"'Z)-tetraki s(octadeca- 9, 12-di enoate) (OF-Deg-Lin); TT3; Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene-l,3,5-tricarboxamide; Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbox amido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl 8-((2-hydroxyethyl)(8- (nonyloxy)-8-oxooctyl)amino)octanoate; IM-001; IS-001 (provided as compound 24 in W02024 / 003759 incorporated herein by reference) and combinations thereof.

[0165] In certain embodiments, the cationic lipid is biodegradable. In various embodiments, the cationic lipid is not biodegradable. In some embodiments, the cationic lipid is cleavable. In certain embodiments, the cationic lipid is not cleavable.

[0166] Cationic lipids are described in further detail in Dong et al. (PNAS. 111(11):3955-60, 2014); Fenton et al. (Adv. Mater. 28:2939. 2016); U.S. Pat. No. 9,512,073; and U.S. Pat. No. 10,201,618, each of which is incorporated herein by reference.B. PEGylated Lipid

[0167] The PEGylated lipid component provides control over particle size and stability of the nanoparticle. The addition of such components may prevent complex aggregation and provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid pharmaceutical composition to target tissues (Klibanov et al., FEBS Letters 268(l):235-7. 1990). These components may be selected to rapidly exchange out of the pharmaceutical composition in vivo (see, e.g., U.S. Pat. No. 5,885,613).

[0168] Contemplated PEGylated lipids include, but are not limited to, a polyethylene glycol (PEG) chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 (e.g., C8, CIO, C12, C14, C16, or C18) length, such as a derivatized ceramide (e.g., N-octanoyl- sphingosine-l-[succinyl(m ethoxypoly ethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); l,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE- PEG); l,2-dilauroyl-sn-glycero-3 -phosphoethanolamine-poly ethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycero-polyethelene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; a PEG-dialkyoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (ALC-0159); and combinations thereof.

[0169] In certain embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In certain embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000,C8 PEG2000, or ALC-0159 (2-[(poly ethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000.C. Cholesterol-Based Lipid

[0170] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNPs comprise one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example: DC-Choi (N,N-dimethyl-N- ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23: 139; U.S. Pat. 5,744,335), imidazole cholesterol ester (“ICE”; WO2011 / 068810), sitosterol (22,23- dihydrostigmasterol), P-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3B-hydroxy-5,24-cholestadiene); lanosterol (8,24-lanostadien-3b-ol); 7- dehydrocholesterol (A5,7-cholesterol); dihydrolanosterol (24,25-dihydrolanosterol); zymosterol (5a-cholesta-8,24-dien-3B-ol); lathosterol (5a-cholest-7-en-3B-ol); diosgenin ((3p,25R)-spirost-5- en-3-ol); campesterol (campest-5-en-3B-ol); campestanol (5a-campestan-3b-ol); 24-methylene cholesterol (5,24(28)-cholestadien-24-methylen-3B-ol); cholesteryl margarate (cholest-5-en-3B-yl heptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNPs is cholesterol.D. Helper Lipid

[0171] A helper lipid enhances the structural stability of the LNP and helps the LNP in endosome escape. It improves uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is a zwitterionic lipid, which has fusogenic properties for enhancing uptake and release of the drug payload. Examples of helper lipids are l,2-dioleoyl-SN-glycero-3- phosphoethanolamine (DOPE); l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2- dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); l,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (DEPE); and l,2-dioleoyl-sn-glycero-3 -phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, l,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-Distearoylphosphatidylethanolamine (DSPE), and l,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE).

[0172] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine(POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelins, ceramides, cerebrosides, gangliosides, 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), or a combination thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0173] In various embodiments, the present LNPs comprise (i) a cationic lipid selected from OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL- HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.E. Molar Ratios of the Lipid Components

[0174] The molar ratios of the herein components are important for the LNPs’ effectiveness in delivering mRNA. The molar ratio of the cationic lipid, the PEGylated lipid, the cholesterol -based lipid, and the helper lipid is A: B: C: D, where A + B + C + D = 100%. In some embodiments, the molar ratio of the cationic lipid in the LNPs relative to the total lipids (i.e., A) is 35-55%, such as 35-50% (e.g., 38-42% such as 40%, or 45-50%). In some embodiments, the molar ratio of the PEGylated lipid component relative to the total lipids (i.e., B) is 0.25-2.75% (e.g., 1-2% such as 1.5%). In some embodiments, the molar ratio of the cholesterol -based lipid relative to the total lipids (i.e., C) is 20-50% (e.g., 27-30% such as 28.5%, or 38-43%). In some embodiments, the molar ratio of the helper lipid relative to the total lipids (i.e., D) is 5-35% (e.g., 28-32% such as 30%, or 8-12%, such as 10%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs contain a molar ratio of the cationic lipid to the helper lipid that is more than 1.

[0175] In certain embodiments, the LNP of the disclosure comprises: i) a cationic lipid at a molar ratio of 35% to 55% or 40% to 50% (e.g., a cationic lipid at a molar ratio of 35%, 36%, 37%, 38%, 39%, 40%, 41% 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%); ii) a polyethylene glycol (PEG) conjugated (PEGylated) lipid at a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., a PEGylated lipid at a molar ratio of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%); iii) a cholesterol-based lipid at a molar ratio of 20% to 45%, 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., a cholesterol-based lipid at a molar ratio of 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%, or 50%); and iv) a helper lipid at a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., a helper lipid at a molar ratio of 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%, or 35%), wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0176] In certain embodiments, the LNP comprises: a cationic lipid at a molar ratio of 40%; a PEGylated lipid at a molar ratio of 1.5%; a cholesterol -based lipid at a molar ratio of 28.5%; and a helper lipid at a molar ratio of 30%.

[0177] In certain embodiments, the PEGylated lipid is dimyristoyl -PEG2000 (DMG- PEG2000).

[0178] In various embodiments, the cholesterol-based lipid is cholesterol.

[0179] In some embodiments, the helper lipid is l,2-dioleoyl-SN-glycero-3- phosphoethanolamine (DOPE).

[0180] In certain embodiments, the LNP comprises: OF-02 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0181] In certain embodiments, the LNP comprises: cKK-ElO at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0182] In certain embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0183] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0184] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0185] In certain embodiments, the LNP comprises: SM-102 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0186] In certain embodiments, the LNP comprises: ALC-0315 at a molar ratio of 35% to 55%; ALC-0159 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0187] In certain embodiments, the LNP comprises: OF-02 at a molar ratio of 40%; DMG- PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0188] In certain embodiments, the LNP comprises: cKK-ElO at a molar ratio of 40%; DMG- PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0189] In certain embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0190] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 (at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0191] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0192] In certain embodiments, the LNP comprises: 9-heptadecanyl 8-{(2-hydroxyethyl)[6- oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) at a molar ratio of 50%; 1,2-distearoyl- w- glycero-3 -phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 38.5%; and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) at a molar ratio of 1.5%.

[0193] In certain embodiments, the LNP comprises: (4-hydroxybutyl)azanediyl]di(hexane- 6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 46.3%; 1,2-distearoyl-sw-glycero- 3 -phosphocholine (DSPC) at a molar ratio of 9.4%; cholesterol at a molar ratio of 42.7%; and 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.6%.

[0194] In certain embodiments, the LNP comprises: (4-hydroxybutyl)azanediyl]di(hexane- 6,1 -diyl) bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 47.4%; 1,2-distearoyl-sw-glycero- 3 -phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 40.9%; and 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.7%.

[0195] In certain embodiments, the LNP comprises: IM-OOlat a molar ratio of 40%; DMG- PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[0196] To calculate the actual amount of each lipid to be put into an LNP formulation, the molar amount of the cationic lipid is first determined based on a desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP. Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the molar ratio selected. These molar amounts are then converted to weights using the molecular weight of each lipid.

[0197] Accordingly, in some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding any of the modified H5 influenza HA polypeptides disclosed herein encapsulated in a LNP, wherein the LNP comprises a cationic lipid. In some embodiments, the cationic lipid comprises or is OF-02. In some embodiments, the cationic lipid comprises or is cKK-ElO. In some embodiments, the cationic lipid comprises or is GL-HEPES- E3-E10-DS-3-E18-1. In some embodiments, the cationic lipid comprises or is GL-HEPES-E3- E12-DS-4-E10. In some embodiments, the cationic lipid comprises or is GL-HEPES-E3-E12-DS- 3-E14. In some embodiments, the cationic lipid comprises or is (4-hydroxybutyl)azanediyl] di(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC-0315). In some embodiments, the cationic lipid comprises or is IM-001. In some embodiments, the cationic lipid comprises or is IS-001.

[0198] In some embodiments, the LNP encapsulating the artificial mRNA of the present disclosure further comprises a PEGylated lipid, a cholesterol-based lipid, and a helper lipid. In some embodiments, the PEGylated lipid comprises or is DMG-PEG2000. In some embodiments, the cholesterol-based lipid comprises or is cholesterol. In some embodiments, the helper lipid comprises or is DOPE. In some embodiments, the LNP comprises the cationic lipid at a molar ratio between about 35% and about 55%, the PEGylated lipid at a molar ratio between about 0.25% and about 2.75%, the cholesterol-based lipid at a molar ratio between about 20% and about 45%, and the helper lipid at a molar ratio between about 5% and about 35%, wherein all of themolar ratios are relative to the total lipid content of the LNP. In some embodiments, the LNP comprises the cationic lipid at a molar ratio of about 40%, the PEGylated lipid at a molar ratio of about 1.5%, the cholesterol -based lipid at a molar ratio of about 28.5%, and the helper lipid at a molar ratio of about 30%, wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0199] In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the modified H5 influenza HA polypeptide of SEQ ID NO: 2 or SEQ ID NO: 14 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS- 4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%. In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 3 encapsulated in a LNP, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%.III. Processes for Making LNP Vaccines

[0200] The LNPs can be prepared by various techniques presently known in the art. For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion that results in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0201] Various methods are described in Patent Application Pub. Nos. US 2011 / 0244026, US 2016 / 0038432, US 2018 / 0153822, US 2018 / 0125989, and US 2021 / 0046192 and can be used for making LNP vaccines. One exemplary process entails encapsulating mRNA by mixing it with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles, as described in Patent Application Pub. No. US 2016 / 0038432. Another exemplary process entails encapsulating mRNA by mixing pre-formed LNPs with mRNA, as described in Patent Application Pub. No. US 2018 / 0153822.

[0202] In some embodiments, the process of preparing mRNA-loaded LNPs includes a step of heating one or more of the solutions to a temperature greater than ambient temperature, the one or more solutions being the solution comprising the pre-formed lipid nanoparticles, the solution comprising the mRNA and the mixed solution comprising the LNP-encapsulated mRNA. In some embodiments, the process includes the step of heating one or both of the mRNA solution and the pre-formed LNP solution, prior to the mixing step. In some embodiments, the process includes heating one or more of the solutions comprising the pre-formed LNPs, the solution comprising the mRNA and the solution comprising the LNP-encapsulated mRNA, during the mixing step. In some embodiments, the process includes the step of heating the LNP- encapsulated mRNA, after the mixing step. In some embodiments, the temperature to which one or more of the solutions is heated is or is greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C, including all values and subranges therebetween. In some embodiments, the temperature to which one or more of the solutions is heated ranges from about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60-70°C, including all values and subranges therebetween. In some embodiments, the temperature is about 65°C.

[0203] Various methods may be used to prepare an mRNA solution suitable for the present invention. In some embodiments, mRNA may be directly dissolved in a buffer solution described herein. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution may be generated by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water or a buffer at a concentration at or greater than about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml, including all values and subranges therebetween.

