Modified influenza a hemagglutinin polypeptides and nucleic acids and uses thereof

Abstract

This application relates to modified influenza A hemagglutinin polypeptides nucleic acids, such as messenger ribonucleic acids (mRNAs), encoding the same, as well as compositions comprising the same, 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.

Description

Attorney Docket No. 0171.0129-PCTMODIFIED INFLUENZA A HEMAGGLUTININ POLYPEPTIDES AND NUCLEIC ACIDS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and relies on the filing date of, European Application No. 24306468.0, filed 6 September 2024, the entire disclosure of which is herein incorporated by reference.FIELD

[0002] This application relates to modified influenza A hemagglutinin polypeptides nucleic acids, such as messenger ribonucleic acids (mRNAs), encoding the same, as well as compositions comprising the same, 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.BACKGROUND

[0003] Influenza viruses cause substantial morbidity and mortality in humans across the globe, leading to the death of over half a million individuals annually. Before SARS-CoV-2 emerged, influenza was the leading cause of respiratory illness. Influenza A viruses infect a wide variety of birds and mammals, including humans, pigs, ferrets, and chickens. Vaccination is the most common preventative measure utilized. Currently approved influenza vaccines are either inactivated vaccines, containing entire virions or virions subjected to treatment with agents that dissolve lipids (“split” vaccines), purified glycoproteins expressed in cell culture (“subunit vaccines”), or live attenuated virus vaccines, which are often produced in cell culture or eggs. Other types of vaccines, such as nucleic acid-based or viral vector-based vaccines, are also being developed. Since COVID-19, messenger RNAs (mRNA) have emerged as a new class of highly efficacious nucleic acids in the field of vaccines.

[0004] Influenza hemagglutinin (HA) is a prototypical class I fusion protein and a major component of current influenza vaccines. HA is a metastable trimeric glycoprotein and undergoes conformational changes from the so-called prefusion state to a postfusion state. However, the production of class I fusion proteins, like HA, by recombinant protein expression is challengingAttorney Docket No. 0171.0129-PCT because of their general intrinsic instability, low expression levels, and failure to form correctly folded trimers. Recent data from clinical trials also suggest that mRNA vaccine encoding influenza HA elicit sub-optimal neutralization response, which could impact its efficacy when used as a vaccine.

[0005] Accordingly, there is an urgent need to develop vaccines, either recombinant vaccine vectors or nucleic acid vaccines, that can deliver an influenza A HA with improved expression, quality, and stability.SUMMARY

[0006] Disclosed herein are modified influenza A HA polypeptides having characteristics, such as increased immunogenic properties, improved stability in the prefusion conformation, improved expression, reduced sialic acid binding, and / or reduced antigenicity to non-neutralizing antibodies, that would enable elicitation of higher neutralization response that can translate into better vaccine efficacy, or lower reactogenicity when a lower dosage is use. Accordingly, in one aspect, provided herein is an artificial messenger ribonucleic acid (mRNA) encoding a modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide comprises at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide. In some embodiments, the at least two cysteine substitutions are at amino acid positions 36 and 398 and / or 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least two cysteine substitutions are at amino acid positions 235 and 262 and / or 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptide comprises amino acid substitutions V36C and S398C as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptide comprises amino acid substitutions G219C and R234C as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptide comprises amino acid substitutions S235C and N262C as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptide comprises amino acid substitutions 1411C and D424C as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptide is from a H1N1 influenza virus. In some embodiments, the H1N1 influenza virus is A / West Virginia / 30 / 2022. In some embodiments, theAttorney Docket No. 0171.0129-PCT modified influenza A HA polypeptide is from a H3N2 influenza virus. In some embodiments, the H3N2 influenza virus is A / Darwin / 06 / 2021. In some embodiments, the modified influenza A HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In some embodiments, the modified influenza A HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. 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: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. In some embodiments, the artificial mRNA comprises the nucleic acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

[0007] In some embodiments, the artificial mRNA of the present disclosure 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'-methoxy ribonucleotide, optionally wherein the pseudouridine is a Nl-methylpseudouridine.

[0008] Also provided herein, in some embodiments, is a composition comprising the artificial mRNA 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 someAttorney Docket No. 0171.0129-PCT 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 of about 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 mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 3, 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 artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 5, 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 artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 7, 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 artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 9, 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.

[0009] In a further aspect, provided herein is a modified influenza A HA polypeptide comprising one or more amino acid substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the one or more amino acid substitutions comprises at least two cysteine substitutions. In some embodiments, the at least two cysteine substitutions are at amino acid positions 36 and 398 and / or 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, at least two cysteine substitutions are at amino acid positions 235 and 262 and / or 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptide comprises amino acid substitutions V36C and S398C as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptide comprises amino acid substitutions G219C and R234C as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptide comprises amino acidAttorney Docket No. 0171.0129-PCT substitutions S235C and N262C as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptide comprises amino acid substitutions 1411C and D424C as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptide is from a H1N1 influenza virus. In some embodiments, the H1N1 influenza virus is A / West Virginia / 30 / 2022. In some embodiments, the modified influenza A HA polypeptide is from a H3N2 influenza virus. In some embodiments, the H3N2 influenza virus is A / Darwin / 06 / 2021. In some embodiments, the modified influenza A HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In some embodiments, the modified influenza A HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

[0010] Also provided herein, in some embodiments, is a trimeric influenza A HA polypeptide complex comprising three copies of any of the modified influenza A 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 a corresponding wild-type influenza A HA polypeptide without the amino acid substitutions. In some embodiments, the trimeric influenza A HA polypeptide complex of the present disclosure has a comparable immunogenicity as a trimeric influenza A HA polypeptide complex prepared from a corresponding wild-type influenza A HA polypeptide without the amino acid substitutions. In some embodiments, the immunogenicity is measured using a hemagglutination-inhibition assay.

[0011] Further provided is an artificial nucleic acid encoding any of the modified influenza A 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 influenza A HA polypeptides disclosed herein, 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.Attorney Docket No. 0171.0129-PCT

[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. In some embodiments, the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and the vaccine further comprises an mRNA encoding an influenza H3 HA polypeptide and an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage. In some embodiments, the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and the vaccine further comprises an mRNA encoding an influenza H3 HA polypeptide, an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage, an mRNA encoding an influenza N2 neuraminidase (NA) polypeptide, an mRNA encoding an influenza N 1 NA polypeptide, and an mRNA encoding an influenza NA polypeptide from the Influenza B / Victoria lineage. In some embodiments, the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and the vaccine further comprises an mRNA encoding a wild-type influenza H3 HA polypeptide and an mRNA encoding a modified influenza HA polypeptide from an influenza B / Victoria lineage. In some embodiments, the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and the vaccine further comprises an mRNA encoding an influenza Hl HA polypeptide and an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage. In some embodiments, the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and the vaccine further comprises an mRNA encoding an influenza Hl HA polypeptide, an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage, an mRNA encoding an influenza N2 NA polypeptide, an mRNA encoding an influenza N1 NA polypeptide, and an mRNA encoding an influenza NA polypeptide from the Influenza B / Victoria lineage. In some embodiments, the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and the vaccine further comprises an influenza H3 HA polypeptide and an influenza HA polypeptide from an influenza B / Victoria lineage. In some embodiments, theAttorney Docket No. 0171.0129-PCT vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and the vaccine further comprises an influenza H3 HA polypeptide, an influenza HA polypeptide from an influenza B / Victoria lineage, an influenza N2 NA polypeptide, an influenza N1 NA polypeptide, and an influenza NA polypeptide from the Influenza B / Victoria lineage. In some embodiments, the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and the vaccine further comprises an influenza Hl HA polypeptide and an influenza HA polypeptide from an influenza B / Victoria lineage. In some embodiments, the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and the vaccine further comprises an influenza Hl HA polypeptide, an influenza HA polypeptide from an influenza B / Victoria lineage, an influenza N2 NA polypeptide, an influenza N 1 NA polypeptide, and an influenza NA polypeptide from the Influenza B / Victoria lineage.

[0014] Also provided herein, in another aspect, is 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 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 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 vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0015] In a further aspect, provided herein is an in vitro method of preparing any of the trimeric influenza A 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 influenza A HA polypeptide complex. In some embodiments, the method further comprises a step of purifying the trimeric influenza A HA polypeptide complex from the cell culture medium.Attorney Docket No. 0171.0129-PCTBRIEF DESCRIPTION OF THE DRAWING

[0016] 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.

[0017] FIG. 1 depicts the structure of an influenza hemagglutinin (HA) trimer in the prefusion conformation. RBS: receptor binding site; FP: fusion peptide; RR1: refolding region 1; CH: central helix; RR2: refolding region 2.

[0018] FIG. 2A-2B depict aggregated results categorized by types of modification in each A / West Virginia / 30 / 2022 (FIG. 2A) and A / Darwin / 06 / 2021 (FIG. 2B) strain.

[0019] FIG. 3 depicts in vitro expression of representative modified Hl HA polypeptides. The number on top of each vertical bar corresponds to the construct ID provided in Table 3.

[0020] FIG. 4A-4C depict in vitro characterization of representative modified Hl HA polypeptides. The number on top of each vertical bar corresponds to the construct ID provided in Table 3. FIG. 4A: binding of the lateral patch (head) monoclonal antibody 045-09-2B05; FIG. 4B: binding of the stem antibody CR9114; FIG. 4C: binding of the FluA20 antibody.

[0021] FIG. 5 depicts in vitro expression of representative modified H3 HA polypeptides. The number on top of each vertical bar corresponds to the construct ID provided in Table 4.

[0022] FIG. 6A-6C depict in vitro characterization of representative modified H3 HA polypeptides. The number on top of each vertical bar corresponds to the construct ID provided in Table 4. FIG. 6A: binding of the lateral patch (head) monoclonal antibody 045-09-2B05; FIG. 6B: binding of the stem antibody CR9114; FIG. 6C: binding of the FluA20 antibody.

[0023] FIG. 7A depicts fusogenicity (ability to mediate cell-cell fusion) of two representative modified influenza Hl HA polypeptides (“Hl WV_512” and “Hl WV_466”) as compared to the corresponding wild-type HA polypeptide (“Hl WV_WT”) and a negative control (“Hl WV_HA0”) which contains a substitution in the HA1-HA2 cleavage site and is fusion deficient. FIG. 7B depicts fusogenicity (ability to mediate cell-cell fusion) of two representative modified influenza H3 HA polypeptides (“H3 Darwin / 6_542” and “H3 Darwin / 6_571”) as compared to the corresponding wild-type HA polypeptide (“H3 Darwin / 6_WT”) and a negative control (“H3 Darwin / 6_HA0”) which contains a substitution in the HA1-HA2 cleavage site and is fusion deficient.Attorney Docket No. 0171.0129-PCT

[0024] FIG. 8A-8D depict the immunogenicity of representative modified influenza Hl and H3 HA polypeptides delivered as LNP-formulated mRNA in mice as compared to the corresponding wild-type HA polypeptide from A / West Virginia / 30 / 2022 (FIG. 8A-8B) or A / Darwin / 06 / 2021 (FIG. 8C-8D). See Table 3 and Table 4 for the construct designs listed on the top of the graphs.DETAILED DESCRIPTION

[0025] 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.

[0026] 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.Definitions

[0027] 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.

[0028] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0029] 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 asAttorney Docket No. 0171.0129-PCT 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.

[0030] 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.Attorney Docket No. 0171.0129-PCT

[0031] 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 (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0032] 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).

[0033] As used herein, the term “antigen” refers 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)Attorney Docket No. 0171.0129-PCT or to an 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 antigenspecific 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 T cell 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 influenza A HA polypeptides described herein.

[0034] As used herein, an “artificial nucleic acid molecule” or the like may typically be understood to be a nucleic acid, 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 molecule 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 (e.g., mRNA), 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 molecule” or the like (e.g., “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.Attorney Docket No. 0171.0129-PCT

[0035] The phrase “as indexed by reference to the amino acid sequence of SEQ ID NO: 1” or “as indexed by reference to the amino acid sequence of SEQ ID NO: 2” 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 corresponding wild-type influenza HA polypeptide sequence, such as the HA polypeptide sequence of A / West Virginia / 30 / 2022 (SEQ ID NO: 1) or A / Darwin / 06 / 2021 (SEQ ID NO: 2)), 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 / West Virginia / 30 / 2022 HA amino acid sequence (SEQ ID NO: 1) or the A / Darwin / 06 / 2021 HA amino acid sequence (SEQ ID NO: 2). Sequences are aligned to the full-length HA protein sequence (including signal peptide, transmembrane and cytoplasmic tail domains) of A / West Virginia / 30 / 2022 (SEQ ID NO: 1) or A / Darwin / 06 / 2021 (SEQ ID NO: 2). The N-terminal methionine of the signal peptide is residue 1. Accordingly, the phrase “amino acid position x as indexed by reference to the amino acid sequence of SEQ ID NO: 1” or “amino acid position x as indexed by reference to the amino acid sequence of SEQ ID NO: 2” is used herein to designate the position / identity of an amino acid residue in a polypeptide of interest (e.g., a modified influenza A HA polypeptide) by referring to the corresponding amino acid at position x in the HA polypeptide sequence of A / West Virginia / 30 / 2022 (SEQ ID NO: 1) or A / Darwin / 06 / 2021 (SEQ ID NO: 2).

[0036] 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.

[0037] 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,Attorney Docket No. 0171.0129-PCT 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.”

[0038] 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. coliY insect cells, yeast cells or plant cells.

[0039] 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).

[0040] As used herein, the term “head region” refers to a segment of an influenza A HA polypeptide that is encompassed by approximately amino acid residues 59-292 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 67- 293 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. Variation in the numbering of the amino acid residues may exist depending on isolates. Morphologically, the head region may be defined as the globular shaped domain of HA.

[0041] As used herein, the term “hemagglutinin polypeptide” or “HA polypeptide” or “HA” 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 aAttorney Docket No. 0171.0129-PCT 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.).

[0042] As used herein, “Hl” refers to an influenza virus subtype 1 hemagglutinin (HA). Type A influenza viruses are divided into Groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refers to classification of a virus based on the sequences of two proteins on the surface of the virus HA and neuraminidase (NA). Currently, there are 18 recognized HA subtypes (H1-H18). Hl is thus distinct from the other HA subtypes, including H2-H18.

[0043] As used herein, “H3” refers to an influenza virus subtype 3 HA. H3 is thus distinct from the other HA subtypes, including Hl, H2 and H4-H18.

[0044] As used herein, “Nl” refers to an influenza virus subtype 1 NA. Type A influenza viruses are divided into Groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refers to classification of a virus based on the sequences of two proteins on the surface of the virus HA and NA. Currently, there are 11 recognized NA subtypes (Nl-Nl l). Nl is thus distinct from the other NA subtypes, including N2-N11.

[0045] As used herein, “N2” refers to an influenza virus subtype 2 neuraminidase (NA). N2 is thus distinct from the other NA subtypes, including Nl and N3-N11.

[0046] 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.

[0047] 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 influenza A 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 “hostAttorney Docket No. 0171.0129-PCT 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., 5. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, microalgae (including both eukaryotic algae, such as Chlamydomonas, Chlorella, Nannochloropsis, Thraustochytriales (e.g., Schizochytrium sp.), diatoms (e.g., Phaeodactylum), and prokaryotic cyanobacteria, also known as blue-green algae such as Arthrospira), 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 3 A 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).

[0048] 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.

[0049] As used here, an “mRNA vaccine” refers to a type of vaccine that uses messenger RNA (mRNA) to produce an immune response.

[0050] 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.

[0051] 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,Attorney Docket No. 0171.0129-PCT 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.

[0052] 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).

[0053] 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.

[0054] As used herein, the term “receptor-binding site” or “RBS” comprises contiguous or non-contiguous amino acid residues of the head region of an influenza HA polypeptide, whichAttorney Docket No. 0171.0129-PCT include amino acids involved in direct binding of sialic acid on the target cell receptor proteins. Amino acid residues that make up a “receptor-binding site” or “RBS” of an influenza HA polypeptide may be described from crystal structures of HA polypeptides complexed with sialic acid analogs and identifying amino acid residues within a certain proximity to the analog or may be described in reference to an HA polypeptide sequence from a particular viral strain (e.g., AAV est Virginia / 30 / 2022 or A / Darwin / 06 / 2021). Thus, in some embodiments, the “receptor-binding site” or “RBS” of a modified HA polypeptide as described herein may be determined using a reference HA polypeptide sequence. In some embodiments, the “receptor-binding site” or “RBS” of a modified HA polypeptide as described herein may be determined using the crystal structures of HA polypeptide sequence in complex with human and avian receptor analogs (e.g., LSTa, LSTc). An exemplary reference crystal structure of HA polypeptide sequence in complex with LSTc includes A / Puerto Rico / 8 / 1934 (H1N1) (PDB ID 1RVZ). In some embodiments, the RBS may be defined as the epitope bound by the broadly neutralizing monoclonal antibody CH65 (see e.g., Whittle et al., Proc. Natl. Acad. Sci. USA, 2011, 108: 14216-14221). Alternatively or additionally, the RBS may be defined as an area including all amino acid residues within 15 Angstroms of a universally conserved tryptophan (W) corresponding to amino acid position 167 of SEQ ID NO: 1 or amino acid position 169 of SEQ ID NO: 2 (see e.g., Xu et al., Nat. Struct. Mol. Biol., 2013, 20(3):363-370).

[0055] As used herein, a “recombinant vaccine” refers to a type of vaccine that uses genetic engineering to produce antigens from a pathogen (e.g., influenza virus) using a harmless organism, such as yeast or bacteria.

[0056] As used herein, the term “stem region” refers to a discontinuous region of an influenza A HA polypeptide, the region comprising approximately amino acid residues 18-58 and 293-519 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 28-67 and 295-520 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. Variation in the numbering of the amino acid residues may exist depending on isolates. Morphologically, the stem region may be defined as the elongated domain that emerges from the globular head.

[0057] 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,Attorney Docket No. 0171.0129-PCT 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.”

[0058] 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 the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0059] 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

[0060] 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 site (RBS), also known as receptor-binding domain (RBD), and mostAttorney Docket No. 0171.0129-PCT 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.

[0061] 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.

[0062] 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.

[0063] The amino acid sequences of a large number of influenza HA polypeptides from different influenza A viruses 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 amino acid sequences of the wild-type HA polypeptide from a representative influenza A virus subtype Hl, i.e., influenza A virus A / West Virginia / 30 / 2022, strain, and a representative influenza A virus subtype H3, i.e., influenza A virus A / Darwin / 06 / 2021 strain are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.Attorney Docket No. 0171.0129-PCTHA polypeptide of influenza A / West Virginia / 30 / 2022 (subtype Hl):MKAILVVMLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDK HNGKLCKLRGVAPLHLGQCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCY PGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDNGVTAACSHAGARSFYKN EIWEVKKGKSYPKINQTYINDKGKEVEVEWGIHHPPTITDQESEYQNADAYVFV GTSRYSKKFKPEIAARPKVRDQAGRMNYYWTEVEPGDKITFEATGNEVAPRYAF TMEKEAGSGIIISDTPVHDCNATCQTPEGAINTSEPFQNVHPITIGKCPKYVRSTKE REATGERNVPSIQSRGEFGAIAGFIEGGWTGMVDGWYGYHHQNDQGSGYAADE KSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHEEKRIENENKKVDDGFEDVW TYNAEEEVEEENERTEDYHDSNVKNEYEKVRHQEKNNAKEIGNGCFEFYHKCD NTCMESVKNGTYDYPKYSEEAKENREKIDGVKEDSTRIYQIEAIYSTVASSEVEV VSEGAISFWMCSNGSEQCRICI (SEQ ID NO: 1).HA polypeptide of influenza A / Darwin / 06 / 2021 (subtype H3):MKTIIAESNIECEVFAQKIPGNDNSTATECEGHHAVPNGTIVKTITNDRIEVTNATE EVQNSSIGEICGSPHQIEDGGNCTEIDAEEGDPQCDGFQNKEWDEFVERSRANSN CYPYDVPDYASERSEVASSGTEEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRE NWETSENNIYPAQNVTMPNKEQFDKEYIWGVHHPDTDKNQISEFAQSSGRITVST KRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDIEEINSTGNEIAPRGYFKIRSGKS SIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTEKEATGMR NVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADEKSTQAAI DQINGKENREIGKTNEKFHQIEKEFSEVEGRVQDEEKYVEDTKIDEWSYNAEEEV AEENQHTIDETDSEMNKEFEKTKKQERENAEDMGNGCFKIYHKCDNACIGSIRN ETYDHNVYRDEAENNRFQIKGVEEKSGYKDWIEWISFAMSCFEECIAEEGFIMW ACQKGNIRCNICI (SEQ ID NO: 2).

[0064] For the purposes of the present disclosure (unless context indicates otherwise), the amino acid positions in the modified influenza A HA polypeptides are given with reference to the amino acid sequence of the full length wild-type HA polypeptide of A / West Virginia / 30 / 2022 set forth in SEQ ID NO: 1 or A / Darwin / 06 / 2021 set forth in SEQ ID NO: 2. However, it should beAttorney Docket No. 0171.0129-PCT noted, and one of skill in the art will understand, that different influenza A 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 or SEQ ID NO: 2. 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 influenza A HA polypeptide sequences is intended even if that residue is not at the same precise numbered position, for example if a given influenza A HA polypeptide sequence is shorter or longer than SEQ ID NO: 1 or SEQ ID NO: 2, or has insertions or deletions as compared to SEQ ID NO: 1 or SEQ ID NO: 2.Modified Influenza A HA Polypeptides

[0065] The present disclosure provides modified influenza A HA polypeptides in which one or more mutations (e.g., substitutions) have been introduced in the amino acid sequence relative to the amino acid sequence of the corresponding wild-type influenza A HA polypeptide. In some embodiments, the modified influenza A HA polypeptides disclosed herein possess certain beneficial characteristics, such as increased immunogenic properties, improved stability in the prefusion conformation, improved expression, reduced sialic acid binding, and / or reduced antigenicity to non-neutralizing antibodies, as compared to the corresponding wild-type influenza A HA polypeptide. In some embodiments, the modified influenza A 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. Also provided are artificial nucleic acid molecules that encode the modified influenza A HA polypeptides disclosed herein.

[0066] The modified influenza A HA polypeptides of the present disclosure comprise one or more amino acid mutations in the head region and / or the stem region as compared to the amino acid sequence of the corresponding wild-type influenza A HA polypeptide. The introduced amino acid mutations in the modified influenza A HA polypeptides of the disclosure include amino acid substitutions, deletions, or additions. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the only mutations introduced in the amino acid sequence of the modified influenza A HA polypeptides of the disclosure are amino acid substitutions relative to the correspondingAttorney Docket No. 0171.0129-PCT wild-type influenza A HA polypeptide, and may include conservative and / or non-conservative substitutions.

[0067] 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, 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.

[0068] In some embodiments, the substitutions may also include 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, e-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, amino acid substitutions at a specific amino acid position 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 influenza A HA polypeptides of the present disclosure.