[0204] In some embodiments, an mRNA stock solution is mixed with a buffer solution using a pump. Exemplary pumps include but are not limited to gear pumps, peristaltic pumps and centrifugal pumps. Typically, the buffer solution is mixed at a rate greater than that of the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least lx, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 15x, or 20x greater than the rate of the mRNA stock solution. In some embodiments, a buffer solution is mixed at a flow rate ranging between about 100-6000 ml / minute(e.g., about 100-300 ml / minute, 300-600 ml / minute, 600-1200 ml / minute, 1200-2400 ml / minute, 2400-3600 ml / minute, 3600-4800 ml / minute, 4800-6000 ml / minute, or 60-420 ml / minute, including all values and subranges therebetween). In some embodiments, a buffer solution is mixed at a flow rate of, or greater than, about 60 ml / minute, 100 ml / minute, 140 ml / minute, 180 ml / minute, 220 ml / minute, 260 ml / minute, 300 ml / minute, 340 ml / minute, 380 ml / minute, 420 ml / minute, 480 ml / minute, 540 ml / minute, 600 ml / minute, 1200 ml / minute, 2400 ml / minute, 3600 ml / minute, 4800 ml / minute, or 6000 ml / minute, including all values and subranges therebetween.

[0205] In some embodiments, an mRNA stock solution is mixed at a flow rate ranging between about 10-600 ml / minute (e.g., about 5-50 ml / minute, about 10-30 ml / minute, about 30-60 ml / minute, about 60-120 ml / minute, about 120-240 ml / minute, about 240-360 ml / minute, about 360-480 ml / minute, or about 480-600 ml / minute, including all values and subranges therebetween). In some embodiments, an mRNA stock solution is mixed at a flow rate of or greater than about 5 ml / minute, 10 ml / minute, 15 ml / minute, 20 ml / minute, 25 ml / minute, 30 ml / minute, 35 ml / minute, 40 ml / minute, 45 ml / minute, 50 ml / minute, 60 ml / minute, 80 ml / minute, 100 ml / minute, 200 ml / minute, 300 ml / minute, 400 ml / minute, 500 ml / minute, or 600 ml / minute, including all values and subranges therebetween.

[0206] The process of incorporation of a desired mRNA into a lipid nanoparticle is referred to as “loading.” Exemplary methods are described in Lasic et al., FEBS Let. (1992) 312:255-8. The LNP -incorporated nucleic acids may be completely or partially located in the interior space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the exterior surface of the lipid nanoparticle membrane. The incorporation of an mRNA into lipid nanoparticles is also referred to herein as “encapsulation” wherein the nucleic acid is entirely or substantially contained within the interior space of the lipid nanoparticle.

[0207] Suitable LNPs may be made in various sizes. In some embodiments, decreased size of lipid nanoparticles is associated with more efficient delivery of an mRNA. Selection of an appropriate LNP size may take into consideration the site of the target cell or tissue and to some extent the application for which the lipid nanoparticle is being made.

[0208] A variety of methods known in the art are available for sizing of a population of lipid nanoparticles. Preferred methods herein utilize Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 pl of an LNP sample are mixed with 990 pl of 10% trehalose. This solution is loaded into a cuvette and then put into the Zetasizer machine. The z-average diameter (nm), or cumulants mean, is regarded as the average size for the LNPs in the sample. The Zetasizer machine can also be used to measure the poly dispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. Average LNP diameter may be reduced by sonication of formed LNP. Intermittent sonication cycles may be alternated with quasi-elastic light scattering (QELS) assessment to guide efficient lipid nanoparticle synthesis.

[0209] In some embodiments, the majority of purified LNPs, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, including all values and subranges therebetween, of the LNPs, have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm, including all values and subranges therebetween). In some embodiments, substantially all (e.g., greater than 80 or 90%) of the purified lipid nanoparticles have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm, including all values and subranges therebetween).

[0210] In certain embodiments, the LNP has an average diameter of 30-200 nm. In various embodiments, the LNP has an average diameter of 80-150 nm.

[0211] In some embodiments, the LNPs in the present composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm, including all values and subranges therebetween.

[0212] In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, including all values and subranges therebetween, of the LNPs in the present composition have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm, including all values and subranges therebetween) or about 50- 70 nm (e.g., 55-65 nm) are particular suitable for pulmonary delivery via nebulization.

[0213] In some embodiments, the dispersity, or measure of heterogeneity in size of molecules (PDI), of LNPs in a pharmaceutical composition provided by the present invention is less than about 0.5. In some embodiments, an LNP has a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08, including all values and subranges therebetween. The PDI may be measured by a Zetasizer machine as described above.

[0214] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, including all values and subranges therebetween, of the purified LNPs in a pharmaceutical composition provided herein encapsulate an mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in a pharmaceutical composition encapsulate an mRNA within each individual particle. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of 50% to 99%; or greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 99%, including all values and subranges therebetween. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91%, 92%, 93%, 94%, or 95%, including all values and subranges therebetween).

[0215] In some embodiments, an LNP has a N / P ratio of between 1 and 10. In some embodiments, a lipid nanoparticle has a N / P ratio above 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8, including all values and subranges therebetween. In further embodiments, a typical LNP herein has an N / P ratio of 4.

[0216] In some embodiments, a pharmaceutical composition according to the present invention contains at least about 0.5 pg, 1 pg, 5 pg, 10 pg, 100 pg, 500 pg, or 1000 pg, including all values and subranges therebetween, of encapsulated mRNA. In some embodiments, a pharmaceutical composition contains about 0.1 pg to 1000 pg, at least about 0.5 pg, at least about 0.8 pg, at least about 1 pg, at least about 5 pg, at least about 8 pg, at least about 10 pg, at least about 50 pg, at least about 100 pg, at least about 500 pg, or at least about 1000 pg, including all values and subranges therebetween, of encapsulated mRNA.

[0217] In some embodiments, mRNA can be made by chemical synthesis or by in vitro transcription (IVT) of a DNA template. For example, in an IVT process, a cDNA template is used to produce an mRNA transcript and the DNA template is degraded by a DNase. The transcript is purified by depth filtration and tangential flow filtration (TFF). The purified transcript is further modified by adding a cap and a tail, and the modified RNA is purified again by depth filtration and TFF.

[0218] The mRNA is then prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of the LNPs. An amphiphilic solution for dissolving the four lipid components of the LNPs may be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer may be, for example, a citrate, phosphate, acetate, or succinate buffer and may have a pH of about 3.0-7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5, including all values and subranges therebetween. The buffer may contain other components such as a salt (e.g., sodium, potassium, and / or calcium salts). In particular embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, pH 4.5.

[0219] An exemplary, nonlimiting process for making an mRNA-LNP composition involves mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled homogeneous manner, where the ratio of lipids:mRNA is maintained throughout the mixing process. In this illustrative example, the mRNA is presented in an aqueous buffer containing citric acid monohydrate, tri-sodium citrate dihydrate, and sodium chloride. The mRNA solution is added to the solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). The lipid mixture of four lipids (e.g., a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid) is dissolved in ethanol. The aqueous mRNA solution and the ethanol lipid solution are mixed at a volume ratio of 4: 1 in a “T” mixer with a near “pulseless” pump system. The resultant mixture is then subjected for downstream purification and buffer exchange. The buffer exchange may be achieved using dialysis cassettes or a TFF system. TFF may be used to concentrate and buffer-exchange the resulting nascent LNP immediately after formation via the T-mix process. The diafiltration process is a continuous operation, keeping the volume constant by adding appropriate buffer at the same rate as the permeate flow.A dministration

[0220] The immunogenic compositions or vaccines of the present disclosure can be formulated for administration in any way known in the art of drug delivery, for example, orally, parenterally, intravenously, intramuscularly, subcutaneously, intradermally, transdermally, intrathecally, submucosally, sublingually, rectally, vaginally, etc. In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for sublingual administration, intramuscular administration, intradermal administration, subcutaneous administration, intravenous administration, intranasal administration, administration by inhalation, or intraperitoneal administration.

[0221] In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular. In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for sublingual administration. In some embodiments, the immunogenic composition or vaccine is formulated for intramuscular injection. The immunogenic composition or vaccine of the present disclosure may also be formulated for intranasal or inhalation administration. The immunogenic composition or vaccine of the present disclosure can also be formulated for any other intended route of administration.

[0222] In some embodiments, the immunogenic composition or vaccine of the present disclosure is formulated for intradermal injection, intranasal administration or intramuscular injection. General considerations in the formulation and manufacture of pharmaceutical agents for administration by these routes may be found, for example, in Remington’s Pharmaceutical Sciences, 19thed., Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference. At present the oral or nasal spray or aerosol route (e.g., by inhalation) are most commonly used to deliver therapeutic agents directly to the lungs and respiratory system. In some embodiments, the immunogenic composition or vaccine of the present disclosure is administered using a device that delivers a metered dosage of the vaccine composition. Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patent No. 4,886,499, U.S. Patent No. 5,190,521, U.S. Patent No. 5,328,483, U.S. Patent No. 5,527,288, U.S. PatentNo. 4,270,537, U.S. Patent No. 5,015,235, U.S. Patent No. 5,141,496, U.S. PatentNo. 5,417,662, all of which are incorporated herein by reference. Intradermal compositions may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO 1999 / 34850, incorporated herein by reference, and functional equivalents thereof.

[0223] Also suitable are jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis. Jet injection devices are described for example in U.S. Patent No. 5,480,381, U.S. PatentNo. 5,599,302, U.S. PatentNo. 5,334,144, U.S. Patent No. 5,993,412, U.S. PatentNo. 5,649,912, U.S. PatentNo. 5,569,189, U.S. PatentNo. 5,704,911, U.S. PatentNo. 5,383,851, U.S. Patent No. 5,893,397, U.S. Patent No. 5,466,220, U.S. Patent No. 5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Patent No. 5,520,639, U.S.Patent No. 4,596,556, U.S. Patent No. 4,790,824, U.S. Patent No. 4,941,880, U.S. Patent No. 4,940,460, WO1997 / 37705, and WO1997 / 13537, all of which are incorporated herein by reference. Additionally, conventional syringes may be used in the classical Mantoux method of intradermal administration.

[0224] Preparations for parenteral administration typically include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0225] The immunogenic compositions or vaccines of the present disclosure may be packaged in a container, such as a prefilled syringe, a vial, or an autoinjector. In some embodiments, the immunogenic compositions or vaccines of the present disclosure are packaged in a prefilled syringe. In some embodiments, the immunogenic compositions or vaccines of the present disclosure are packaged in a vial. In some embodiments, the immunogenic compositions or vaccines of the present disclosure are packaged in an autoinjector. In other embodiments, the immunogenic compositions or vaccines of the present disclosure are packaged cartridges for patient-friendly autoinjector and infusion pump devices.

[0226] Prefilled syringes provide several advantages over other types of packages, such as convenience, affordability, accuracy, sterility, and safety. Accordingly, in some embodiments, provided herein is a pre-filled syringe comprising about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL volume of any of the immunogenic compositions or vaccines disclosed herein.Methods of Use

[0227] Also provided herein are methods of administering the immunogenic compositions or vaccines described herein to a subject. The methods may be used to vaccinate a subject to prevent an influenza A virus infection in the subject, to decrease the subject’s likelihood of getting an influenza A virus infection, or to reduce the subject’s likelihood of getting serious illness from an influenza A virus infection. Likewise, the present disclosure provides any of the immunogeniccompositions or vaccine compositions described herein for use in vaccinating a subject against an influenza A virus infection. Also disclosed is use of any of the immunogenic compositions as described herein for the manufacture of a vaccine for use in vaccinating a subject against an influenza A virus infection. In some embodiments, the vaccination method or use comprises administering to a subject in need thereof an immunologically effective amount of any of the vaccines described herein.

[0228] As used herein, the term “immunologically effective amount” or “therapeutically effective amount” means an amount sufficient to immunize a subject. In some embodiments, the immunologically effective amount or therapeutically effective amount is capable of eliciting protective immunity against an infectious disease, which include, but are not limited to, an increase of antibody titers and / or T cell immunity against an infectious disease. In some embodiments, an immunologically effective amount or therapeutically effective amount of the vaccine or immunogenic composition as disclosed herein increases protective immunity in a subject by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, including all values and subranges therebetween, when compared with a subject who is not administered with the vaccine or immunogenic composition as disclosed herein.