[0069] Accordingly, provided herein is a modified influenza A HA polypeptide comprising a head region and a stem region, wherein the modified influenza A HA polypeptide comprises oneAttorney Docket No. 0171.0129-PCT or more modifications selected from proline mutations, disulfide bridge forming mutations, interface stabilizing mutations, pH sensor inactivating mutations, and / or sialic acid binding interfering mutations as described in detail herein below. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise at least one of two of the aforementioned modifications, such as at least one proline mutation and at least one disulfide bridge forming mutation, or at least one disulfide bridge forming mutation and at least one pH sensor inactivating mutation. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise at least one of three of the aforementioned modifications, such as at least one proline mutation, at least one disulfide bridge forming mutation, and at least one pH sensor inactivating mutation. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise at least one of four of the aforementioned modifications, such as at least one proline mutation, at least one disulfide bridge forming mutation, at least one interface stabilizing mutation, and at least one pH sensor inactivating mutation. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise at least one of each of the five aforementioned modifications.

[0070] In some embodiments, the one or more aforementioned modifications stabilize the modified influenza A HA polypeptides of the disclosure in a prefusion conformation. Monoclonal antibodies to well-defined, structural epitopes of the influenza A HA polypeptide, such as stem region- specific antibodies (e.g., CR9114), vestigial esterase (VE) specific antibodies (e.g., CR8071), and receptor binding site (RBS) specific antibodies (e.g., R95-1D05), can be used to investigate the conformation of the modified influenza A HA polypeptides disclosed herein using flow cytometry. The binding of an RBS-specific antibody, such as R95-1D05, indicates the total surface expression and integrity of sialic acid binding pocket on the surface expressed HA polypeptides. The binding of a VE-specific antibody, such as CR8071, and a stem-region- specific antibody, such as CR9114, indicates that the surface expressed HA polypeptide is in a prefusion conformation. The binding ratio of a stem-region- specific antibody, such as CR9114, to an RBS- specific antibody, such as R95-1D05 (e.g., CR9114 MFER95-1D05 MFI) indicates the ratio of prefusion, closed HA polypeptides to total HA polypeptides presented on the cell surface, with a higher ratio indicating that a higher percentage of the surface expressed HA polypeptides is in a prefusion, locked conformation. Accordingly, in some embodiments, stabilization of the prefusion conformation is measured by an increased binding of the modified influenza A HA polypeptide toAttorney Docket No. 0171.0129-PCT a stem region- specific antibody (e.g., CR9114) as compared to the corresponding wild-type influenza A HA polypeptide. In certain embodiments, stabilization of the prefusion conformation is measured by an increased (e.g., at least two-fold higher) binding ratio of a stem region- specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) as compared to the corresponding wild-type influenza A HA polypeptide. In some embodiments, stabilization of the prefusion conformation is measured by an increased binding of the modified influenza A HA polypeptide to a stem region-specific antibody (e.g., CR9114) and an increased binding ratio (e.g., two or greater) of a stem region- specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) as compared to the corresponding wild-type influenza A HA polypeptide.

[0071] Morphologically, the head region may be defined as the globular shaped domain of HA and the stem region may be defined as the elongated domain that emerges from the globular head. A schematic structure of an influenza HA trimer is provided in FIG. 1. In some embodiments, the head region of a modified influenza A HA polypeptide according to the present disclosure can be defined as being a segment of the modified influenza A HA polypeptide at approximately amino acid positions 59-292 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 67-293 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the stem region, being a discontinuous region of the HA polypeptide, can be defined as comprising approximately amino acid residues 18-58 and 293-519 of the modified influenza A HA polypeptide as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 28-67 and 295-520 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0072] Wild-type influenza A HA polypeptides comprise a signal peptide at their N-terminus (e.g., corresponding to amino acids 1-15 of SEQ ID NO: 1), which directs transport of the HA polypeptide during production and generally is not present in the final polypeptide that is, for instance, used in a vaccine. Accordingly, in some embodiments, the modified influenza A HA polypeptides of the present disclosure do not comprise a signal peptide. In other embodiments, the modified influenza A HA polypeptides of the present disclosure comprise a signal peptide that is native to the wild-type influenza A HA polypeptide to which the one or more modifications are introduced. In some embodiments, the modified influenza A HA polypeptides of the present disclosure comprise a signal peptide that is heterologous to the wild-type influenza A HA polypeptide to which the one or more modifications are introduced. In some embodiments, suchAttorney Docket No. 0171.0129-PCT a heterologous signal peptide is an influenza HA signal peptide derived from an influenza A HA polypeptide that is different from the wild-type influenza A HA polypeptide to which the one or more modifications are introduced. In some embodiments, such a heterologous signal peptide is an influenza HA signal peptide derived from an influenza B HA polypeptide. In some embodiments, the heterologous signal peptide is from a non-influenza source. Any signal peptide known in the art that can direct transport of the HA polypeptide during production can be used. For recombinant production of the modified influenza A HA polypeptides of the disclosure in insect cells, for instance, any signal peptides from both mammals and viruses can be used to guide protein secretion in insect cells.

[0073] The influenza A HA polypeptides to which the one or more modifications according to the present disclosure are introduced can be from any wild-type influenza A viruses known in the art or discovered in the future, including, but not limited to, subtypes H1N1 and H3N2. In some embodiments, the modified influenza A HA polypeptides of the present disclosure are generated from a wild-type HA polypeptide of an influenza A virus subtype H1N1. In some embodiments, the modified influenza A HA polypeptides of the present disclosure are generated from a wildtype HA polypeptide of an influenza A virus subtype H3N2. In some embodiments, the modified influenza A HA polypeptides of the present disclosure are generated from a wild-type HA polypeptide of the influenza A virus A / West Virginia / 30 / 2022 strain. In some embodiments, the modified influenza A HA polypeptides of the present disclosure are generated from a wild-type HA polypeptide of the influenza A virus A / Darwin / 06 / 2021 strain. It should be understood that any H1N1 or H3N2 influenza A strain not specifically mentioned herein can be the source of the HA polypeptide for generation of the modified influenza A HA polypeptides according to the present disclosure.Proline Mutations

[0074] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides provided herein comprise at least one proline mutation (e.g., substitution) in the stem region (comprising approximately amino acid residues 18-58 and 293-519 of the modified influenza A HA polypeptide as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 28-67 and 295-520 as indexed by reference to the amino acid sequence of SEQ ID NO: 2). The introduction of the at least one proline mutation (e.g., substitution) is designed to break at least one helicalAttorney Docket No. 0171.0129-PCT structure, such as an a-helix, that appears in the stem region of an influenza A HA polypeptide in a postfusion conformation. Without wishing to be bound by any theory, proline is the only naturally occurring amino acid in which the side chain is bonded to the backbone nitrogen, forming a five-membered pyrrolidine ring. This pyrrolidine ring restricts the rotation of the N-Ca bond, decreasing the backbone conformational entropy of the unfolded form of the protein relative to other naturally occurring amino acids. Thus, without intending to be bound by any theory, introducing a proline substitution may increase the stability of a protein by decreasing the entropic difference between the unfolded and the folded form.

[0075] The stem region of influenza A HA polypeptides consists of a fusion peptide (FP), a N-terminal refolding region 1 (RR1), a central helix (CH), and a C -terminal refolding region 2 (RR2) (see FIG. 1). In some embodiments, the at least one proline mutation (e.g., substitution) is introduced in the fusion peptide of the stem region. In some embodiments, the at least one proline mutation (e.g., substitution) is introduced in the N-terminal refolding region 1 of the stem region. In some embodiments, the at least one proline mutation (e.g., substitution) is introduced in the central helix of the stem region. In some embodiments, the at least one proline mutation (e.g., substitution) is introduced in the C-terminal refolding region 2 of the stem region.

[0076] In some embodiments, the modified influenza A HA polypeptides of the present disclosure comprise one or more proline substitutions at amino acid positions 346, 380, 404, 405, 408, 409, 413, 414, 415, 417, 418, 419, and / or 421 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Specifically, relative to a corresponding wild-type influenza A HA polypeptide, the influenza A HA polypeptide may comprise one or more prefusion conformationstabilizing proline substitutions at amino acid positions 346, 380, 404, 405, 408, 409, 413, 414, 415, 417, 418, 419, and / or 421 as indexed by reference to the amino acid sequence of SEQ ID NO:1.

[0077] In some embodiments, the modified influenza A HA polypeptides of the present disclosure comprise one or more proline substitutions at amino acid positions 347, 381, 405, 409, 410, 414, 415, 416, 418, 419, 420, and / or 422 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. Specifically, relative to a corresponding wild-type influenza A HA polypeptide, the influenza A HA polypeptide may comprise one or more prefusion conformation- stabilizing proline substitutions at amino acid positions 347, 381, 405, 409, 410, 414, 415, 416, 418, 419, 420, and / or 422 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.Attorney Docket No. 0171.0129-PCT

[0078] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides of the present disclosure comprise one or more of the proline substitutions selected from L346P, A380P, N404P, T405P, T408P, A409P, E413P, F414P, N415P, L417P, E418P, K419P, and / or I421P, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the present disclosure comprise one or more of the proline substitutions selected from I347P, A381P, N405P, H409P, Q410P, E414P, F415P, S416P, V418P, E419P, G420P, and / or V422P, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2.Disulfide Bridge Forming Mutations

[0079] In some embodiments, the modified influenza A HA polypeptides provided herein comprise at least two cysteine mutations (e.g., substitutions) at appropriate amino acid positions such that the introduction of the at least two cysteine mutations (e.g., substitutions) form disulfide bonds in a trimer complex formed with the modified influenza A HA polypeptide. Such mutations are referred to as “disulfide bridge forming mutations” throughout the present disclosure. Without wishing to be bound by any theory, the introduced disulfide bonds stabilize the trimer complex in a prefusion conformation, even after exposure to low pH. For stabilizing the prefusion conformation of a trimer complex formed with the modified influenza A HA polypeptide, the amino acid pair chosen for substitution to cysteine should be in close proximity in the prefusion conformation but distant in the postfusion conformation. Such amino acid pair can be identified, for example, by visual inspection of a crystal structure of an influenza A HA polypeptide in a prefusion conformation. Several influenza A HA crystal structures, including those of H1N1 and swine-origin viruses, are available in the RCSB Protein Data Bank (RCSB PDB; rcsb.org / ; Berman et al., Nucleic Acids Research, 2000, 28:235-242). Such amino acid pairs can also be identified by more quantitative selection using computational protein design software, such as BioLuminate™ (Schrodinger LLC, New York, 2015), Discovery Studio™ (Accelrys, San Diego, 2015), MOE™ (Chemical Computing Group Inc., Montreal, 2015), and Rosetta™ (University of Washington, Seattle, 2015).

[0080] In some embodiments, the modified influenza A HA polypeptides provided herein comprise at least two cysteine substitutions that are appropriately positioned so that the at leastAttorney Docket No. 0171.0129-PCT two cysteine substitutions form disulfide bonds linking the HA1 and HA2 subunits of the modified influenza A HA polypeptide in an HA trimer to stabilize the prefusion conformation and reduce or eliminate the postfusion triggering of the HA trimer. In some embodiments, the modified influenza A HA polypeptides provided herein comprise at least two cysteine substitutions that are appropriately positioned so that the at least two cysteine substitutions form a disulfide bridge between a loop region of HA1 and a helix of HA2 of the modified influenza A HA polypeptide in an HA trimer to stabilize the prefusion conformation and reduce or eliminate the postfusion triggering of the HA trimer. In some embodiments, the modified influenza A HA polypeptides provided herein comprise at least two cysteine substitutions that are appropriately positioned so that the at least two cysteine substitutions form disulfide bonds between two adjacent HA2 subunits of the modified influenza A HA polypeptide in an HA trimer to stabilize the prefusion conformation and reduce or eliminate the postfusion triggering of the HA trimer. In some embodiments, the modified influenza A HA polypeptides provided herein comprise at least two cysteine substitutions that are appropriately positioned so that the at least two cysteine substitutions form disulfide bonds between two adjacent HAI protomers of the modified influenza A HA polypeptide in an HA trimer to stabilize the prefusion conformation and reduce or eliminate the postfusion triggering of the HA trimer.

[0081] In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 36 and 398, 37 and 391, 117 and 419, 217 and 232, 219 and 234, 222 and 416, 226 and 230, 235 and 256, and / or 402 and 441, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 36 and 398 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0082] In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 45 and 451, 46 and 392, 46 and 451, 122 and 421, 123 and 420, 123 and 421, 127 and 420, 219 and 234, 221 and 236, 223 and 237, 228 and 232, 234 and 262, 235 and 262, 411 and 424, 411 and 428, 469 and 479, as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modifiedAttorney Docket No. 0171.0129-PCT influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 235 and 262 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0083] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions selected from V36C and S398C, L37C and K391C, E117C and K419C, F217C and A232C, G219C and R234C, R222C and H416C, K226C and E230C, P235C and K256C, and / or K402C and E441C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions selected from I45C and H451C, T46C and Q392C, T46C and H451C, A122C and R421C, S123C and G420C, S123C and R421C, L127C and G420C, T219C and G234C, S221C and R236C, K223C and P237C, A228C and N232C, G234C and N262C, S235C and N262C, 1411C and D424C, 1411C and Y428C, and / or K469C and G479C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0084] Specifically, relative to a corresponding wild-type influenza A HA polypeptide, the influenza A HA polypeptides comprising disulfide bridge-forming cysteine substitutions at amino acid positions 36 and 398 (e.g., V36C and S398C), or amino acid positions 219 and 234 (e.g., G219C and R234C), wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1, were found to be stabilized in the prefusion conformation. Accordingly, in some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 36 and 398 and / or 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 36 and 398 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HAAttorney Docket No. 0171.0129-PCT polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise amino acid substitutions V36C and S398C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise amino acid substitutions G219C and R234C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0085] Likewise, relative to a corresponding wild-type influenza A HA polypeptide, the influenza A HA polypeptides comprising disulfide bridge-forming cysteine substitutions at amino acid positions 235 and 262 (e.g., S235C and N262C), or amino acid positions 411 and 424 (e.g., 1411C and D424C), wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 2 were found to be stabilized in the prefusion conformation. Accordingly, in some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 235 and 262 and / or 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 235 and 262 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the disclosure comprise at least two cysteine substitutions at amino acid positions 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise amino acid substitutions S235C and N262C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptides of the disclosure comprise amino acid substitutions 1411C and D424C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1.Interface Stabilizing Mutations

[0086] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides provided herein comprise one or more amino acid mutations (e.g., substitutions) in the head region (comprising approximately aminoAttorney Docket No. 0171.0129-PCT acid positions 59-292 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 67-293 as indexed by reference to the amino acid sequence of SEQ ID NO: 2) and / or stem region (comprising approximately amino acid residues 18-58 and 293- 519 of the modified influenza A HA polypeptide as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 28-67 and 295-520 as indexed by reference to the amino acid sequence of SEQ ID NO: 2), wherein the one or more amino acid mutations (e.g., substitutions) stabilize the modified influenza A HA polypeptides in a prefusion conformation through interface stabilization. Such mutations are also referred to as “interface stabilizing mutations” throughout the present disclosure. Without wishing to be bound by any theory, interface stabilization can be achieved through various ways, such as cavity filling and formation of polar interaction with a neighboring residue (e.g., formation of a hydrogen bond or salt bridge). Accordingly, in some embodiments, the one or more amino acid mutations (e.g., substitutions) that stabilize the modified influenza A HA polypeptides in a prefusion conformation through interface stabilization comprise at least one cavity filling mutation (e.g., substitution) in the stem region. In some embodiments, the one or more amino acid mutations (e.g., substitutions) that stabilize the modified influenza A HA polypeptides in a prefusion conformation through interface stabilization comprise one or more amino acid mutations (e.g., substitutions) in the head and / or stem region to form a polar interaction with a neighboring amino acid residue. In some embodiments, the polar interaction comprises a hydrogen bond. In some embodiments, the polar interaction comprises a salt bridge.

[0087] The term “cavity filling mutation” refers to a mutation that results in a substitution of an amino acid residue in a parental polypeptide, such as a wild-type influenza A HA polypeptide (e.g., HA polypeptide of A / West Virginia / 30 / 2022 or A / Darwin / 06 / 2021) 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 (e.g., substitutions) can contribute to stabilizing the pH-sensitive interfaces between the head and stem regions and thus, stabilize the prefusion conformation of the modified influenza A HA polypeptide. The cavities in the prefusion conformation of a wild-type influenza A HA polypeptide can be identified, for example, by visual inspection of a crystal structure of an influenza A HA polypeptide in a prefusion conformation. Several influenza A HA crystal structures, including those of H1N1 and swine-origin viruses, are available in the RCSB Protein Data Bank (RCSBAttorney Docket No. 0171.0129-PCTPDB; rcsb.org / ; Berman et al., Nucleic Acids Research, 2000, 28:235-242). Such amino acid pairs can also be identified by more quantitative selection using computational protein design software, such as BioLuminate™ (Schrodinger LLC, New York, 2015), Discovery Studio™ (Accelrys, San Diego, 2015), MOE™ (Chemical Computing Group Inc., Montreal, 2015), and Rosetta™ (University of Washington, Seattle, 2015). The amino acids to be substituted for cavity filling mutations typically include small aliphatic amino acids (e.g., Glycine (G), Alanine (A), and Valine (V)) or small polar amino acids (e.g., Serine (S) and Threonine (T)). They may also include amino acids that are buried in the prefusion conformation but exposed to solvent in the postfusion conformation. The amino acid substitutions to be introduced can be large aliphatic amino acids (e.g., Isoleucine (I), Leucine (L) and Methionine (M)) or large aromatic amino acids (e.g., Histidine (H), Phenylalanine (F), Tyrosine (Y) and Tryptophan (W)) or an amino acid with a basic side chain at neutral pH, (e.g., Arginine (R), Lysine (K), and Histidine (H)). For example, Eysine (K) mutations can be introduced in the stem region to fill the cavity among central helices and to form polar interaction with a neighboring residue.

[0088] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides of the disclosure comprise at least one interface stabilizing mutations, such as cavity filling substitution, at amino acid position 395, 402, 412, 433, and / or 443 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the disclosure comprise at least one interface stabilizing mutations, such as cavity filling substitution, at amino acid position 396, 403, and / or 413 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0089] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides of the present disclosure comprise one or more of the cavity filling substitutions selected from K395M, K402L, K412M, L433I, L443M, L443Y, and / or L443K, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the present disclosure comprise one or more of the cavity filling substitutions selected from K396M, K403L, and K413M, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2.Attorney Docket No. 0171.0129-PCT

[0090] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the one or more amino acid mutations (e.g., substitutions) that stabilize the modified influenza A HA polypeptide in the prefusion conformation through interface stabilization comprise one or more substitutions at amino acid positions 395, 402, 412, 433, and / or 443 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the one or more amino acid mutations (e.g., substitutions) that stabilize the modified influenza A HA polypeptide in the prefusion conformation through interface stabilization comprise one or more substitutions at amino acid positions 396, 403, and / or 413 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the one or more amino acid mutations that stabilize the modified influenza A HA polypeptide in the prefusion conformation through interface stabilization comprise one or more substitutions selected from K395M, K402L, K412M, L433I, L443M, L443Y, and / or L443K, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid mutations that stabilize the modified influenza A HA polypeptide in the prefusion conformation through interface stabilization comprise one or more substitutions selected from K396M, K403L, and K413M, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2. pH Sensor Inactivating Mutations

[0091] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides provided herein comprise one or more amino acid mutations (e.g., one or more amino acid substitutions) in the head region (comprising approximately amino acid positions 59-292 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 67-293 as indexed by reference to the amino acid sequence of SEQ ID NO: 2) and / or stem region (comprising approximately amino acid residues 18-58 and 293-519 of the modified influenza A HA polypeptide as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 28-67 and 295-520 as indexed by reference to the amino acid sequence of SEQ ID NO: 2), wherein the introduction of the one or more amino acid mutations inactivate one or more pH sensors in the head and / or stem region. Such mutations are also referred to as “pH sensor inactivating mutations” or “pH sensor knockout mutations” throughout the present disclosure. The transition of an influenza A HA polypeptide from the high-energy, metastable prefusion state to a postfusionAttorney Docket No. 0171.0129-PCT conformation is triggered by low pH. Thus, without wishing to be bound by any theory, inactivating one or more pH sensors of the influenza A HA polypeptides may contribute to stabilizing the prefusion conformation of the modified influenza A HA polypeptide. Any amino acid residue or combination of residues acting as a pH sensor in the influenza A HA polypeptide known in the art or identified in the future can be substituted to inactivate such a pH sensor. For example, pH sensors in the stem region and / or at the head interface can be inactivated by substituting Histidine (H) with a hydrophobic residue such as Leucine (L) or Methionine (M).

[0092] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the one or more amino acid mutations (e.g., substitutions) that inactivate one or more pH sensors in the head and / or stem region of the modified influenza A HA polypeptides of the disclosure are at amino acid positions 370, 416, 455, and / or 486 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wildtype influenza A HA polypeptide, the one or more amino acid mutations (e.g., substitutions) that inactivate one or more pH sensors in the head and / or stem region of the modified influenza A HA polypeptides of the disclosure are at amino acid positions 34, 371, 451, 456, 487, and / or 504 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0093] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides of the present disclosure comprise one or more pH sensor-inactivating substitutions selected from H370F, H370P, H370W, H370Y, H416F, H416P, H416Y, H455Q, H486F, H486P, H486Q, and / or H486Y, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wild-type influenza A HA polypeptide, the modified influenza A HA polypeptides of the present disclosure comprise one or more pH sensor-inactivating substitutions selected from H34Q, H371F, H371P, H371W, H371Y, H451F, H451Q, H451W, H487F, H487P, H487Q, H487Y, H504F, H504W, H504Y, and / or T456V, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0094] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the one or more amino acid substitutions that stabilize the modified influenza A HA polypeptide in the prefusion conformation through pH sensor inactivation comprise one or more substitutions at amino acid positions 370, 416, 455, and / or 486 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to a corresponding wild-Attorney Docket No. 0171.0129-PCT type influenza A HA polypeptide, the one or more amino acid substitutions that stabilize the modified influenza A HA polypeptide in the prefusion conformation through pH sensor inactivation comprise one or more substitutions at amino acid positions 34, 371, 451, 456, 487, and / or 504 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0095] In some embodiments, the one or more amino acid mutations that stabilize the modified influenza A HA polypeptide in the prefusion conformation through pH sensor inactivation comprise one or more substitutions selected from H370F, H370P, H370W, H370Y, H416F, H416P, H416Y, H455Q, H486F, H486P, H486Q, and / or H486Y, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more amino acid mutations that stabilize the modified influenza A HA polypeptide in the prefusion conformation through pH sensor inactivation comprise one or more substitutions selected from H34Q, H371F, H371P, H371W, H371Y, H451F, H451Q, H451W, H487F, H487P, H487Q, H487Y, H504F, H504W, H504Y, and / or T456V, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2.Sialic Acid Binding Interfering Mutations

[0096] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides provided herein comprise at least one amino acid mutation (e.g., substitution) in the head region (comprising approximately amino acid positions 59-292 as indexed by reference to the amino acid sequence of SEQ ID NO: 1 or approximately amino acid residues 67-293 as indexed by reference to the amino acid sequence of SEQ ID NO: 2), wherein the at least one amino acid mutation reduces sialic acid binding of the modified influenza A HA polypeptide as compared to a corresponding wild-type influenza A HA polypeptide without the at least one amino acid mutation (e.g., substitution). Such mutations are also referred to as “sialic acid binding interfering mutations” throughout the present disclosure. In some embodiments, the at least one amino acid mutation (e.g., substitution) that reduces sialic acid binding of the modified influenza A HA polypeptide does not impair the integrity of the epitope targeted by RBS neutralizing antibodies.