[0229] Accordingly, in some embodiments, the disclosure provides a method of immunizing a subject comprising administering to the subject in need thereof any of the immunogenic compositions or vaccines described herein. In some embodiments, the disclosure provides a method of immunizing a subject comprising administering to the subject in need thereof an immunologically effective amount any of the immunogenic compositions or vaccines described herein. As used herein, “immunize” or “immunizing” means to induce in a subject a protective immune response against an influenza A virus infection. Likewise, the present disclosure provides any of the immunogenic compositions or vaccine compositions described herein for use in immunizing a subject against an influenza A virus infection. Also disclosed is use of any of the immunogenic compositions as described herein, for the manufacture of a vaccine for use in immunizing a subject against an influenza A virus infection.

[0230] In some embodiments, the method or use prevents influenza A virus infection or disease caused by the influenza A virus infection in the subject. In some embodiments, the methodor use decreases the subject’s likelihood of getting an influenza A virus infection by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, including all values and subranges therebetween, when compared with a subject who is not administered with the vaccine or immunogenic composition as disclosed herein. In some embodiments, the method or use reduces the subject’s likelihood of getting serious illness from the influenza A virus infection by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, including all values and subranges therebetween, when compared with a subject who is not administered with the vaccine or immunogenic composition as disclosed herein. In some embodiments, the method or use raises a protective immune response in the subject. In some embodiments, the protective immune response is an antibody response.

[0231] For pandemic influenza vaccines and immunogenic compositions, like vaccines and immunogenic compositions comprising the modified H5 influenza HA polypeptides or nucleic acids (e.g., mRNA) encoding the same, as described herein, the hemagglutinin inhibition (HI) antibody titer may be an acceptable surrogate marker of activity that is reasonable likely to predict clinical benefit. For example, seroprotection based on HI antibody titers can be used as a surrogate parameter for protection provided by a given pandemic influenza vaccine or immunogenic composition. As used herein, “seroprotection” refers to a HI antibody titer of at least 1 :40 following administration of an immunogenic composition or vaccine as described herein. Typically, the HI antibody titer is measured using a HI assay, such as the HI assay used in Example 4 of this application. Seroprotection may be measured at an appropriate time following administration of an immunogenic composition or vaccine, depending on variables such as the dosage, administration schedule, and / or subject. In certain embodiments, seroprotection is measured at least 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, 55, 65, 70, 75, 80, 85, 90, 95, or 100 days following administration of an immunogenic composition or vaccine, including all values and subranges therebetween. In certain embodiments, seroprotection is measured at day 43 following administration of an immunogenic composition or vaccine.

[0232] In some embodiments, the method or use induces influenza seroprotection in a subject by at least day 22, day 24, day 30, or day 43 following the administration of a first dose of an immunogenic composition or vaccine as described herein. In some embodiments, the method or use induces influenza seroprotection in at least about 40%, 45%, 50%, 60%, 65%, 70%, or 75%, of subjects, including all values and subranges therebetween, as measured at day 22 following the administration of a first dose of an immunogenic composition or vaccine as described herein. In some embodiments, the method or use induces influenza seroprotection in at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% of subjects, including all values and subranges therebetween, as measured at day 43 following the administration of a first dose of an immunogenic composition or vaccine as described herein. In some embodiments, the method or use induces influenza seroprotection in at least about 90-97%, 90-94%, 94-97%, or 96-97.2% as measured at day 43 following the administration of a first dose of an immunogenic composition or vaccine as described herein. In some embodiments, the method or use induces influenza seroprotection in at least about 70% of subjects aged 18-64 or 18-60 following the administration of a first dose of an immunogenic composition or vaccine as described herein, optionally, wherein the seroprotection is measured at day 43 following administration of the first dose of the immunogenic composition or vaccine as described herein. In some embodiments, the method or use induces influenza seroprotection in at least about 60% of subjects at least 65 years of age following the administration of a first dose of an immunogenic composition or vaccine as described herein, optionally, wherein the seroprotection is measured at day 43 following administration of the first dose of the immunogenic composition or vaccine as described herein.

[0233] Seroconversion is an immunological parameter used to measure the antibody responses in subjects who receive a vaccine or immunogenic composition. As used herein, “seroconversion” refers to 1) a change in HI antibody titer from a pre-vaccination HI antibody titer of less than 1 : 10 to a post-vaccination HI antibody titer of at least 1 :40 or 2) a change in HI antibody titer from a pre-vaccination HI antibody titer of at least 1 : 10 to a post-vaccination HI antibody titer at least four times higher than the pre-vaccination HI antibody titer. Typically, the HI antibody titer is measured using a HI assay, such as the HI assay used in Example 4 of this application. Seroconversion may be measured at an appropriate time following administration of an immunogenic composition or vaccine, depending on variables such as the dosage, administration schedule, and / or subject. In certain embodiments, seroconversion is measured at least 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, 55, 65, 70, 75, 80, 85, 90, 95, or 100 days following administration of an immunogenic composition or vaccine, including all values and subranges therebetween. In certain embodiments, seroconversion is measured at day 43 following administration of an immunogenic composition or vaccine.

[0234] In some embodiments, the method or use induces influenza seroconversion in at least about 30%, 40%, 50%, 60%, or 70%, of subjects, including all values and subranges therebetween, at day 22 following the administration of a first dose of an immunogenic composition or vaccine as described herein. In some embodiments, the method or use induces influenza seroconversion in at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% of subjects, including all values and subranges therebetween, at day 43 following the administration of a first dose of an immunogenic composition or vaccine as described herein. In some embodiments, the method or use induces influenza seroconversion in at least about 75-97%, 75-85%, 85-97%, or 90- 97.2% of subjects at day 43 following the administration of a first dose of an immunogenic composition or vaccine as described herein. In some embodiments, the method or use induces influenza seroconversion in at least about 40% of subjects aged 18-64 or 18-60 following the administration of a first dose of an immunogenic composition or vaccine as described herein, optionally, wherein the seroconversion is measured at day 43 following administration of the first dose of the immunogenic composition or vaccine as described herein. In some embodiments, the method or use induces influenza seroprotection in at least about 30% of subjects at least 65 years of age following the administration of a first dose of an immunogenic composition or vaccine as described herein, optionally, wherein the seroconversion is measured at day 43 following administration of the first dose of the immunogenic composition or vaccine as described herein.

[0235] In some embodiments, the HI antibody geometric mean titer (GMT) following administration of an immunogenic composition or vaccine, as described herein, is at least 9, 10, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, or 100, including all values and subranges therebetween. Typically, the HI antibody titer is measured using a HI assay, such as the HI assay used in Example 4 of this application. The GMT is commonly used in the art to measure antibody titers, which typically do not follow a linear scale. The geometric mean is typically calculated by taking the logarithm (e.g., base 10 logarithm) of each titer value, calculating the average of the log-transformed titer values, and converting the log-based average back to theoriginal scale. In certain embodiments, the HI antibody GMT is measured at least 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, 55, 65, 70, 75, 80, 85, 90, 95, or 100 days following administration of an immunogenic composition or vaccine, including all values and subranges therebetween. In certain embodiments the HI antibody GMT is about 5-75, 9-60, 10-55, 9.7 to 17.1, 26.5 to 41.2, or 31.3 to 54.9. In certain embodiments, the HI antibody GMT is measured at day 43 following administration of an immunogenic composition or vaccine.

[0236] Also provided, in some embodiments, is a method of reducing one or more symptoms of an influenza A virus infection comprising administering to a subject in need thereof any of the immunogenic compositions or vaccines described herein. In some embodiments, provided herein is a method of reducing one or more symptoms of an influenza A virus infection comprising administering to a subject in need thereof a prophylactically effective amount of any of the immunogenic compositions or vaccines described herein.

[0237] The present disclosure provides any of the immunogenic compositions or vaccine compositions described herein for use in reducing one or more symptoms of an influenza A virus infection. Also disclosed is any of the immunogenic compositions as described herein, for the manufacture of a vaccine for use in reducing one or more symptoms of an influenza A virus infection in a subject.

[0238] In some embodiments, the method or use of the present disclosure reduces one or more symptoms of an influenza A virus infection by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, including all values and subranges therebetween, when compared with a subject who is not administered with the vaccine or immunogenic composition as disclosed herein.

[0239] In some embodiments, the immunogenic composition or vaccine, and an optional adjuvant, may be administered prior to or after development of one or more symptoms of the influenza A virus infection. That is, in some embodiments, the immunogenic compositions or vaccines described herein may be administered prophylactically to prevent the influenza A virus infection or ameliorate the symptoms of a potential influenza A virus infection.

[0240] In some embodiments, the subject is at risk of infection if the subject will be in contact with other individuals or other animals known or suspected to have been infected with an influenza A virus infection and / or if the subject will be present in a location in which influenza A virus infection is known or thought to be prevalent or endemic. In some embodiments, the immunogenic composition or vaccine is administered to a subject suffering from an influenza A virus infection, or the subject is displaying one or more symptoms commonly associated with an influenza A virus infection. In some embodiments, the subject is known or believed to have been exposed to an influenza A virus infection.

[0241] Vaccines or immunogenic compositions in accordance with the present disclosure may be administered in any amount or dose appropriate to achieve a desired outcome. In some embodiments, the desired outcome is induction of a lasting adaptive immune response against the influenza A virus. In some embodiments, the desired outcome is reduction in intensity, severity, and / or frequency, and / or delay of onset of one or more symptoms associated with influenza A virus infection. In some embodiments, the desired outcome is to provide vaccines with consistent RNA quality. The dose required may vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration.

[0242] In some embodiments, the immunogenic compositions or vaccines described herein are administered to subjects, wherein the subjects can be any member of the animal kingdom. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human subject is an avian (e.g., a chicken or a bird). In some embodiments, the non-human subject is a reptile, an amphibian, a fish, an insect, and / or a worm. In some embodiments, the non-human subject is a mammal (e.g., a ferret, a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig).

[0243] In some embodiments, the immunogenic compositions or vaccines described herein are administered to a human subject. In some embodiments, a human subject is 6 months of age or older, 6 months through 35 months of age, at least two years of age, at least 3 years of age, 36 months through 8 years of age, 9 years of age or older, at least 6 months of age and less than 5 years of age, at least 6 months of age and less than 18 years of age, or at least 3 years of age and less than 18 years of age. In some embodiments, the human subject is an infant (less than 36 months). In some embodiments, the human subject is a child or adolescent (less than 18 years ofage). In some embodiments, the human subject is a child of at least 6 months of age and less than 5 years of age. In some embodiments, the human subject is at least 5 years of age and less than 60 years of age. In some embodiments, the human subject is at least 5 years of age and less than 65 years of age. In some embodiments, the human subject is elderly (at least 60 years of age or at least 65 years of age). In some embodiments, the human subject is at least 65 years of age. In some embodiments, the human subject is a non-elderly adult (at least 18 years of age and less than 65 years of age or at least 18 years of age and less than 60 years of age).

[0244] The methods and uses of the immunogenic compositions or vaccines described herein include administration of a single dose to a subject (i.e., no booster dose). In some embodiments, the methods and uses of the immunogenic compositions or vaccines described herein include prime-boost vaccination strategies. Prime-boost vaccination comprises administering a priming immunogenic composition or vaccine and then, after a period of time has passed, administering to the subject a boosting immunogenic composition or vaccine. The immune response is “primed” upon administration of the priming immunogenic composition or vaccine and is “boosted” upon administration of the boosting immunogenic composition or vaccine. The priming immunogenic composition or vaccine can include an immunogenic composition or vaccine as described herein and an optional adjuvant. Likewise, the boosting immunogenic composition or vaccine can include an immunogenic composition or vaccine as described herein and an optional adjuvant. The priming immunogenic composition or vaccine can be, but need not be, the same as the boosting immunogenic composition or vaccine. Administration of the boosting immunogenic composition or vaccine is generally weeks or months after administration of the priming immunogenic composition or vaccine, preferably about 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks. In certain embodiments, the recipient of the prime-boost vaccination is a naive subject, typically a naive infant or child.