[0097] In some embodiments, the at least one amino acid substitution that reduces sialic acid binding of the modified influenza A HA polypeptide is at amino acid position 108, 150, 204, 208, 240, and / or 242 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution that reduces sialic acid binding of theAttorney Docket No. 0171.0129-PCT modified influenza A HA polypeptide is at amino acid position 114, 152, 210, and / or 242 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0098] In some embodiments, the at least one sialic acid binding interfering mutation in the modified influenza A HA polypeptide of the disclosure comprises the amino acid substitution selection from Y108I, T150A, T150D, D204K, L208A, L208D, Q240A, and / or G242N, wherein the amino acid position is as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one sialic acid binding interfering mutation in the modified influenza A HA polypeptide of the disclosure comprises the amino acid substitution selection from Y114I, S152A, L210A, L210D, and / or I242A, wherein the amino acid position is as indexed by reference to the amino acid sequence of SEQ ID NO: 2.Combinations of mutations

[0099] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides of the present disclosure comprise two or more (e.g., three, four, or five) of the modifications (e.g., amino acid substitutions) selected from: a) at least one proline mutation (e.g., substitution) in the stem region as defined elsewhere herein, wherein the at least one proline mutation breaks at least one helical structure in the stem region of the modified influenza A HA polypeptide in a postfusion conformation; b) at least two cysteine mutations (e.g., substitutions), wherein the at least two cysteine mutations form a disulfide bridge in a trimer complex formed with the modified influenza A HA polypeptide; c) one or more amino acid mutations (e.g., substitutions) in the head and / or stem region as defined elsewhere herein, wherein the one or more amino acid mutations stabilize the modified influenza A HA polypeptide in a prefusion conformation through interface stabilization; d) one or more amino acid mutations (e.g., substitutions) in the head and / or stem region as defined elsewhere herein, wherein the one or more amino acid mutations inactivate one or more pH sensors in the head and / or stem region; and e) at least one amino acid mutation (e.g., substitution) in the head region as defined elsewhere herein, wherein the at least one amino acid mutation reduces sialic acid binding of the modified influenza A HA polypeptide as compared to a corresponding wild-type influenza A HA polypeptide without the at least one amino acid mutation (e.g., substitution). For instance, one or more pH sensor inactivating mutations (e.g., substitutions) may be combined with one or more interface stabilizing mutations (e.g., substitutions), or one or more proline mutations (e.g.,Attorney Docket No. 0171.0129-PCT substitutions) may be combined with one or more cysteine mutations (e.g., substitutions) that stabilize the modified influenza A HA polypeptide in the prefusion conformation.

[0100] For example, in some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein are combined with one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more interface stabilizing mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more interface stabilizing mutations (e.g., substitutions) disclosed herein are combined with one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein are combined with one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein.

[0101] More than two different types of modifications can also be combined. For instance, in some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein and one or more interface stabilizing mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein and one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein. InAttorney Docket No. 0171.0129-PCT some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein and one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein and one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more interface stabilizing mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein.

[0102] In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein, one or more interface stabilizing mutations (e.g., substitutions) disclosed herein, and one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein, one orAttorney Docket No. 0171.0129-PCT more interface stabilizing mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein, one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein, one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein, one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interfering mutations disclosed herein.

[0103] In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein, one or more interface stabilizing mutations (e.g., substitutions) disclosed herein, and one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein, one or more interface stabilizing mutations (e.g., substitutions) disclosed herein, one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein, one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein.

[0104] In some embodiments, one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein are combined with one or more interface stabilizing mutations (e.g., substitutions) disclosed herein, one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interfering mutations (e.g., substitutions)Attorney Docket No. 0171.0129-PCT disclosed herein. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more disulfide bridge forming mutations (e.g., substitutions) disclosed herein, one or more interface stabilizing mutations (e.g., substitutions) disclosed herein, one or more pH sensor inactivating mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interfering mutations (e.g., substitutions) disclosed herein.

[0105] In particular embodiments, the influenza A HA polypeptides provided herein comprise: a) at least one proline substitution relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least one proline substitution is at amino acid position 346, 380, 404, 405, 408, 409, 413, 414, 415, 417, 418, 419, and / or 421 as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) at least two cysteine substitutions relative to a corresponding wildtype influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 36 and 398, 37 and 391, 117 and 419, 217 and 232, 219 and 234, 222 and 416, 226 and 230, 235 and 256, and / or 402 and 441, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; c) at least one interface stabilization amino acid substitution relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least one interface stabilization amino acid substitution is at amino acid position 395, 402, 412, 433, and / or 443 as indexed by reference to the amino acid sequence of SEQ ID NO: 1; d) one or more pH sensor knock-out amino acid substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the one or more pH sensor knock-out amino acid substitutions are at amino acid position 370, 416, 455, and / or 486 as indexed by reference to the amino acid sequence of SEQ ID NO: 1; and / or e) at least one amino acid substitution relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least one amino acid substitution is at amino acid position 108, 150, 204, 208, 240, and / or 242 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In other embodiments, the influenza A HA polypeptides provided herein comprise: a) at least one proline substitution relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least one proline substitution is at amino acid position 347, 381, 405, 409, 410, 414, 415, 416, 418, 419, 420, and / or 422 as indexed by reference to the amino acid sequence of SEQ ID NO: 2; b) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 45 and 451, 46 and 392, 46 and 451, 122 and 421, 123 and 420, 123 and 421, 127 and 420, 219 and 234, 221 and 236, 223Attorney Docket No. 0171.0129-PCT and 237, 228 and 232, 234 and 262, 235 and 262, 411 and 424, 411 and 428, 469 and 479, as indexed by reference to the amino acid sequence of SEQ ID NO: 2; c) at least one interface stabilization amino acid substitution relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least one interface stabilization amino acid substitution is at amino acid position 396, 403, and / or 413 as indexed by reference to the amino acid sequence of SEQ ID NO: 2; d) one or more pH sensor knock-out amino acid substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the one or more pH sensor knock-out amino acid substitutions are at amino acid position 34, 371 , 451 , 456, 487, and / or 504 as indexed by reference to the amino acid sequence of SEQ ID NO: 2; and / or e) at least one amino acid substitution relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least one amino acid substitution is at amino acid position 114, 152, 210, and / or 242 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.Exemplary Modified Influenza A HA polypeptides

[0106] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions at:1) amino acid position 395;2) amino acid position 402;3) amino acid position 412;4) amino acid position 433;5) amino acid position 443;6) amino acid positions 25 and 45;7) amino acid positions 395 and 447;8) amino acid positions 395, 447, 449, and 450;9) amino acid positions 395, 447, 449, 450, and 36;10) amino acid positions 395, 447, and 370;11) amino acid positions 395 and 447;12) amino acid positions 412 and 442;13) amino acid positions 217 and 232;14) amino acid positions 219 and 234;15) amino acid positions 226 and 230;Attorney Docket No. 0171.0129-PCT16) amino acid positions 235 and 256;17) amino acid positions 117 and 419;18) amino acid positions 222 and 426;19) amino acid positions 402 and 441;20) amino acid positions 37 and 391;21) amino acid positions 36 and 398;22) amino acid position 370;23) amino acid position 416;24) amino acid position 455;25) amino acid position 486;26) amino acid position 380;27) amino acid positions 409 and 413;28) amino acid position 413;29) amino acid positions 413 and 418;30) amino acid positions 413 and 419;31) amino acid positions 414 and 417;32) amino acid position 421;33) amino acid po sition 419;34) amino acid position 346;35) amino acid position 404;36) amino acid positions 404 and 413;37) amino acid position 415;38) amino acid position 405;39) amino acid position 408;40) amino acid position 204;41) amino acid position 242;42) amino acid position 208;43) amino acid position 240;44) amino acid position 150; or45) amino acid position 108, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.Attorney Docket No. 0171.0129-PCT

[0107] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions at:1) amino acid positions 122 and 421;2) amino acid positions 228 and 232;3) amino acid position 381;4) amino acid position 414;5) amino acid positions 414 and 419;6) amino acid positions 414 and 420;7) amino acid positions 415 and 418;8) amino acid positions 234 and 262;9) amino acid position 34;10) amino acid position 371 ;11) amino acid position 409;12) amino acid position 451 ;13) amino acid position 487;14) amino acid position 504;15) amino acid positions 45 and 451 ;16) amino acid position 242;17) amino acid position 347;18) amino acid positions 411 and 424;19) amino acid positions 411 and 428;20) amino acid positions 223 and 237;21) amino acid positions 396 and 448;22) amino acid positions 396, 448, and 371;23) amino acid positions 396, 448, 450, and 451;24) amino acid positions 396, 448, 450, 451, and 45;25) amino acid position 396;26) amino acid positions 396 and 448;27) amino acid position 403;28) amino acid position 413;Attorney Docket No. 0171.0129-PCT29) amino acid positions 413 and 443;30) amino acid positions 469 and 479;31) amino acid positions 127 and 420;32) amino acid position 210;33) amino acid position 444;34) amino acid position 405;35) amino acid positions 405 and 414;36) amino acid positions 410 and 414;37) amino acid positions 123 and 420;38) amino acid positions 123 and 421;39) amino acid position 152;40) amino acid positions 221 and 236;41) amino acid positions 235 and 262;42) amino acid position 416;43) amino acid positions 46 and 451 ;44) amino acid positions 46 and 392;45) amino acid positions 219 and 234;46) amino acid position 456;47 ) amino acid po sition 418;48) amino acid position 422; or49) amino acid position 114 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0108] As shown herein, such one or more amino acid substitution(s) can stabilize the modified influenza A HA polypeptide in a prefusion (closed) conformation, as, e.g., measured by an increased binding of the modified influenza A HA polypeptide to a stem region- specific antibody (e.g., CR9114) as compared to the corresponding wild-type influenza A HA polypeptide. In some embodiments, stabilization of the modified influenza A HA polypeptide in the prefusion conformation is measured by determining the binding ratio of a stem region- specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) as compared to the corresponding wildtype influenza A HA polypeptide. In some embodiments, the binding ratio of stem region- specific antibody (e.g., CR9114) to RBS-specific antibody (e.g., R95-1D05) is at least two-fold higher forAttorney Docket No. 0171.0129-PCT the modified influenza A HA polypeptide as compared to the corresponding wild-type influenza A HA polypeptide. In some embodiments, stabilization of the prefusion conformation is measured by determining the binding of the modified influenza A HA polypeptide to a stem region- specific antibody (e.g., CR9114) and by determining the binding ratio of a stem region- specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) as compared to the corresponding wild- type influenza A HA polypeptide.