[0245] Accordingly, in some embodiments, the immunogenic composition or vaccine of the present disclosure is administered as part of a prime-boost vaccination strategy to a subject, such as an infant or child or an adult of 18 years or older. In some embodiments, the prime-boost vaccination strategy comprises administering a boosting immunogenic composition or vaccine about 2-4 weeks, such as about 2 weeks, about 3 weeks, or about 4 weeks, after administration of a priming immunogenic composition or vaccine. In some embodiments, the boosting immunogenic composition or vaccine is the same as the priming immunogenic composition orvaccine. In some embodiments, the boosting immunogenic composition or vaccine is different from the priming immunogenic composition or vaccine.

[0246] The immunogenic composition or vaccine can be administered using any suitable route of administration, including, for example, parenteral delivery, as discussed above. In some embodiments, the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.Other Applications

[0247] The modified H5 influenza HA polypeptides disclosed herein may have other applications, such as use in an in vitro method for preparation of a trimeric H5 influenza HA polypeptide complex. Accordingly, provided herein is an in vitro method of preparing a trimeric H5 influenza HA polypeptide complex, the method comprising expressing an artificial nucleic acid molecule encoding any of the modified H5 influenza HA polypeptides disclosed herein in a host cell to prepare the trimeric H5 influenza HA polypeptide complex. In some embodiments, the artificial nucleic acid molecule encoding the modified H5 influenza HA polypeptide is part of a vector. In other embodiments, the expression of the modified H5 influenza HA polypeptide is by culturing the host cell in cell culture medium. Thus, in some embodiments, provided herein is an in vitro method of preparing a trimeric H5 influenza HA polypeptide complex, the method comprising culturing the host cell in a cell culture medium, and expressing the trimeric H5 influenza HA polypeptide complex. In some embodiments, the in vitro method disclosed herein further comprises a step of purifying the trimeric H5 influenza HA polypeptide complex from the cell culture medium.

[0248] In certain embodiments, the trimeric H5 influenza HA polypeptide complex prepared according to the in vitro method disclosed herein is more stable in prefusion conformation as compared to a trimeric H5 influenza HA polypeptide complex prepared from the corresponding wild-type H5 influenza HA polypeptide without the one or more modifications. In some embodiments, stability in prefusion conformation is measured by an increased binding of the trimeric H5 influenza HA polypeptide complex to a stem region-specific antibody (e.g., CR9114) as compared to a trimeric H5 influenza HA polypeptide complex prepared from the corresponding wild-type H5 influenza HA polypeptide without the one or more modifications. In certain embodiments, stability in prefusion conformation is measured by an increased binding ratio of a stem region-specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) ascompared to a trimeric H5 influenza HA polypeptide complex prepared from the corresponding wild-type H5 influenza HA polypeptide without the one or more modifications. In some embodiments, stability in prefusion conformation is measured by an increased binding of the trimeric H5 influenza HA polypeptide complex to a stem region-specific antibody (e.g., CR9114) and an increased binding ratio of a stem region-specific antibody (e.g., CR9114) to an RBS- specific antibody (e.g., R95-1D05) as compared to a trimeric H5 influenza HA polypeptide complex prepared from the corresponding wild-type H5 influenza HA polypeptide without the one or more modifications.

[0249] In certain embodiments, the trimeric H5 influenza HA polypeptide complex prepared according to the in vitro method disclosed herein is more immunogenic as compared to a trimeric H5 influenza HA polypeptide complex prepared from the corresponding wild-type H5 influenza HA polypeptide without the one or more modifications. Immunogenicity can be measured using any methods known in the art. For instance, in some embodiments, immunogenicity is measured using hemagglutination inhibition assay (HAI).First Set of Representative Embodiments of the Present Disclosure

[0250] Embodiment 1 : An artificial messenger ribonucleic acid (mRNA) encoding a modified H5 influenza hemagglutinin (HA) polypeptide, wherein the modified H5 influenza HA polypeptide comprises an amino acid substitution at amino acid position 447 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0251] Embodiment 2.: The artificial mRNA of Embodiment 1, wherein the amino acid substitution at amino acid position 447 is M447I.

[0252] Embodiment 3: The artificial mRNA of Embodiment 1 or 2, wherein the modified H5 influenza HA polypeptide is from an influenza A virus subtype H5N1.

[0253] Embodiment 4: The artificial mRNA of Embodiment 1 or 2, wherein the modified H5 influenza HA polypeptide is from influenza A virus strain A / Astrakhan / 3212 / 2020.

[0254] Embodiment 5: The artificial mRNA of any one of Embodiments 1-4, wherein the modified H5 influenza HA polypeptide comprises a polybasic cleavage site at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, optionally wherein the polybasic cleavage site comprises the amino acid sequence of SEQ ID NO: 8.

[0255] Embodiment 6: The artificial mRNA of any one of Embodiments 1-5, wherein the modified H5 influenza HA polypeptide further comprises one or more mutations in the polybasic cleavage site such that the polybasic cleavage site is converted to a monobasic cleavage site, optionally wherein the polybasic cleavage site is converted to the monobasic cleavage site by removing one or more, optionally all but one, basic amino acids, optionally wherein the polybasic cleavage site is converted to the monobasic cleavage site by replacing the polybasic cleavage site with the amino acid sequence TR.

[0256] Embodiment 7: The artificial mRNA of any one of Embodiments 1-6, wherein the modified H5 influenza HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0257] Embodiment 8: The artificial mRNA of Embodiment 7, wherein the modified H5 influenza HA polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0258] Embodiment 9: The artificial mRNA of any one of Embodiments 1-8, comprising a 5'- cap structure and / or a 3'-poly(A) sequence.

[0259] Embodiment 10: The artificial mRNA of any one of Embodiments 1-9, comprising at least one chemically modified nucleotide and / or a phosphorothioate bond.

[0260] Embodiment 11 : The artificial mRNA of Embodiment 10, wherein the at least one chemically modified nucleotide comprises a pseudouridine, a 2'-fluoro ribonucleotide, or a 2'- methoxy ribonucleotide, optionally wherein the pseudouridine is a N1 -methylpseudouridine.

[0261] Embodiment 12: The artificial mRNA of any one of Embodiments 1-11, comprising a nucleic acid sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 3.

[0262] Embodiment 13: The artificial mRNA of Embodiment 12, comprising the nucleic acid sequence of SEQ ID NO: 3.

[0263] Embodiment 14: A composition comprising the artificial mRNA of any one of Embodiments 1-13 encapsulated in a lipid nanoparticle (LNP).

[0264] Embodiment 15: The composition of Embodiment 14, wherein the LNP comprises a cationic lipid.

[0265] Embodiment 16: The composition of Embodiment 15, wherein the cationic lipid comprises or is OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, (4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyldecanoate) (ALC-0315), or IM-001.

[0266] Embodiment 17: The composition of Embodiment 15 or 16, wherein the LNP further comprises a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0267] Embodiment 18: The composition of Embodiment 17, wherein: a) the PEGylated lipid comprises or is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2000); and / or b) the cholesterol -based lipid comprises or is cholesterol; and / or c) the helper lipid comprises or is dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0268] Embodiment 19: The composition of Embodiment 17 or 18, wherein: a) the cationic lipid is present at a molar ratio between about 35% and about 55%;; b) the PEGylated lipid is present at a molar ratio between about 0.25% and about 2.75%; c) the cholesterol-based lipid is present at a molar ratio between about 20% and about 45%; and d) the helper lipid is present at a molar ratio between about 5% and about 35%, wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0269] Embodiment 20: The composition of Embodiment 19, wherein: a) the cationic lipid is present at a molar ratio of about 40%; b) the PEGylated lipid is present at a molar ratio of about 1.5%; c) the cholesterol-based lipid is present at a molar ratio of about 28.5%; and d) the helper lipid is present at a molar ratio of about 30%, wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0270] Embodiment 21 : The composition of Embodiment 19, wherein the artificial mRNA encodes the modified H5 influenza HA polypeptide of SEQ ID NO: 2, and wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%.

[0271] Embodiment 22: The composition of any one of Embodiments 14-21, wherein the composition is an immunogenic composition.

[0272] Embodiment 23: A modified H5 influenza hemagglutinin (HA) polypeptide comprising an amino acid substitution M447I as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0273] Embodiment 24: The modified H5 influenza HA polypeptide of Embodiment 23, wherein the modified H5 influenza HA polypeptide is from an influenza A virus subtype H5N1.

[0274] Embodiment 25: The modified H5 influenza HA polypeptide of Embodiment 23, wherein the modified H5 influenza HA polypeptide is from influenza A virus strain A / Astrakhan / 3212 / 2020.

[0275] Embodiment 26: The modified H5 influenza HA polypeptide of any one of Embodiments 23-25, wherein the modified H5 influenza HA polypeptide comprises a polybasic cleavage site at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, optionally wherein the polybasic cleavage site comprises the amino acid sequence of SEQ ID NO: 8.

[0276] Embodiment 27: The modified H5 influenza HA polypeptide of Embodiment 26, wherein the modified H5 influenza HA polypeptide further comprises one or more mutations in the polybasic cleavage site such that the polybasic cleavage site is converted to a monobasic cleavage site, optionally wherein the polybasic cleavage site is converted to the monobasic cleavage site by removing one or more, optionally all but one, basic amino acids, optionally wherein the polybasic cleavage site is converted to the monobasic cleavage site by replacing the polybasic cleavage site with the amino acid sequence TR.

[0277] Embodiment 28: The modified H5 influenza HA polypeptide of any one of Embodiments 23-27, comprising the amino acid sequence of SEQ ID NO: 2.

[0278] Embodiment 29: A trimeric influenza A HA polypeptide complex, comprising three copies of the modified H5 influenza HA polypeptide of any one of Embodiments 23-28.

[0279] Embodiment 30: The trimeric influenza A HA polypeptide complex of Embodiment29, wherein the trimeric influenza A HA polypeptide complex is more immunogenic as compared to a trimeric influenza A HA polypeptide complex prepared from a control H5 influenza HA polypeptide comprising the amino acid of SEQ ID NO: 1.

[0280] Embodiment 31 : The trimeric influenza A HA polypeptide complex of Embodiment30, wherein immunogenicity is measured using a hemagglutination-inhibition assay.

[0281] Embodiment 32: An artificial nucleic acid encoding the modified H5 influenza HA polypeptide of any one of Embodiments 23-28.

[0282] Embodiment 33: The artificial nucleic acid of Embodiment 32, wherein the artificial nucleic acid comprises at least one chemically modified nucleotide and / or a phosphorothioate bond.

[0283] Embodiment 34: A vector comprising the artificial nucleic acid of Embodiment 32 or 33.

[0284] Embodiment 35: The vector of Embodiment 34, wherein the vector is a messenger RNA (mRNA) production vector.

[0285] Embodiment 36: A host cell comprising the vector of Embodiment 34 or 35.

[0286] Embodiment 37: A composition comprising the modified H5 influenza HA polypeptide of any one of Embodiments 23-28, the trimeric influenza A HA polypeptide complex of any one of Embodiments 29-31, the artificial nucleic acid of Embodiment 32 or 33, or the vector of Embodiment 34 or 35.

[0287] Embodiment 38: The composition of Embodiment 37, wherein the composition is an immunogenic composition.

[0288] Embodiment 39: A vaccine comprising the immunogenic composition of Embodiment 22 or 38, and a pharmaceutically acceptable carrier.

[0289] Embodiment 40: The vaccine of Embodiment 39, further comprising an adjuvant.

[0290] Embodiment 41 : A method of immunizing a subject, the method comprising administering to the subject in need thereof the vaccine of Embodiment 39 or 40.

[0291] Embodiment 42: The method of Embodiment 41, wherein the method prevents an influenza A virus infection in the subject, decreases the subject’s likelihood of getting an influenza A virus infection, or reduces the subject’s likelihood of getting serious illness from an influenza A virus infection.

[0292] Embodiment 43 : The method of Embodiment 41 or 42, wherein the subject is a human.

[0293] Embodiment 44: The method of Embodiment 43, wherein the human is 6 months of age or older, less than 18 years of age, at least 6 months of age and less than 18 years of age, at least 18 years of age and less than 65 years of age, at least 6 months of age and less than 5 years of age, at least 5 years of age and less than 65 years of age, at least 60 years of age, or at least 65 years of age.