[0109] In some embodiments, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions at amino acid positions 36 and 398 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions at amino acid positions 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions at amino acid positions 235 and 262 as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions at amino acid positions 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0110] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions:1) K395M;2) K402L;3) K412M;4) L433I;5) L443M;6) L443Y;7) L443K;8) H25Q and H45N;9) K395I and E447I;10) K395I, E447I, E449M, and R450M;11) K395I, E447I, E449M, R450M, and V36M;12) K395I, E447I, and H370W;Attorney Docket No. 0171.0129-PCT ) K395M and E447L; ) K412M and L442I; ) F217C and A232C; ) G219C and R234C; ) K226C and E230C; ) P235C and K256C; ) E117C and K419C; ) G221ins, R222C, G222ins, and H416C;) K402C and E441C; ) L37C and K391C; ) V36C and S398C; ) H370F; ) H370P; ) H370W; ) H370Y; ) H416F; ) H416P; ) H416Y; ) H455Q; ) H486F; ) H486P; ) H486Q; ) H486Y; ) A380P; ) A409P and E413P; ) E413P; ) E413P and E418P; ) E413P and K419P; ) F414P and L417P; ) I421P; ) K419P;Attorney Docket No. 0171.0129-PCT44) L346P;45) N404P;46) N404P and E413P;47) N415P;48) T405P;49) T408P;50) D204K;51) G242N;52) L208A;53) L208D;54) Q240A;55) T150A;56) T150D; or57) Y108I, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0111] In some embodiments, relative to a corresponding wild- type influenza A HA polypeptide, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions:1) A122C and R421C;2) A228 and N232C;3) A381P;4) E414P;5) E414P and E419P;6) E414P and G420P;7) F415P and -V418P;8) G234C and N262C;9) H34Q;10) H371F;11) H371P;12) H371W;Attorney Docket No. 0171.0129-PCT ) H371Y; ) H409P; ) H451F; ) H451Q; ) H451W; ) H487F; ) H487P; ) H487Q; ) H487Y; ) H504F; ) H504W; ) H504Y; ) I45C and H451C; ) I242A; ) I347P; ) I411C and D424C; ) I411C and Y428C; ) K223C and P237C; ) K396I and E448I; ) K396I, E448I, and H371 W; ) K396I, E448I, Q450M, and H45 IM; ) K396I, E448I, Q450M, H45 IM, and I45M;) K396M; ) K396M and E448L; ) K403L; ) K413M; ) K413M and L443I; ) K469C and G479C; ) L127C and G420C; ) L210A; ) L210D;Attorney Docket No. 0171.0129-PCT44) L444D;45) L444K;46) L444M;47) L444Y;48) N405P;49) N405P and E414P;50) Q410P and E414E;51) S123C and G420C;52) S123C and R421C;53) S152A;54) S221C and R236C;55) S235C and N262C;56) S416P;57) T46C and H451C;58) T46C and Q392C;59) T219C and G234C;60) T456V;61) V418P;62) V422P; or63) Y114I, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0112] In some embodiments, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions V36C and S398C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions G219C and R234C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza A HA polypeptides provided herein comprise amino acid substitutions S235C and N262C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified influenza A HA polypeptides provided herein comprise amino acidAttorney Docket No. 0171.0129-PCT substitutions 1411C and D424C, wherein the amino acid positions are as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0113] A representative modified influenza A HA polypeptide according to the present disclosure may have the amino acid sequence set forth in SEQ ID NO: 3:MKAILVVMLYTFTTANADTLCIGYHANNSTDTVDTCLEKNVTVTHSVNLLEDK HNGKLCKLRGVAPLHLGQCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCY PGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDNGVTAACSHAGARSFYKN EIWEVKKGKSYPKINQTYINDKGKEVEVEWGIHHPPTITDQESEYQNADAYVFV GTSRYSKKFKPEIAARPKVRDQAGRMNYYWTEVEPGDKITFEATGNEVAPRYAF TMEKEAGSGIIISDTPVHDCNATCQTPEGAINTSEPFQNVHPITIGKCPKYVRSTKE REATGERNVPSIQSRGEFGAIAGFIEGGWTGMVDGWYGYHHQNDQGSGYAADE KSTQNAIDKITNKVNCVIEKMNTQFTAVGKEFNHEEKRIENENKKVDDGFEDVW TYNAEEEVEEENERTEDYHDSNVKNEYEKVRHQEKNNAKEIGNGCFEFYHKCD NTCMESVKNGTYDYPKYSEEAKENREKIDGVKEDSTRIYQIEAIYSTVASSEVEV VSEGAISFWMCSNGSEQCRICI (SEQ ID NO: 3).

[0114] Another representative modified influenza A HA polypeptide according to the present disclosure may have the amino acid sequence set forth in SEQ ID NO: 5:MKAIEVVMEYTFTTANADTECIGYHANNSTDTVDTVEEKNVTVTHSVNEEEDK HNGKECKERGVAPEHEGQCNIAGWIEGNPECESESTARSWSYIVETSNSDNGTCY PGDFINYEEEREQESSVSSFERFEIFPKTSSWPNHDSDNGVTAACSHAGARSFYKN EIWEVKKGKSYPKINQTYINDKGKEVEVEWGIHHPPTITDQESEYQNADAYVFV CTSRYSKKFKPEIAACPKVRDQAGRMNYYWTEVEPGDKITFEATGNEVAPRYAF TMEKEAGSGIIISDTPVHDCNATCQTPEGAINTSEPFQNVHPITIGKCPKYVRSTKE REATGERNVPSIQSRGEFGAIAGFIEGGWTGMVDGWYGYHHQNDQGSGYAADE KSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHEEKRIENENKKVDDGFEDVW TYNAEEEVEEENERTEDYHDSNVKNEYEKVRHQEKNNAKEIGNGCFEFYHKCD NTCMESVKNGTYDYPKYSEEAKENREKIDGVKEDSTRIYQIEAIYSTVASSEVEV VSEGAISFWMCSNGSEQCRICI (SEQ ID NO: 5).

[0115] A further representative modified influenza A HA polypeptide according to the present disclosure may have the amino acid sequence set forth in SEQ ID NO: 7:Attorney Docket No. 0171.0129-PCTMKTIIALSNILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATE LVQNSSIGEICGSPHQILDGGNCTLIDALLGDPQCDGFQNKEWDLFVERSRANSN CYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRL NWLTSLNNIYPAQNVTMPNKEQFDKLYIWGVHHPDTDKNQISLFAQSSGRITVST KRSQQAVIPNIGCRPRIRDIPSRISIYWTIVKPGDILLICSTGNLIAPRGYFKIRSGKS SIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMR NVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAI DQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLV ALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRN ETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAMSCFLLCIALLGFIMW ACQKGNIRCNICI (SEQ ID NO: 7).

[0116] A yet another representative modified influenza A HA polypeptide according to the present disclosure may have the amino acid sequence set forth in SEQ ID NO: 9:MKTIIALSNILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATE LVQNSSIGEICGSPHQILDGGNCTLIDALLGDPQCDGFQNKEWDLFVERSRANSN CYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRL NWLTSLNNIYPAQNVTMPNKEQFDKLYIWGVHHPDTDKNQISLFAQSSGRITVST KRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKS SIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMR NVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAI DQINGKLNRLIGKTNEKFHQCEKEFSEVEGRVQCLEKYVEDTKIDLWSYNAELL VALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIR NETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAMSCFLLCIALLGFIM W ACQKGNIRCNICI (SEQ ID NO: 9).

[0117] Accordingly, in some embodiments, the influenza A HA polypeptides of the present disclosure 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: 3. In some embodiments, the influenza A HA polypeptides of the present disclosure 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 toAttorney Docket No. 0171.0129-PCT the amino acid sequence of SEQ ID NO: 5. In some embodiments, the influenza A HA polypeptides of the present disclosure 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: 7. In some embodiments, the influenza A HA polypeptides of the present disclosure 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: 9. In some embodiments, the influenza A HA polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the influenza A HA polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 5. In some embodiments, the influenza A HA polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 7. In some embodiments, the influenza A HA polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0118] Similar to wild- type influenza A HA polypeptides, the modified influenza A HA polypeptides disclosed herein are capable of forming a trimeric HA complex through symmetry operations. Thus, in some embodiments, provided herein is a trimeric influenza A HA polypeptide complex comprising three copies of any of the modified influenza A HA polypeptides according to the present disclosure. In certain embodiments, the trimeric influenza A HA polypeptide complex of the present disclosure has improved stability in the prefusion conformation as compared to a trimeric influenza HA polypeptide complex prepared from the corresponding wildtype influenza A HA polypeptide without the amino acid substitutions. 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 influenza A HA polypeptide complex of the present disclosure to a stem region- specific antibody (e.g., CR9114) as compared to a trimeric influenza A HA polypeptide complex prepared from the corresponding wild-type influenza A HA polypeptide without the amino acid 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 influenza A HA polypeptide complex prepared from the corresponding wild-type influenza A HA polypeptide without the amino acid substitutions.Attorney Docket No. 0171.0129-PCT

[0119] 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 a corresponding wild-type influenza A HA polypeptide without the amino acid substitutions. In some embodiments, the trimeric influenza A HA polypeptide complex of the present disclosure has a comparable immunogenicity as a trimeric influenza A HA polypeptide complex prepared from a corresponding wild-type influenza A HA polypeptide without the amino acid substitutions. Immunogenicity can be measured using any methods known in the art. In some embodiments, the immunogenicity is measured using a hemagglutination-inhibition assay (HAI)Positional variation of mutations or substitutions

[0120] While modified influenza A HA polypeptides with particular amino acid mutations or substitutions at exemplary amino acid positions are provided herein, the present disclosure also contemplates amino acid mutations or substitutions in close proximity to the specified amino acid positions. Therefore, in some embodiments, the present disclosure also comprises amino acid positions within three residues (e.g., within one or two residues) of an amino acid position specified herein. For example, the disclosure of a mutation or substitution at amino acid position 36 of influenza A HA polypeptide may also include a mutation or substitution at position 33, 34, 35, 37, 38 or 39, respectively.

[0121] This is illustrated by the exemplified modified influenza A HA polypeptides described herein, in particular with respect to mutations or substitutions resulting in disulfide bridge formation or proline stabilization. For instance, amino acid substitutions to cystines at amino acid positions 122 and 421, 123 and 420, as well as 123 and 421, of SEQ ID NO: 2 all result in disulfide bridge formation. Likewise, amino acid substitutions to cystines at amino acid positions 45 and 451 and 46 and 451 of SEQ ID NO: 2 all result in disulfide bridge formation. Similarly, amino acid substitutions to proline at either amino acid position 416 or 418 of SEQ ID NO: 2 stabilized the exemplified influenza A HA polypeptides in the prefusion conformation. Another example is amino acid substitutions to proline at amino acid positions 414 and 419 or, alternatively, at amino acid positions 414 and 420 of SEQ ID NO: 2, which achieved the same or a similar effect.Nucleic Acid Construction and Expression

[0122] The present disclosure further provides artificial nucleic acid molecules encoding the disclosed modified influenza A HA polypeptides. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. The modified influenza A HAAttorney Docket No. 0171.0129-PCT 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 influenza A 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 influenza A HA polypeptide. The synthesized DNA or mRNA sequences encoding the modified influenza A HA polypeptides of the 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.

[0123] Accordingly, in some embodiments, provided herein is an artificial nucleic acid encoding the modified influenza A HA polypeptides of the present disclosure. In some embodiments, 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. In some embodiments, the artificial nucleic acid comprises both DNA and RNA.