[0294] Embodiment 45: The method of any one of Embodiments 41-44, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0295] Embodiment 46: A method of reducing one or more symptoms of an influenza A virus infection, the method comprising administering to a subject in need thereof the vaccine of Embodiment 39 or 40.

[0296] Embodiment 47: An in vitro method of preparing a trimeric influenza A HA polypeptide complex, the method comprising culturing the host cell of Embodiment 36 in a cell culture medium, and expressing the trimeric influenza A HA polypeptide complex.

[0297] Embodiment 48: The in vitro method of Embodiment 47, wherein the trimeric influenza A HA polypeptide complex is more immunogenic as compared to a trimeric influenza A HA polypeptide complex prepared from a control H5 influenza HA polypeptide comprising the amino acid of SEQ ID NO: 1.

[0298] Embodiment 49: The in vitro method of Embodiment 48, wherein immunogenicity is measured using a hemagglutination-inhibition assay.

[0299] Embodiment 50: The in vitro method of any one of Embodiments 47-49, further comprising a step of purifying the trimeric influenza A HA polypeptide complex from the cell culture medium.Second Set of Representative Embodiments of the Present Disclosure

[0300] Embodiment 2.1: An artificial messenger ribonucleic acid (mRNA) encoding a modified H5 influenza hemagglutinin (HA) polypeptide, wherein the modified H5 influenza HA polypeptide comprises an amino acid substitution at amino acid position 447 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0301] Embodiment 2.2: The artificial mRNA of Embodiment 2.1, wherein the amino acid substitution at amino acid position 447 is M447I .

[0302] Embodiment 2.3 : The artificial mRNA of Embodiment 2.1 or 2.2, wherein the modified H5 influenza HA polypeptide is from an influenza A virus subtype H5N1, or is from influenza A virus strain A / Astrakhan / 3212 / 2020.

[0303] Embodiment 2.4: The artificial mRNA of any one of Embodiments 2.1-2.3, wherein:i) the modified H5 influenza HA polypeptide comprises a polybasic cleavage site at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, optionally wherein the polybasic cleavage site comprises the amino acid sequence of SEQ ID NO: 8; or ii) the modified H5 influenza HA polypeptide further comprises one or more mutations in a polybasic cleavage site located at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, optionally wherein the one or more mutations comprise removing one or more, optionally all but one, basic amino acids, optionally wherein the one or more mutations converts the polybasic cleavage site to a monobasic cleavage site.

[0304] Embodiment 2.5: The artificial mRNA of any one of Embodiments 2.1-2.4, wherein the modified H5 influenza HA polypeptide comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0305] Embodiment 2.6: The artificial mRNA of any one of Embodiments 2.1-2.5, comprising: i) a 5'-cap structure and / or a 3'-poly(A) sequence; and / or ii) at least one chemically modified nucleotide and / or a phosphorothioate bond, optionally wherein the at least one chemically modified nucleotide comprises a pseudouridine, a 2'-fluoro ribonucleotide, or a 2'-methoxy ribonucleotide, optionally wherein the pseudouridine is a N1 -methylpseudouridine.

[0306] Embodiment 2.7: The artificial mRNA of any one of Embodiments 2.1-2.6, comprising a nucleic acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 3.

[0307] Embodiment 2.8: A composition comprising the artificial mRNA of any one of Embodiments 2.1-2.7 encapsulated in a lipid nanoparticle (LNP), optionally: i) wherein the LNP comprises a cationic lipid, optionally wherein the cationic lipid comprises or is OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4- E10, GL-HEPES-E3-E12-DS-3-E14, (4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyldecanoate) (ALC-0315), or IM-001; and / orii) wherein the LNP further comprises a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid; optionally wherein: a) the PEGylated lipid comprises or is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000); and / or b) the cholesterol-based lipid comprises or is cholesterol; and / or c) the helper lipid comprises or is dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). and / or wherein: a) the cationic lipid is present at a molar ratio between about 35% and about 55%, or about 40%; b) the PEGylated lipid is present at a molar ratio between about 0.25% and about 2.75%, or about 1.5%; c) the cholesterol-based lipid is present at a molar ratio between about 20% and about 45%, or about 28.5%; and d) the helper lipid is present at a molar ratio between about 5% and about 35%, or about 30%, wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0308] Embodiment 2.9: A modified H5 influenza hemagglutinin (HA) polypeptide comprising an amino acid substitution at amino acid position 447 as indexed by reference to the amino acid sequence of SEQ ID NO: 1, optionally comprising an amino acid substitution M447I as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0309] Embodiment 2.10: The modified H5 influenza HA polypeptide of Embodiment 2.9, wherein: i) the modified H5 influenza HA polypeptide is from an influenza A virus subtype H5N1; or ii) the modified H5 influenza HA polypeptide is from influenza A virus strain A / Astrakhan / 3212 / 2020; or iii) the modified H5 influenza HA polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0310] Embodiment 2.11: A trimeric influenza A HA polypeptide complex, comprising three copies of the modified H5 influenza HA polypeptide of Embodiment 2.9 or 2.10, optionally wherein the trimeric influenza A HA polypeptide complex is more immunogenic as compared to a trimeric influenza A HA polypeptide complex prepared from a control H5 influenza HA polypeptide comprising the amino acid of SEQ ID NO: 1, further optionally wherein immunogenicity is measured using a hemagglutination-inhibition assay.

[0311] Embodiment 2.12: A vaccine comprising the artificial mRNA of Embodiments 2.1-2.7, the composition of Embodiment 2.8, the modified H5 influenza HA polypeptide of Embodiment 2.9 or 2.10, or the trimeric influenza A HA polypeptide complex of Embodiment 2.11, and a pharmaceutically acceptable carrier, optionally further comprising an adjuvant.

[0312] Embodiment 2.13: The vaccine of Embodiment 2.12 for use in a method of immunizing a subject or reducing one or more symptoms of an influenza A virus infection.

[0313] Embodiment 2.14: An in vitro method of preparing a trimeric influenza A HA polypeptide complex, the method comprising culturing a host cell comprising the artificial mRNA of any one of Embodiments 2.1-2.7, and expressing the trimeric influenza A HA polypeptide complex, optionally further comprising a step of purifying the trimeric influenza A HA polypeptide complex from the cell culture medium.

[0314] Embodiment 2.15: The in vitro method of Embodiment 2.14, wherein the trimeric influenza A HA polypeptide complex is more immunogenic as compared to a trimeric influenza A HA polypeptide complex prepared from a control H5 influenza HA polypeptide comprising the amino acid of SEQ ID NO: 1, optionally wherein immunogenicity is measured using a hemagglutination-inhibition assay.

[0315] Embodiment 2.16: The vaccine of Embodiment 2.12 for use in a method for treatment of the human or animal body.

[0316] Embodiment 2.17: The vaccine of Embodiment 2.12 for use in a method for immunization of the human or animal body.

[0317] Embodiment 2.18: The vaccine of Embodiment 2.12 for use in a method for immunizing a subject from or reducing one or more symptoms of an influenza virus infection.

[0318] Embodiment 2.19: The vaccine of Embodiment 2.12 for use in a method for immunizing a subject from or reducing one or more symptoms of an influenza A virus infection.

[0319] Embodiment 2.20: Use of any one or any combination of (i) the artificial mRNA of Embodiments 2.1-2.7, (ii) the composition of Embodiment 2.8, (iii) the modified H5 influenza HA polypeptide of Embodiment 2.9 or 2.10, (iv) the trimeric influenza A HA polypeptide complex of Embodiment 2.11, optionally with a pharmaceutically acceptable carrier, for the manufacture of medicament for immunizing a subject from or reducing one or more symptoms of an influenza A virus infection.EXAMPLES

[0320] The following examples are to be considered illustrative and not limiting on the scope of the present disclosure described above.Example 1. Design and Generation of a Modified H5 Influenza Hemagglutinin (HA) Polypeptide

[0321] This example describes the design and generation of a representative modified H5 influenza HA polypeptide.

[0322] The HA polypeptide of influenza A virus strain A / Astrakhan / 3212 / 2020, an influenza A virus subtype H5, contains 567 amino acids and has the amino acid sequence set forth in SEQ ID NO: 1 :A7 / A7E / . / . / .N / ES7.FWSDQICIGYHANNSTEQVDTIMEI<NVTVTHAQDILEI<T HNGKLCDLNGVKPLILKDCSVAGWLLGNPMCDEFIRVPEWSYIVERANP ANDLCYPGSLNDYEELKHLLSRINHFEKILIIPKSSWPNHETSLGVSAACPY QGAPSFFRNVVWLII<I<NDAYPTII<ISYNNTNREDLLILWGIHHSNNAEEQT NLYKNPTTYISVGTSTLNQRLVPKIATRSQVNGQRGRMDFFWTILKPDDA IHFESNGNFIAPEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSM PFHNIHPLTIGECPKYVKSNKLVLATGLRNSPLREKRRKRGLFGAIAGFIE GGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDK MNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENER TLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRN GTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTAASSLALAIMMAGL SLWMCSNGSLQCRICI (SEQ ID NO: 1).

[0323] The first 16 amino acids of SEQ ID NO: 1 (in italic) constitute the signal peptide of the HA polypeptide and are not part of the mature protein. The amino acids at positions 341-345 of SEQ ID NO: 1 (KRRKR (SEQ ID NO: 8); in bold) constitute the polybasic cleavage site of the HA polypeptide, which makes influenza A virus A / Astrakhan / 3212 / 2020 strain highly pathogenic. In order to work with zoonotic highly pathogenic avian influenza (HP Al) strains, the polybasic cleavage site of the wild-type HA polypeptide generally needs to be mutated to create low pathogenic variants with a monobasic cleavage site, like the HA polypeptides of seasonal influenza viruses. To this end, the polybasic cleavage site of SEQ ID NO: 1 was mutated to a monobasic cleavage site by removing all but one basic amino acid and introducing a further modification in front of the remaining basic amino acid. Thus, the amino acid sequence LREKRRKRG (SEQ ID NO: 9; polybasic cleavage site in bold) in the wild-type HA polypeptide was modified to LRE77?G (SEQ ID NO: 10; monobasic cleavage site in bold and italic). The amino acid sequence of this monobasic version of the HA polypeptide of A / Astrakhan / 3212 / 2020 strain is provided below with the signal peptide in italic and the monobasic cleavage site in bold and italic.Af / A7E / . / . / .N / HS7.FWSDQICIGYHANNSTEQVDTIMEI<NVTVTHAQDILEI<T HNGKLCDLNGVKPLILKDCSVAGWLLGNPMCDEFIRVPEWSYIVERANP ANDLCYPGSLNDYEELKHLLSRINHFEKILIIPKSSWPNHETSLGVSAACPY QGAPSFFRNVVWLII<I<NDAYPTII<ISYNNTNREDLLILWGIHHSNNAEEQT NLYKNPTTYISVGTSTLNQRLVPKIATRSQVNGQRGRMDFFWTILKPDDA IHFESNGNFIAPEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSM PFHNIHPLTIGECPKYVKSNKLVLATGLRNSPLRE77?GLFGAIAGFIEGGW QGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFH DSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDY PQYSEEARLKREEISGVKLESIGTYQILSIYSTAASSLALAIMMAGLSLWM CSNGSLQCRICI (SEQ ID NO: 11).

[0324] An amino acid substitution was introduced into this monobasic version of the HA polypeptide at the amino acid position corresponding to amino acid position 447 of SEQ ID NO: 1 (which corresponds to amino acid position 444 of SEQ ID NO: 11) by replacing Met at this position with He, which resulted in a modified H5 influenza HA polypeptide with the amino acid sequence set forth in SEQ ID NO: 2. This mutation created a cavity filling effect in the stem regionof the HA polypeptide (FIG. 1), leading to an HA trimer that is more stable in the prefusion conformation. This modified H5 influenza HA polypeptide is also referred to herein as the M447I mutant.

[0325] An mRNA sequence encoding the M447I mutant on the monobasic cleavage backbone was then generated and the nucleic acid sequence is set forth in SEQ ID NO: 3.Example 2. In vitro Characterization of a Modified H5 Influenza HA Polypeptide

[0326] This example describes the in vitro conformation assessment of the modified H5 influenza HA polypeptide described in Example 1 (i.e., the M447I mutant).

[0327] Monoclonal antibodies to well-defined, structural epitopes were selected against the surface of H5 influenza HA polypeptide to ensure that the protein was properly folded and presented at the cell surface. A stem-region-specific antibody (CR9114), a receptor binding domain (RBD)-specific antibody (65C6), and an interprotomer interface binding antibody (FluA- 20) were used to investigate the conformation of the M447I mutant on the surface of cells using flow cytometry. The binding of 65C6 indicates the total surface expression and integrity of sialic acid binding pocket on surface expressed HA polypeptides. The binding of CR9114 indicates that the surface expressed HA polypeptides successfully presented a properly folded stem region and are in a prefusion state. The binding of FluA-20 indicates that the HA trimer is breathing and in an open state, whereas the absence of FluA-20 binding indicates that the HA trimer is in more of a closed state. For the purposes of this example, MFI stands for median fluorescence intensity and was calculated as median fluorescence intensity x percent positive cells for monoclonal antibody binding.

[0328] To assess the surface expression and presentation of both the wild-type (mono-base) HA polypeptide of A / Astrakhan / 3212 / 2020 (baseline, also called H5_Base) and the M447I mutant, 239FT cells were transfected with a single DNA plasmid (pDNA) or lipid nanoparticle (LNP)- encapsulated mRNA (mRNA) encoding each individual HA polypeptide. The LNP used to formulate the mRNA in this example contained GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid. One day after transfection, cells were harvested, washed, and stained for analysis by flow cytometry. All cells were first stained with live / dead dye to assess cell viability. 293FT cells expressing each construct were then divided into four staining reactions and stained with primary antibodies including CR9114, 65C6, and FluA-20 or left in staining buffer alone (Fc control). Allreactions were then stained with a secondary antibody against human IgG conjugated to Alexa Fluor 488. Cells were washed in staining buffer following each of the previously described steps. Cells were then analyzed for monoclonal antibody binding using a Cytek Aurora flow cytometer with a five-laser configuration. Data was analyzed using FLOWJO™ 10.8.1 (FlowJo LLC). Flow cytometry gating was conducted by gating on single, live, 293FT cell capable of binding to monoclonal antibodies of interest against the stem, RBS, and FluA-20 antibody.

[0329] The percent total 293FT cells positive for monoclonal antibody binding measured by flow cytometry is shown in FIG. 2A-2B. The graphs depict the percentage of live cells that bound to each individual monoclonal antibody targeting HA from 30,000 cells. Black bars depict 65C6 (RBD) binding, horizontal stripes depict CR9114 (stem) binding, vertical lines depict FluA-20 (interprotomer interface) binding, and bricks depict secondary antibody binding in the absence of a primary antibody (background). Construct names are listed on the x-axis and percent values are listed on top of the bar graphs. pDNA denotes that constructs were transfected as plasmid DNA and mRNA denotes that constructs were transfected with LNP-encapsulated mRNA. The wildtype HA polypeptide from A / Astrakhan / 3212 / 2020 (H5_Base) was used as the baseline. As shown in FIG. 2A-2B, the M447I mutant has high RBS and stem -region binding, similar to that of the wild-type (mono-base) A / Astrakhan / 3212 / 2020 HA polypeptide, indicating that it is conformationally correct at prefusion state. As also shown in FIG. 2A-2B, the M447I mutant has less binding with FluA-20 (interprotomer interface), suggesting a more closed head conformation than the wild-type A / Astrakhan / 3212 / 2020 HA polypeptide.

[0330] The assessment of surface expression levels of the M447I mutant on 293FT cells measured by flow cytometry is shown in FIG. 3A-3B. The graphs depict the MFI of live cells that bound to each individual monoclonal antibody targeting HA from 30,000 cells. Black bars depict 65C6 (RBD) binding, horizontal stripes depict CR9114 (stem) binding, vertical lines depict FluA- 20 (interprotomer interface) binding, and bricks depict secondary antibody binding in the absence of a primary antibody (background). Construct names are listed on the x-axis. pDNA denotes that constructs were transfected as plasmid DNA and mRNA denotes that constructs were transfected with LNP-encapsulated mRNA. As shown in FIG. 3A-3B, the M447I mutant exhibited a comparable surface expression level as the wild-type (mono-base) A / Astrakhan / 3212 / 2020 HA polypeptide and had less binding with FluA-20 (interprotomer interface).

[0331] The assessment of the ratio of stem to head binding monoclonal antibodies against the M447I mutant, measured by flow cytometry for both pDNA and mRNA transfections, is shown in FIG. 4. The graph depicts the normalized ratio of stem to head binding antibodies MFI for each construct. Construct names are listed on the x-axis. Data were calculated as (MFI of CR9114 binding / MFI of 65C6 binding) per construct. MFI is median fluorescence intensity x percent positive cells for monoclonal antibody binding. H5_Base is the wild-type HA polypeptide from A / Astrakhan / 3212 / 2020. pDNA denotes that constructs were transfected as plasmid DNA and mRNA denotes that constructs were transfected with LNP-encapsulated mRNA. As shown in FIG. 4, the M447I mutant has an improved stem to RBS binding antibody ratio compared to the wild-type (mono-base) HA polypeptide from A / Astrakhan / 3212 / 2020, suggesting that this modified H5 influenza HA polypeptides is presented in a more prefusion, closed conformation than the wild-type HA polypeptide from A / Astrakhan / 3212 / 2020.Example 3. In vitro Expression of a Modified H5 Influenza HA Polypeptide

[0332] This example describes the in vitro expression of the modified H5 influenza HA polypeptide in mRNA-transfected human skeletal muscle cells. In this example, mRNA encoding the M447I mutant or the wild-type (polybasic) HA polypeptide from A / Astrakhan / 3212 / 2020 was formulated in LNP containing GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid.

[0333] Human skeletal muscle (HSkM) cells were seeded the day prior to transfection and were incubated overnight at 37°C / 5% CO2 in a humidified incubator. The following day, mRNA- LNPs were thawed and diluted in 10% trehalose to lOx the final concentration before dilution to lx in cell growth medium. Cell media was replaced with treatment media, and the cells were incubated for 20+0.5 hours at 37°C / 5% CO2 in a humidified incubator.

[0334] At the experimental endpoint, cells were fixed at room temperature with 4% paraformaldehyde, washed with PBS, permeabilized with di gitonin, and blocked with 10% goat serum for 1 hour at room temperature. Cells were subsequently incubated overnight with an antibody specific for influenza A HA at 4°C overnight. The following day, cells were washed and incubated with an Alexa-647 conjugated secondary antibody, CellMask Blue, and NucBlue for 1 hour at room temperature. Image acquisition was performed on an Operetta CLS high content imaging microscope using a 20x Water objective. Images were analyzed by segmenting cellsbased on NUCBLUE™ (Hoechst) / CELLMASK™ Blue (Invitrogen) staining, and the mean cellular MFI was reported.

[0335] As shown in FIG. 5, the in vitro expression of the M447I mutant (“monobasic M447I (D680-643)” in left panel and “D680-643 (M447I LRETR)” in right panel) was increased compared to the wild-type (polybasic) HA polypeptide from A / Astrakhan / 3212 / 2020 (“WT Polybasic (D680-667)” in left panel and “D680-667 (WT Polybasic)” in right panel). The graph on the left side shows the HA expression in HSkM cells transfected with mRNA formulated in LNP and the graph on the right side shows the expression as normalized to the wild-type control.Example 4. Immunogenicity of a Modified H5 Influenza HA Polypeptide from an Influenza H5N8 Strain

[0336] This example describes the immunogenicity of the modified H5 influenza HA polypeptide in mice. In this example, mRNA encoding the M447I mutant or the wild-type (polybasic) HA polypeptide from A / Astrakhan / 3212 / 2020 was formulated in LNP containing GL- HEPES-E3-E12-DS-4-E10 as the cationic lipid.

[0337] Immunogenicity was assessed with one dose, which was 1 pg HA per animal. In this study, 6-8-week-old BALB / C mice were immunized by the intramuscular route with the indicated test article and controls as summarized in FIG. 6 (6 animals per group). Mice were primed on day 0 and boosted on day 21 with the same test article and controls by the same immunization route. Animals were bled on days 20 and 35 to assess the neutralizing antibody titers against the A / Astrakhan / 3212 / 2020 influenza virus by the hemagglutination inhibition (HAI) assay, more specifically HIH (hemagglutination inhibition horse) assay since horse red blood cells were used.

[0338] The HIH assay was conducted as follows. The sera samples were treated with receptor- destroying-enzyme (RDE) by diluting one-part serum with four-parts enzyme and incubated 18- 20 hours in a 37°C water bath. The enzyme was inactivated by 35-45-minute incubation at 56°C. Treated sera was diluted 2-fold for a dilution range of 1 : 10 to 1 :2,560 for testing against the H5N8 low pathogenic avian influenza strain A / Astrakhan / 3212 / 2020. The assay was performed in V- bottom 96-well plates using 4 HAU of chicken embryonated egg amplified virus and 0.5% horse red blood cell (RBC) (serum starting dilution = 1 : 10). The HIH titer was determined as the highest dilution of serum resulting in complete inhibition of hemagglutination and is reported by the dilution titer.

[0339] As shown in FIG. 6, the M447I mutant (“M447I”) induced an increased HA inhibition geometric mean titer (GMT HI titers) at both day 20 and day 35 post-injection (filled shape) as compared to the wild-type (polybasic) HA polypeptide from A / Astrakhan / 3212 / 2020 (“WT (poly)”). All mice receiving the M447I mutant (M447I) reached 1:40 HIH threshold post prime unlike the wild-type (polybasic) HA polypeptide from A / Astrakhan / 32 / 12 / 2020 (“WT (poly)”).Example 5. Evaluation of the Safety and Immunogenicity of a Pandemic influenza H5 mRNA Vaccine

[0340] This example describes the evaluation of the safety and immunogenicity of a pandemic influenza H5 mRNA vaccine in healthy adults 18-64 years old or healthy adults at least 65 years old. In this example, a codon-optimized mRNA encoding the M447I mutant was encapsulated in LNP containing GL-HEPES-E3-E12-DS-4-E10 as the cationic lipid and formulated as an mRNA vaccine for intramuscular (IM) injection.