[0124] A representative codon-optimized mRNA sequence encoding the influenza A HA polypeptides of SEQ ID NO: 3 is set forth in SEQ ID NO: 4:AUGAAGGCUAUCUUGGUCGUCAUGUUGUACACCUUCACCACCGCUAACGC UGAUACCUUGUGCAUCGGUUACCACGCUAACAACUCCACUGAUACCGUCGA UACCUGUUUGGAGAAGAACGUCACCGUCACCCACUCUGUCAACUUGUUGG AGGAUAAGCACAACGGUAAGUUGUGCAAGUUGAGGGGUGUCGCUCCCUUG CACUUGGGUCAGUGCAACAUCGCUGGUUGGAUCUUGGGCAACCCCGAGUG CGAAUCCUUGUCCACUGCUCGAUCCUGGUCCUACAUCGUUGAAACGAGCAA CUCCGAUAACGGUACUUGCUACCCCGGUGAUUUCAUCAACUACGAGGAGU UGAGGGAACAGUUGUCCUCUGUCUCCUCCUUCGAGAGGUUCGAGAUUUUC CCCAAGACCUCCUCAUGGCCCAACCAUGAUUCCGAUAACGGUGUUACUGCU GCUUGCUCUCAUGCUGGUGCUAGGUCCUUCUACAAGAACUUGAUCUGGUU GGUCAAGAAGGGUAAGUCCUACCCCAAGAUCAACCAGACCUACAUCAACGAttorney Docket No. 0171.0129-PCTAUAAGGGUAAGGAGGUCUUGGUGUUGUGGGGUAUCCACCACCCCCCUACCAUCACCGAUCAGGAGUCCCUCUACCAGAACGCUGAUGCUUAUGUGUUCGUUGGUACUUCCAGGUACUCCAAGAAGUUCAAGCCCGAGAUCGCUGCUAGGCCCAAGGUCAGGGAUCAGGCUGGUAGGAUGAACUACUACUGGACCUUGGUCGAGCCCGGUGAUAAGAUCACCUUCGAGGCUACCGGAAACUUGGUUGCCCC UAGGUACGCUUUCACCAUGGAGAAGGAAGCUGGUUCUGGUAUCAUCAUCU CCGAUACCCCUGUCCACGAUUGCAACGCUACCUGCCAGACCCCCGAAGGUG CUAUCAACACCUCCUUGCCCUUCCAGAACGUCCAUCCGAUCACGAUCGGUA AGUGCCCCAAGUAUGUUCGCUCUACCAAGUUGAGGUUGGCUACCGGUUUG AGGAACGUUCCCUCCAUCCAGUCCAGGGGUUUGUUUGGUGCUAUCGCUGG UUUCAUCGAGGGUGGUUGGACCGGUAUGGUCGAUGGUUGGUACGGUUACC ACCAUCAGAACGAUCAGGGUUCUGGUUACGCUGCUGAUUUGAAGUCUACC CAGAACGCUAUCGAUAAGAUCACCAACAAGGUCAACUGUGUUAUCGAGAA GAUGAACACCCAGUUCACCGCUGUCGGUAAGGAGUUCAACCACUUGGAGA AGAGGAUCGAGAACUUGAACAAGAAGGUCGAUGAUGGUUUCUUGGAUGUC UGGACCUACAACGCUGAGUUGUUGGUCUUGUUGGAGAACGAGAGGACCUU GGAUUACCACGAUUCCAACGUCAAGAACUUGUACGAGAAGGUCAGGCACC AGUUGAAGAACAAUGCCAAGGAGAUCGGGAACGGUUGCUUCGAGUUCUAC CACAAGUGUGAUAACACCUGUAUGGAGUCCGUUAAGAACGGUACCUACGA UUACCCCAAGUACUCCGAGGAGGCUAAGUUGAACAGGGAGAAGAUCGAUG GUGUCAAGUUGGAUUCCACCAGGAUCUACCAGAUCUUGGCGAUCUACUCU ACUGUCGCUAGUUCCUUGGUCUUGGUUGUUUCCCUUGGUGCUAUCUCCUU CUGGAUGUGCUCCAACGGUUCCUUGCAGUGCAGGAUCUGCAUCUAAUAA (SEQ ID NO: 4).

[0125] A representative codon-optimized mRNA sequence encoding the influenza A HA polypeptide of SEQ ID NO: 5 is set forth in SEQ ID NO: 6:AUGAAGGCUAUCUUGGUCGUCAUGUUGUACACCUUCACCACCGCUAACGCUGAUACCUUGUGCAUCGGUUACCACGCUAACAACUCCACUGAUACCGUCGAUACCGUCUUGGAGAAGAACGUCACCGUCACCCACUCUGUCAACUUGUUGG AGGAUAAGCACAACGGUAAGUUGUGCAAGUUGAGGGGUGUCGCUCCCUUG CACUUGGGUCAGUGCAACAUCGCUGGUUGGAUCUUGGGCAACCCCGAGUGAttorney Docket No. 0171.0129-PCTCGAAUCCUUGUCCACUGCUCGAUCCUGGUCCUACAUCGUUGAAACGAGCAACUCCGAUAACGGUACUUGCUACCCCGGUGAUUUCAUCAACUACGAGGAGUUGAGGGAACAGUUGUCCUCUGUCUCCUCCUUCGAGAGGUUCGAGAUUUUCCCCAAGACCUCCUCAUGGCCCAACCAUGAUUCCGAUAACGGUGUUACUGCUGCUUGCUCUCAUGCUGGUGCUAGGUCCUUCUACAAGAACUUGAUCUGGUUGGUCAAGAAGGGUAAGUCCUACCCCAAGAUCAACCAGACCUACAUCAACGAUAAGGGUAAGGAGGUCUUGGUGUUGUGGGGUAUCCACCACCCCCCUACCAUCACCGAUCAGGAGUCCCUCUACCAGAACGCUGAUGCUUAUGUGUUCGUUUGUACUUCCAGGUACUCCAAGAAGUUCAAGCCCGAGAUCGCUGCUUGUCCCAAGGUCAGGGAUCAGGCUGGUAGGAUGAACUACUACUGGACCUUGGUCGAGCCCGGUGAUAAGAUCACCUUCGAGGCUACCGGAAACUUGGUUGCCCCUAGGUACGCUUUCACCAUGGAGAAGGAAGCUGGUUCUGGUAUCAUCAUCUCCGAUACCCCUGUCCACGAUUGCAACGCUACCUGCCAGACCCCCGAAGGUGCUAUCAACACCUCCUUGCCCUUCCAGAACGUCCAUCCGAUCACGAUCGGUAAGUGCCCCAAGUAUGUUCGCUCUACCAAGUUGAGGUUGGCUACCGGUUUGAGGAACGUUCCCUCCAUCCAGUCCAGGGGUUUGUUUGGUGCUAUCGCUGGUUUCAUCGAGGGUGGUUGGACCGGUAUGGUCGAUGGUUGGUACGGUUACCACCAUCAGAACGAUCAGGGUUCUGGUUACGCUGCUGAUUUGAAGUCUACCCAGAACGCUAUCGAUAAGAUCACCAACAAGGUCAACUCGGUUAUCGAGAAGAUGAACACCCAGUUCACCGCUGUCGGUAAGGAGUUCAACCACUUGGAGAAGAGGAUCGAGAACUUGAACAAGAAGGUCGAUGAUGGUUUCUUGGAUGUCUGGACCUACAACGCUGAGUUGUUGGUCUUGUUGGAGAACGAGAGGACCUUGGAUUACCACGAUUCCAACGUCAAGAACUUGUACGAGAAGGUCAGGCACCAGUUGAAGAACAAUGCCAAGGAGAUCGGGAACGGUUGCUUCGAGUUCUACCACAAGUGUGAUAACACCUGUAUGGAGUCCGUUAAGAACGGUACCUACGAUUACCCCAAGUACUCCGAGGAGGCUAAGUUGAACAGGGAGAAGAUCGAUGGUGUCAAGUUGGAUUCCACCAGGAUCUACCAGAUCUUGGCGAUCUACUCUACUGUCGCUAGUUCCUUGGUCUUGGUUGUUUCCCUUGGUGCUAUCUCCUU CUGGAUGUGCUCCAACGGUUCCUUGCAGUGCAGGAUCUGCAUCUAAUAA (SEQ ID NO: 6).Attorney Docket No. 0171.0129-PCT

[0126] A representative codon-optimized mRNA sequence encoding the influenza A HA polypeptide of SEQ ID NO: 7 is set forth in SEQ ID NO: 8:AUGAAAACCAUCAUCGCCCUGAGCAACAUCCUGUGCCUGGUCUUCGCCCAGAAGAUCCCAGGCAACGACAACAGCACGGCCACCCUCUGCCUGGGGCACCACGCCGUCCCAAACGGGACGAUCGUGAAGACGAUCACAAACGACCGGAUCGAGGUGACAAACGCCACCGAGCUGGUGCAGAACUCCAGCAUCGGGGAGAUCUGCGGAAGCCCCCACCAGAUCCUGGACGGGGGCAACUGCACACUGAUCGACGCCCUGCUGGGCGACCCCCAGUGCGACGGGUUCCAGAACAAAGAGUGGGACCUGUUCGUGGAGAGGAGCAGAGCCAACAGCAACUGCUACCCCUACGACGUCCCCGACUACGCCAGCCUGAGAAGCCUGGUGGCCAGCAGCGGGACGCUGGAGUUCAAGAACGAGAGCUUCAACUGGACAGGGGUCAAGCAGAACGGCACCAGCUCAGCGUGCAUCAGGGGGAGCAGCAGCAGCUUCUUCAGCCGGCUGAACUGGCUGACCUCACUGAACAACAUCUACCCCGCCCAAAACGUCACCAUGCCCAACAAAGAGCAGUUCGACAAGCUCUACAUCUGGGGGGUGCACCACCCCGACACCGACAAGAACCAGAUCAGCCUGUUCGCCCAGUCAAGCGGGCGGAUCACCGUCAGCACCAAGCGGAGCCAGCAGGCGGUGAUCCCCAACAUCGGCUGUCGGCCCCGCAUCCGCGACAUCCCGAGCCGGAUCAGCAUCUACUGGACCAUCGUGAAGCCCGGAGACAUCCUGCUGAUCUGCAGCACCGGGAACCUGAUCGCCCCCCGGGGGUACUUCAAGAUCAGGAGCGGAAAGAGCAGCAUCAUGAGGAGCGACGCCCCGAUCGGGAAGUGCAAAAGCGAGUGCAUCACCCCAAACGGGAGCAUCCCCAACGACAAGCCGUUCCAGAACGUCAACCGCAUCACGUACGGGGCCUGCCCCCGGUACGUGAAGCAGAGCACGCUGAAGCUGGCCACCGGCAUGCGGAACGUGCCCGAGAAGCAGACCCGGGGCAUCUUCGGCGCCAUCGCCGGCUUCAUCGAGAACGGCUGGGAGGGCAUGGUGGACGGGUGGUACGGGUUCCGGCACCAGAACAGCGAGGGCCGCGGGCAGGCGGCGGACCUGAAAAGCACACAGGCCGCCAUCGACCAGAUCAACGGCAAGCUGAACCGGCUGAUCGGGAAGACCAACGAGAAGUUCCACCAGAUCGAGAAGGAGUUCUCCGAGGUCGAGGGCCGGGUGCAGGACCUGGAGAAGUACGUGGAGGACACCAAGAUCGACCUGUGGAGCUACAACGCCGAGCUGCUCGUGGCCCUCGAGAACCAGCACACCAUCGACCUGACCGACAGCGAGAUGAACAAGCUCUUUGAGAAGACCAAAAAGCAGCUCAGAGAGAACGCAGAGGACAUGGGCAACGGAUGCUUCAAGAUCUACCACAAGUGCGAttorney Docket No. 0171.0129-PCTACAACGCAUGCAUCGGCAGCAUCCGGAACGAAACCUACGACCACAACGUGUACAGGGACGAAGCACUGAACAACCGCUUCCAGAUCAAGGGAGUGGAGCUCAAGAGCGGCUACAAGGACUGGAUUCUGUGGAUCAGCUUCGCCAUGAGCUGCUUCCUCCUGUGCAUCGCCCUGCUGGGCUUCAUCAUGUGGGCCUGCCAGAAGGGCAACAUCAGAUGCAACAUCUGCAUCUAAUAA (SEQ ID NO: 8).]]