[0341] The codon-optimized mRNA used in this study contained the following sequence (the start codon (AUG) and the stop codon (UAA) in bold) with a poly A tail enzymatically added (not shown):GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUA GAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAU UGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACA CGAUGGAGAACAUCGUGCUGCUGCUGGCCAUCGUGUCUCUGGUGAA GUCCGACCAGAUUUGUAUCGGCUACCACGCUAACAACAGCACCGAA CAGGUGGACACCAUCAUGGAGAAGAACGUGACCGUGACCCACGCCC AGGACAUCCUGGAAAAGACCCACAAUGGCAAGCUGUGCGAUCUGAA UGGCGUGAAGCCCCUGAUCCUGAAGGACUGCUCCGUGGCCGGCUGG CUGCUGGGCAAUCCAAUGUGCGAUGAGUUUAUCCGGGUGCCCGAGU GGAGCUACAUCGUGGAGAGAGCCAACCCCGCCAACGACCUGUGCUA CCCCGGCAGCCUGAAUGACUACGAGGAGCUGAAGCAUCUGCUGUCC CGGAUCAAUCACUUCGAGAAGAUCCUGAUCAUUCCCAAGAGCUCCU GGCCUAACCACGAGACAUCCCUGGGCGUGAGCGCCGCCUGUCCUUAC CAGGGGGCCCCCUCCUUUUUCAGGAAUGUGGUGUGGCUGAUCAAGA AGAACGACGCCUACCCCACCAUCAAGAUCAGCUACAACAACACCAACCGGGAGGACCUGCUGAUCCUGUGGGGCAUCCACCACAGCAACAACG CCGAGGAGCAGACCAACCUGUAUAAGAAUCCCACCACCUACAUCUCC GUGGGGACCAGCACCCUGAACCAGAGGCUGGUGCCCAAGAUCGCCACCCGGUCCCAGGUGAACGGCCAGAGGGGCAGAAUGGAUUUCUUCUGGACCAUCCUGAAGCCCGACGAUGCCAUCCACUUUGAGAGCAACGGC AACUUCAUCGCCCCCGAGUACGCCUACAAGAUCGUGAAGAAGGGCG ACUCUACCAUCAUGAAGUCCGGGGUGGAGUACGGCCACUGCAACACCAAGUGCCAGACCCCCGUGGGCGCCAUCAAUAGCAGCAUGCCUUUCCACAAUAUCCACCCCCUGACCAUCGGAGAGUGCCCCAAGUACGUGAA AAGCAACAAGCUGGUGCUGGCCACCGGCCUGAGGAACAGCCCUCUG AGAGAGACUCGGGGCCUGUUCGGCGCCAUCGCCGGCUUCAUCGAGGGCGGCUGGCAGGGGAUGGUGGACGGCUGGUACGGCUACCACCACAG CAACGAGCAGGGCUCCGGCUACGCCGCCGACAAGGAGUCCACCCAGA AGGCCAUCGACGGCGUGACCAACAAGGUGAACUCUAUCAUCGACAAGAUGAACACCCAGUUCGAGGCCGUGGGCCGGGAGUUCAACAACCUG GAAAGGAGGAUCGAGAACCUGAAUAAAAAGAUGGAGGACGGCUUCC UGGACGUGUGGACAUACAACGCCGAGCUGCUGGUGCUGAUCGAGAAUGAGAGAACCCUGGAUUUCCACGACUCCAACGUGAAGAACCUGUAC GACAAGGUGCGGCUGCAGCUGAGGGACAACGCCAAGGAGCUGGGCA AUGGCUGCUUCGAGUUCUACCACAAGUGCGACAACGAAUGCAUGGAGUCCGUGAGGAACGGCACCUACGAUUAUCCCCAGUACAGCGAGGAG GCCAGACUGAAGCGCGAGGAGAUCAGCGGGGUGAAGCUGGAGUCCA UUGGCACCUACCAGAUCCUGAGCAUCUAUAGCACCGCCGCCUCCAGCCUGGCCCUGGCCAUCAUGAUGGCCGGCCUGUCUCUGUGGAUGUGUA GCAACGGCAGCCUGCAGUGCAGGAUCUGCAUCUAAUAACGGGUGGC AUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC(SEQ ID NO: 12).

[0342] The LNP for mRNA encapsulation used in this study contained GL-HEPES-E3-E12- DS-4-E10 at a molar ratio of about 40%, l,2-dimyristoyl-racglycero-3 -methoxypoly ethylene (DMG-PEG2000 or DMG-PEG-2K) at a molar ratio of about 1.5%, cholesterol at a molar ratio ofabout 28.5%, and l,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE) at a molar ratio of about 30%, all of the molar ratios are relative to the total lipid content of the LNP.

[0343] The aim of this study was to assess the safety and immunogenicity of 2 IM injections (21 days apart) of the pandemic influenza H5 mRNA vaccine at 3 dose levels (30 pg, 75 pg, and 150 pg total mRNA) in healthy adults aged 18-64 years old or healthy adults at least 65 years old. The primary objective was to characterize the safety profile of 3 dose levels of the pandemic influenza H5 mRNA vaccine after each and any injection in each age group and the secondary objective was to assess the immune response to 3 dose levels of the pandemic influenza H5 mRNA vaccine or placebo in each age group. The endpoints for the safety study were frequency and severity of immediate unsolicited systemic adverse events (AEs), solicited injection site and systemic reactions, and unsolicited AEs, adverse events of special interest (AESIs), and serious adverse events (SAEs), as well as frequency of out-of-range biological results. The endpoints for the immunogenicity study were hemagglutination inhibition (HAI) and neutralizing antibodies to H5 influenza virus, as well as the breadth of H5 influenza virus coverage.

[0344] Briefly, 272 healthy adult participants were enrolled and randomized and stratified by age in the study as follows:

[0345] In each age group (> 18 to < 65 years or > 65 years), participants were randomized to receive 2 IM injections 21 days apart (i.e., at DOI and D22) of the pandemic influenza H5 mRNA vaccine at a dose of either 30 pg, 75 pg, or 150 pg (total mRNA / 0.5 mL dose), or placebo. The pandemic influenza H5 mRNA was encapsulated in lipid nanoparticle (LNP) containing GL- HEPES-E3-E12-DS-4-E10 as cationic lipid and formulated in 5% sucrose, 50 mM Tris, 150 mM NaCl, 0.4% P188, and 10 pM EDTA. The placebo was 0.9% NaCl, 0.5 mL / dose.

[0346] Nine study visits and 1 telephone call were performed per participant. Vaccination visits (including blood samples) occurred at D01 and D22. Short-term follow-up visits (including blood samples) occurred 2, 8, and 21 days after each injection (i.e., at D03, D09, D22, D24, D30, and D43). Participants were also followed up (including blood samples) at 3 and 6 months after the second injection (i.e., at DI 12 and D202), and at 12 months after the second injection (i.e., at D387) for safety. A graphical design of this study is presented in FIG. 7.

[0347] For the main safety parameters, 95% confidence intervals (Cis) of point estimates were calculated using exact binomial method (Clopper-Pearson method) for single proportions and using the normal approximation for quantitative data. Statistical analysis was conducted for each dose group, by strata of age (i.e., > 18 to < 65 years and aged > 65 years), and by the period of 7 days and / or the period of 21 days after each vaccination, and / or by interval of visits for safety data collected from D01 to the end of the study. No hypotheses were tested. The number of participants with documented safety were used as denominator of the frequencies.

[0348] The secondary immunogenicity parameters were calculated with their 95% Cis using the exact binomial distribution (Clopper-Pearson method) for proportions and using normal approximation of log transformed for geometric mean titers (GMTs) and GMTs ratio. The 95% CI of proportions difference (i.e., difference in seroconversion) was calculated using Wilson Score method without continuity correction. All analyses were conducted by dose group and by age stratum. Reverse cumulative distribution curves (RCDCs) of pre-vaccination titer prior to the first vaccination at D01 and post-vaccination titer at D22, D43, DI 12, and D202 were generated for each dose level.

[0349] Seroprotection rates at different time points (D01, D22, and D43) obtained from both groups are provided in FIG. 8. As shown in FIG. 9A, seroprotection rates exceeded the thresholds required by the FDA’s Center for Biologies Evaluation and Research (“CBER Target”) for both groups (i.e., > 18 to < 65 years and aged > 65 years) at all dose levels (i.e., 30, 75 and 150 pg totalmRNA) of this pandemic flu H5 mRNA vaccine at D43. Likewise, as shown in FIG. 9B, seroconversion rates also exceeded the thresholds required by the FDA’s Center for Biologies Evaluation and Research (“CBER Target”) for both groups (i.e., > 18 to < 65 years and aged > 65 years) at all dose levels (i.e., 30, 75 and 150 pg total mRNA) of this pandemic flu H5 mRNA vaccine at D43. The geometric mean titers (GMTs) obtained from the immunogenicity analysis further showed that there were robust increases in GMTs across all mRNA groups (FIG. 10). As shown in FIG. 10, the highest GMT ratios were observed with the 75 pg dose and the 150 pg dose, although the 30 pg dose still induced greater than 10-fold increase in HIH GMT from day 1 to day 43.

[0350] Safety -wise, fewer solicited reactions were observed in the groups receiving 30 pg total mRNA / dose across both age categories (FIG. 11). Moreover, as shown in FIG. 12A-12B, the proportion of participants reporting reactions post-second dose was not higher than those reporting reactions after the first dose in both groups (i.e., > 18 to < 65 years (FIG. 12A) and aged > 65 years (FIG. 12B)) for all dose levels (i.e., 30, 75 and 150 pg total mRNA). Overall, it was observed that solicited reactions were more frequent in the participants receiving the pandemic flu H5 mRNA vaccine as compared to placebo, with very few Grade 3 reactions, and the lowest dose appeared to produce lower reactogenicity. As to unsolicited adverse events (AE) and unsolicited adverse reaction (AE) within the follow-up period after any vaccine injection (21 days following the first injection and 28 days following the second injection), the data show that there was no immediate AEs or ARs (FIG. 13). Based on these results, it was concluded that the overall safety profile for this pandemic flu H5 mRNA vaccine was acceptable for both age groups.Example 6. Immunogenicity of a Modified H5 Influenza HA Polypeptide from an Influenza H5N1 Strain

[0351] This example describes the immunogenicity of a modified H5 influenza HA polypeptide from the H5N1 A / Indonesia / 05 / 2005 strain in mice. In this example, mRNA encoding the M448I mutant (having the amino acid sequence of SEQ ID NO: 14) or the wild-type HA polypeptide from A / Indonesia / 05 / 2005 was formulated in an LNP containing GL-HEPES-E3- E12-DS-4-E10 as the cationic lipid.

[0352] This example demonstrates the transferability of the M447I mutation from the H5N8 A / Astrakhan / 3212 / 2020 strain to the H5N1 A / Indonesia / 05 / 2005 strain, while maintaining immunogenicity.

[0353] Modified non-replicating (MNR) mRNA encoding either the modified H5 comprising the M448I stabilizing mutation (Agl, A_H5_Ag3482_HA) or wild type (A_H5_Ag4099_HA) H5 from the A / Indonesia / 05 / 2005 strain were encapsulated in the LNP. BALB / c mice (n = 6 per group) received vaccine formulations via IM route at Day 0 and Day 21 at doses of either 1 pg or 0.1 pg per animal. Functional HA-specific antibody responses were evaluated by hemagglutination inhibition (HIH) assay after both first and second immunizations. For the HIH assay briefly, serum samples from the treatment groups were treated with RDE. Treated sera was diluted 2-fold for a dilution range of 1 :10 to 1 :2,560 for testing against the H5N1 strain A / Indonesia / 05 / 2005. The HIH assay was performed in V-bottom 96-well plates using 4 HAU of chicken embryonated egg amplified virus and 1.1% horse red blood cell (RBC) (serum starting dilution = 1 : 10). The HIH titer was determined as the highest dilution of serum resulting in complete inhibition of hemagglutination.

[0354] Mice immunized with either vaccine formulation produced functional antibody responses against H5 from A / Indonesia / 05 / 2005 as measured by HIH (FIG. 14A and 14B). Both the modified H5 with the M448I mutation and wild-type vaccines elicited detectable HIH responses by Day 21 (post-prime) and demonstrated boosted HIH titers after the second dose on Day 35. No background titers were detected in the PBS placebo control group.

[0355] Importantly, the immune responses generated by the vaccines comprising either wildtype H5 mRNA (A_H5_Ag4099_HA) or the mRNA encoding the modified H5 with the M448I mutation (A_H5_Ag3482_HA) were comparable at both 1 pg and 0.1 pg doses after either one or two immunizations. Notably, 50% of mice receiving the vaccine with mRNA encoding the modified H5 with the M448I mutation (A_H5_Ag3482_HA) achieved seroconversion threshold titers (>1 : 10) after the prime dose, compared to only 17% of mice reaching the 1 :20 titer threshold in the wild-type group.

[0356] These results confirm the successful transferability of the M448I mutation from the H5N8 A / Astrakhan / 3212 / 2020 strain to the H5N1 A / Indonesia / 05 / 2005 strain without compromising immunogenicity. These results demonstrate the robustness of this M448I mutation across different H5 strains, supporting its utility in vaccine design across the H5 subtype.

[0357] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill inthe art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the present disclosure and may be practiced within the scope of the appended claims. For example, all constructs, methods, and / or component features, steps, elements, or other aspects thereof can be used in various combinations.

[0358] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the present disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0359] Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0360] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.

Claims

1. We Claim:

1. An artificial nucleic acid comprising a nucleotide sequence encoding a modified H5 influenza hemagglutinin (HA) polypeptide, wherein the modified H5 influenza HA polypeptide comprises an amino acid substitution at amino acid position 447 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

2. The artificial nucleic acid of claim 1, wherein the amino acid substitution at amino acid position 447 is M447I.

3. The artificial nucleic acid of claim 1 or 2, wherein the modified H5 influenza HA polypeptide is from an influenza A virus subtype H5N8 or an influenza A virus subtype H5N1.

4. The artificial nucleic acid of claim 1 or 2, wherein the modified H5 influenza HA polypeptide is from influenza A virus strain A / Astrakhan / 3212 / 2020 or an influenza A virus A / Indonesia / 05 / 2005 strain.

5. The artificial nucleic acid of any one of claims 1-4, wherein the modified H5 influenza HA polypeptide further comprises one or more mutations in a polybasic cleavage site located at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 such that the polybasic cleavage site is converted to a monobasic cleavage site.

6. The artificial nucleic acid of claim 5, wherein the polybasic cleavage site comprises the amino acid sequence KRRKR (SEQ ID NO: 8) and the polybasic cleavage site is converted to the monobasic cleavage site by removing one or more, optionally all but one, basic amino acids.

7. The artificial nucleic acid of claim 5 or 6, wherein the polybasic cleavage site is converted to the monobasic cleavage site by replacing the polybasic cleavage site with the amino acid sequence TR.

8. The artificial nucleic acid of any one of claims 1-7, wherein the modified H5 influenza HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 2.

9. The artificial nucleic acid of claim 8, wherein the modified H5 influenza HA polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

10. The artificial nucleic acid of any one of claims 1-9, wherein the artificial nucleic acid is an artificial messenger RNA (mRNA) optionally comprising a 5'-cap structure and / or a 3'- poly(A) sequence.

11. The artificial nucleic acid of claim 10, wherein the artificial mRNA comprises or consists of a 5' cap structure, such as a 5' cap having the following structure:a 5' untranslated region (5' UTR) having the nucleic acid sequence of SEQ ID NO: 5, a protein coding region having the nucleic acid sequence of SEQ ID NO: 3, a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ ID NO: 6, and a poly A tail.

12. The artificial nucleic acid of claim 10 or 11, wherein the artificial mRNA comprises at least one chemically modified nucleotide and / or a phosphorothioate bond.

13. The artificial nucleic acid of claim 12, wherein the at least one chemically modified nucleotide comprises a pseudouridine, a 2'-fluoro ribonucleotide, or a 2'-methoxy ribonucleotide, optionally wherein the pseudouridine is a N1 -methylpseudouridine.

14. The artificial nucleic acid of any one of claims 1-13, wherein the nucleotide sequence comprises at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 3.

15. The artificial nucleic acid of claim 14, comprising the nucleic acid sequence of SEQ ID NO: 3.

16. A composition comprising the artificial nucleic acid of any one of claims 1-15 encapsulated in a lipid nanoparticle (LNP).

17. The composition of claim 16, wherein the LNP comprises a cationic lipid.

18. The composition of claim 17, wherein the cationic lipid comprises or is OF-02, cKK-ElO, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3- E12-DS-3-E14, (4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2 -hexyl decanoate) (ALC- 0315), or IM-001.

19. The composition of claim 17 or 18, wherein the LNP further comprises a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

20. The composition of claim 19, wherein: a) the PEGylated lipid comprises or is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000); and / or b) the cholesterol-based lipid comprises or is cholesterol; and / or c) the helper lipid comprises or is dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

21. The composition of claim 19 or 20, wherein: a) the cationic lipid is present at a molar ratio between about 35% and about 55%; b) the PEGylated lipid is present at a molar ratio between about 0.25% and about2.75%;c) the cholesterol-based lipid is present at a molar ratio between about 20% and about 45%; and d) the helper lipid is present at a molar ratio between about 5% and about 35%, wherein all of the molar ratios are relative to the total lipid content of the LNP.

22. The composition of claim 21, wherein: a) the cationic lipid is present at a molar ratio of about 40%; b) the PEGylated lipid is present at a molar ratio of about 1.5%; c) the cholesterol-based lipid is present at a molar ratio of about 28.5%; and d) the helper lipid is present at a molar ratio of about 30%, wherein all of the molar ratios are relative to the total lipid content of the LNP.

23. The composition of claim 22, wherein the artificial nucleic acid is an artificial mRNA encoding the modified H5 influenza HA polypeptide of SEQ ID NO: 2, and wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of about 40%, DMG-PEG2000 at a molar ratio of about 1.5%, cholesterol at a molar ratio of about 28.5%, and DOPE at a molar ratio of about 30%.

24. The composition of any one of claims 16-23, wherein the composition is an immunogenic composition.

25. A modified H5 influenza hemagglutinin (HA) polypeptide comprising an amino acid substitution M447I as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

26. The modified H5 influenza HA polypeptide of claim 25, wherein the modified H5 influenza HA polypeptide is from an influenza A virus subtype H5N8 or an influenza A virus subtype H5N 1.

27. The modified H5 influenza HA polypeptide of claim 25, wherein the modified H5 influenza HA polypeptide is from influenza A virus strain A / Astrakhan / 3212 / 2020 or an influenza A virus A / Indonesia / 05 / 2005 strain.

28. The modified H5 influenza HA polypeptide of any one of claims 25-27, wherein the modified H5 influenza HA polypeptide further comprises one or more mutations in a polybasic cleavage site located at amino acid positions 341-345 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 such that the polybasic cleavage site is converted to a monobasic cleavage site.

29. The modified H5 influenza HA polypeptide of claim 28, wherein the polybasic cleavage site comprises the amino acid sequence KRRKR (SEQ ID NO: 8) and the polybasic cleavage site is converted to the monobasic cleavage site by removing one or more, optionally all but one, basic amino acids.

30. The modified H5 influenza HA polypeptide of claim 28 or 29, wherein the polybasic cleavage site is converted to the monobasic cleavage site by replacing the polybasic cleavage site with the amino acid sequence TR.

31. The modified H5 influenza HA polypeptide of any one of claims 25-30, comprising the amino acid sequence of SEQ ID NO: 2.

32. A trimeric influenza A HA polypeptide complex, comprising three copies of the modified H5 influenza HA polypeptide of any one of claims 25-31.

33. The trimeric influenza A HA polypeptide complex of claim 32, wherein the trimeric influenza A HA polypeptide complex is more immunogenic as compared to a trimeric influenza A HA polypeptide complex prepared from a control H5 influenza HA polypeptide comprising the amino acid of SEQ ID NO: 1.

34. The trimeric influenza A HA polypeptide complex of claim 33, wherein immunogenicity is measured using a hemagglutination-inhibition assay.

35. An artificial nucleic acid encoding the modified H5 influenza HA polypeptide of any one of claims 25-31.

36. The artificial nucleic acid of claim 35, wherein the artificial nucleic acid comprises at least one chemically modified nucleotide and / or a phosphorothioate bond.

37. A vector comprising the artificial nucleic acid of claim 35 or 36.

38. The vector of claim 37, wherein the vector is a messenger RNA (mRNA) production vector.

39. A host cell comprising the vector of claim 37 or 38.

40. A composition comprising the modified H5 influenza HA polypeptide of any one of claims 25-31, the trimeric influenza A HA polypeptide complex of any one of claims 32-34, the artificial nucleic acid of claim 35 or 36, or the vector of claim 37 or 38.

41. The composition of claim 40, wherein the composition is an immunogenic composition.

42. A vaccine comprising the immunogenic composition of claim 24 or 41, and a pharmaceutically acceptable carrier.

43. A vaccine comprising the artificial nucleic acid of any one of claims 1-15 encapsulated in a lipid nanoparticle (LNP), wherein the artificial nucleic acid is an artificial mRNA, and wherein the vaccine comprises from about 0.1 pg to about 150 pg, such as from about 5 pg to about 120 pg, from about 10 pg to about 60 pg, or about 15 pg to about 45 pg, of the artificial mRNA.

44. The vaccine of claim 43, wherein the vaccine comprises about 30 pg, about 75 pg, or about 150 pg of the artificial mRNA.

45. The vaccine of claim 43 or 44, wherein the artificial mRNA encodes the modified H5 influenza HA polypeptide of SEQ ID NO: 2, wherein the LNP comprises a cationic lipid, and wherein the cationic lipid comprises GL-HEPES-E3-E12-DS-4-E10.

46. A method of immunizing a subject, the method comprising administering to the subject in need thereof the vaccine of any one of claims 41-45.

47. The method of claim 46, wherein the method prevents an influenza A virus infection in the subject, decreases the subject’s likelihood of getting an influenza A virus infection, or reduces the subject’s likelihood of getting serious illness from an influenza A virus infection.

48. The method of claim 46 or 47, wherein the method induces influenza seroprotection in the subject by at least day 43 following the administration of the vaccine to the subject.

49. The method of any one of claim 46-48, wherein the subject is a human.

50. The method of claim 49, wherein the human is 6 months of age or older, less than 18 years of age, at least 6 months of age and less than 18 years of age, at least 18 years of age and less than 65 years of age, at least 6 months of age and less than 5 years of age, at least 5 years of age and less than 65 years of age, at least 60 years of age, or at least 65 years of age.

51. The method of any one of claims 46-50, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

52. The method of any one of claims 46-51, wherein the vaccine is administered as part of a prime-boost vaccination strategy.

53. The method of claim 52, wherein the prime-boost vaccination strategy comprises administering a boosting vaccine about 2-3 weeks after administration of a priming vaccine.

54. A method of reducing one or more symptoms of an influenza A virus infection, the method comprising administering to a subject in need thereof the vaccine of any one of claims 41-45.

55. An in vitro method of preparing a trimeric influenza A HA polypeptide complex, the method comprising culturing the host cell of claim 39 in a cell culture medium, and expressing the trimeric influenza A HA polypeptide complex.

56. The in vitro method of claim 55, wherein the trimeric influenza A HA polypeptide complex is more immunogenic as compared to a trimeric influenza A HA polypeptide complex prepared from a control H5 influenza HA polypeptide comprising the amino acid of SEQ ID NO: 1.

57. The in vitro method of claim 56, wherein immunogenicity is measured using a hemagglutination-inhibition assay.

58. The in vitro method of any one of claims 55-57, further comprising a step of purifying the trimeric influenza A HA polypeptide complex from the cell culture medium.

59. The vaccine of any one of claims 42-45 for use in a method for treatment of a human or animal body.

60. The vaccine of any one of claims 42-45 for use in a method for immunization of a human or animal body.

61. The vaccine of any one of claims 42-45 for use in a method for immunizing a subject from or reducing one or more symptoms of an influenza virus infection.

62. The vaccine of any one of claims 42-45 for use in a method for immunizing a subject from or reducing one or more symptoms of an influenza A virus infection.

63. Use of any one or any combination of (i) the artificial nucleic acid of any one of claims 1-15, (ii) the composition of any one of claims 16-24, (iii) the modified H5 influenza HA polypeptide of any one of claims 25-31, (iv) the trimeric influenza A HA polypeptide complex of any one of claims 32-34, optionally with a pharmaceutically acceptable carrier, for the manufacture of a medicament for immunizing a subject from or reducing one or more symptoms of an influenza A virus infection.

64. The composition of any one of claims 16-24, wherein the artificial nucleic acid is an artificial mRNA and the artificial mRNA comprises or consists of: a 5' cap structure, such as a 5' cap having the following structure:a 5' untranslated region (5' UTR) having the nucleic acid sequence of SEQ ID NO: 5, a protein coding region having the nucleic acid sequence of SEQ ID NO: 3, a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ ID NO: 6, and a poly A tail.

65. A method of preparing the composition of any one of claims 19-24, comprising the steps of: providing an aqueous buffered solution comprising the artificial nucleic acid molecule of any one of claims 1-15, providing an amphiphilic solution comprising the cationic lipid, the PEGylated lipid, the cholesterol-based lipid, and the helper lipid, and mixing the aqueous buffered solution and the amphiphilic solution at a ratio of 5 : 1 to 3 : 1.

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