[0127] A representative codon-optimized mRNA sequence encoding the influenza A HA polypeptide of SEQ ID NO: 9 is set forth in SEQ ID NO: 10:AUGAAGACCAUCAUCGCGCUGAGCAACAUCCUGUGCCUGGUGUUCGCCCAGAAGAUCCCAGGCAACGACAACAGCACGGCCACCCUCUGCCUGGGCCACCACGCCGUCCCCAACGGGACGAUCGUGAAGACGAUCACAAACGACCGGAUCGAGGUCACCAACGCCACCGAGCUGGUCCAGAACUCCAGCAUCGGCGAGAUCUGCGGAAGCCCCCACCAGAUCCUGGACGGGGGCAACUGCACCCUCAUCGAUGCCCUGCUGGGGGACCCGCAGUGCGACGGCUUCCAGAACAAGGAGUGGGACCUCUUCGUGGAGAGGAGCAGAGCGAACAGCAACUGCUACCCCUACGACGUCCCAGACUACGCCAGCCUGCGGAGCCUGGUCGCCAGCAGCGGGACCCUGGAGUUCAAGAACGAGUCGUUCAACUGGACGGGAGUCAAGCAGAACGGCACCAGUAGCGCGUGCAUCCGCGGCUCCAGCAGCAGCUUCUUCUCCCGGCUGAACUGGCUGACGUCACUCAACAACAUCUACCCCGCCCAAAACGUGACGAUGCCCAACAAGGAGCAGUUCGACAAGCUCUACAUCUGGGGCGUGCACCACCCGGACACGGACAAGAACCAGAUCAGCCUGUUCGCGCAGUCAAGCGGGCGGAUCACGGUGAGCACGAAGCGCAGCCAGCAGGCCGUGAUCCCGAACAUCGGGAGCAGGCCGCGCAUCCGGGACAUCCCGAGCCGCAUCAGCAUCUACUGGACGAUCGUGAAGCCCGGAGACAUCCUGCUGAUCAACAGCACGGGGAACCUCAUCGCCCCGCGCGGAUACUUCAAGAUCCGCAGCGGGAAAAGCUCCAUCAUGCGGAGCGACGCCCCGAUCGGGAAGUGCAAGAGCGAGUGCAUCACCCCGAACGGGAGCAUCCCCAACGACAAGCCCUUCCAGAACGUGAACCGCAUCACCUACGGGGCCUGCCCCCGGUACGUGAAGCAGAGCACGCUGAAGCUGGCGACCGGCAUGCGGAACGUGCCCGAGAAGCAGACCCGGGGCAUCUUCGGCGCCAUCGCCGGCUUCAUCGAGAACGGCUGGGAGGGCAUGGUGGACGGGUGGUACGGGUUCCGCCACCAGAACUCCGAGGGGAGGGGGCAGGCCGCCGACCUCAAGUCCACCCAGGCCGCGAUCGACCAGAUCAACGGGAAGCUCAACAGGCUGAUCGGGAAGACCAACGAAttorney Docket No. 0171.0129-PCTGAAGUUCCACCAGUGUGAGAAGGAGUUCUCCGAGGUGGAGGGGAGAGUGC AGUGUCUCGAGAAGUACGUGGAGGACACCAAGAUCGACCUGUGGAGCUAC AACGCGGAGCUGCUCGUGGCCCUGGAGAACCAGCACACCAUCGACCUCACG GACAGUGAGAUGAACAAGCUCUUUGAGAAGACGAAGAAGCAGCUGCGGGA AAACGCAGAGGACAUGGGCAACGGGUGCUUCAAGAUCUACCACAAGUGCG ACAACGCCUGUAUCGGCAGCAUCCGCAACGAGACGUACGACCACAACGUCU ACCGCGACGAGGCCCUCAACAACAGGUUCCAGAUCAAGGGCGUGGAGCUG AAGUCCGGGUACAAGGACUGGAUUCUCUGGAUCUCCUUCGCGAUGAGCUG CUUCCUGCUGUGCAUCGCCCUGCUGGGCUUCAUCAUGUGGGCCUGCCAGAA GGGCAACAUCAGAUGCAACAUCUGCAUCUAAUAA (SEQ ID NO: 10).

[0128] In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the influenza A 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: 4. In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the influenza A 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: 6. In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the influenza A 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: 8. In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the influenza A 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: 10. In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the influenza A HA polypeptides disclosed herein comprise or consist of the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the influenza A HA polypeptides disclosed herein comprise or consist of the nucleic acid sequence ofAttorney Docket No. 0171.0129-PCTSEQ ID NO: 6. In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the influenza A HA polypeptides disclosed herein comprise or consist of the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the artificial nucleic acid molecules (e.g., mRNAs) encoding the influenza A HA polypeptides disclosed herein comprise or consist of the nucleic acid sequence of SEQ ID NO: 10.

[0129] To express the modified influenza A HA polypeptides of the 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 (San Diego, 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.

[0130] 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.

[0131] 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 aAttorney Docket No. 0171.0129-PCT 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.

[0132] The modified influenza A HA polypeptides 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 influenza A 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 .

[0133] 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 influenza A HA polypeptides of the 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 influenza A HA polypeptides according to the disclosure having a desired conformation (e.g., stabilized prefusion conformation) can be selected.

[0134] Accordingly, in some embodiments, provided herein are artificial nucleic acids encoding any of the modified influenza A HA polypeptides described herein. The artificial nucleicAttorney Docket No. 0171.0129-PCT 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.

[0135] 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.

[0136] 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.

[0137] 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; a polynucleotide 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.

[0138] 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.

[0139] Also disclosed herein are host cells (e.g., mammalian cells, e.g., human cells) comprising the vectors or RNA compositions disclosed herein.

[0140] 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 830Attorney Docket No. 0171.0129-PCT(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).

[0141] 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).

[0142] 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.RNA

[0143] In certain embodiments, the vaccine or immunogenic compositions disclosed herein may comprise one or more self-amplifying ribonucleic acids encoding an influenza A 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.

[0144] In certain embodiments, the RNAs are messenger RNAs (mRNAs) comprising an open reading frame (ORF) encoding an influenza A HA polypeptide as disclosed herein. In certain 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

[0145] 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-methylguanosine 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.Attorney Docket No. 0171.0129-PCT

[0146] 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.

[0147] 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 ARC A 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-methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase.

[0148] 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.

[0149] In certain embodiments, the mRNA of the disclosure comprises a 5' cap of:B. Untranslated Region (UTR)Attorney Docket No. 0171.0129-PCT

[0150] 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.

[0151] 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.

[0152] 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 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, aboutAttorney Docket No. 0171.0129-PCT2,000 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.

[0153] 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).

[0154] 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.

[0155] 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: 11) (U.S. Publication No. 2016 / 0151409, incorporated herein by reference).

[0156] 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. In certain 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).

[0157] In certain embodiments, the 5' UTR is derived from a ribosomal protein Large 32 (L32) gene (U.S. Publication No. 2017 / 0029847, supra).

[0158] 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).Attorney Docket No. 0171.0129-PCT

[0159] In certain embodiments, the 5' UTR is derived from the 5' UTR of an ATP5A1 gene (U.S. Publication No. 2016 / 0166710, supra).

[0160] In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.

[0161] In some embodiments, the 5' UTR comprises a nucleic acid sequence of GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACC GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG UGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 12).

[0162] In some embodiments, the 3' UTR comprises a nucleic acid sequence of CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCC ACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 13).

[0163] 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

[0164] 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 of nucleotides, that are different from an adenosine nucleotide). In certain embodiments, the poly(A) tail comprises the sequence:AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAA (SEQ ID NO: 14).Attorney Docket No. 0171.0129-PCT

[0165] 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”).

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal.

[0171] In various embodiments, the nucleic acid may comprise at least one poly(C) sequence.

[0172] 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 ModificationAttorney Docket No. 0171.0129-PCT

[0173] 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 analogues (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 analogues 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-methyl-inosine, 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 -methoxy aminomethyl-2- thio-uracil, 5’-methoxycarbonylmethyl-uracil, 5-methoxy -uracil, uracil-5-oxyacetic acid methyl ester, uracil- 5 -oxy acetic acid (v), 1-methyl-pseudouracil, queosine, P-D-mannosyl-queosine, phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7- deazaguanosine, 5-methylcytosine, and inosine.

[0174] 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-methyl-l-deaza-pseudouridine, 2-thio-l-methyl- pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl- pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methyluridine, 5-methoxyuridine, and 2'-O-methyl uridine.

[0175] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof.

[0176] In some embodiments, the chemical modification comprises N1 -methylpseudouridine.Attorney Docket No. 0171.0129-PCT

[0177] 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.

[0178] 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.

[0179] The preparation of such analogues 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

[0180] 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.Immunogenic Compositions and Vaccines

[0181] The present disclosure also relates to immunogenic compositions that comprise any of the modified influenza A HA polypeptides disclosed herein or artificial nucleic acids or vectors encoding such modified influenza A HA polypeptides. As used herein, the term “immunogenic composition” refers to a composition that generates an immune response that may or may not be a protective immune response or protective immunity. The term “immune response” refers to aAttorney Docket No. 0171.0129-PCT 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.

[0182] 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.

[0183] 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 E. 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 injectable solutions. The exact formulation should suit the mode of administration. The modified influenzaAttorney Docket No. 0171.0129-PCTA 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.

[0184] Accordingly, in some embodiments, provided herein is a composition comprising any of the modified influenza A HA polypeptides disclosed herein, a trimeric influenza A HA polypeptide complex comprising three copies of any of the modified influenza A HA polypeptides disclosed herein, an artificial nucleic acid encoding any of the modified influenza A 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 mRNAs encode any of the modified influenza A HA polypeptides disclosed herein. In some embodiments, such a composition is an immunogenic composition.

[0185] In some embodiments, also provided herein is an immunogenic composition or vaccine comprising any of the modified influenza A HA polypeptides disclosed herein. In some embodiments, provided herein is an immunogenic composition or vaccine comprising a trimeric influenza A HA polypeptide complex comprising three copies of any of the modified influenza A 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 influenza A 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 influenza A 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).

[0186] In some embodiments, the immunogenic compositions or vaccines comprise other polypeptides in addition to the modified HA A polypeptides disclosed herein. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, more than one polypeptide (e.g., two, three, four, five, six, seven, eight, nine, ten, or more polypeptides). In some embodiments, the immunogenic compositions or vaccines comprise, orAttorney Docket No. 0171.0129-PCT comprise mRNAs that encode, three polypeptides. In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, six polypeptides.

[0187] In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, polypeptides derived from two or more (e.g., three, four, five, six, seven, eight, nine, or ten) influenza viral proteins selected from hemagglutinin (e.g., hemagglutinin 1 (HA1) and hemagglutinin 3 (HA3)) and neuraminidase (NA). In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, one or more (e.g., three, four five, six, seven, eight, or more) polypeptides derived from an HA protein, from an NA protein, and / or from both HA and NA proteins. In some embodiments, the polypeptides are derived from different influenza strains.

[0188] In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, one or more polypeptides of influenza A, B and C viruses. In some embodiments, the HA polypeptides of influenza A viruses are selected from subtypes Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, and H18. In some embodiments, the NA polypeptides of influenza A viruses are selected from subtypes Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, and Ni l.

[0189] In some embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, two, three, four, five, six, seven, eight, nine, or more of (i) one or more HA polypeptides, (ii) one or more NA polypeptides, or (iii) a combination of one or more HA polypeptides and NA polypeptides.

[0190] In certain embodiments, the immunogenic compositions or vaccines comprise, or comprise mRNAs that encode, two, three, four, five, six, seven, eight, nine or more of (i) one or more HA polypeptides, (ii) one or more NA polypeptides, or (iii) a combination of one or more HA polypeptides and NA polypeptides selected from H1N1, H3N2, H2N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2, and H10N7 subtypes and / or B / Yamagata and B / Victoria lineages.

[0191] In some embodiments, the immunogenic composition or vaccine comprises an mRNA encoding an influenza H3 HA polypeptide, an mRNA encoding an influenza Hl HA polypeptide, and an mRNA encoding an influenza B HA polypeptide from the influenza B / Victoria lineage, wherein at least one of the influenza H3 HA polypeptide and the influenza Hl HA polypeptide is a modified influenza A HA polypeptide according to the present disclosure.Attorney Docket No. 0171.0129-PCT

[0192] In some embodiments, the immunogenic composition or vaccine comprises an mRNA encoding a modified influenza A HA polypeptide from a Hl influenza virus, an mRNA encoding a wild-type influenza H3 HA polypeptide, and an mRNA encoding a modified influenza HA polypeptide from an influenza B / Victoria lineage. Exemplary modified influenza HA polypeptides from an influenza B / Victoria lineage are described in, for example, PCT publication No. WO 2025 / 051975, incorporated herein by reference in its entirety.

[0193] In some embodiments, the immunogenic composition or vaccine comprises an mRNA encoding an influenza H3 HA polypeptide, an mRNA encoding an influenza N2 NA polypeptide, an mRNA encoding an influenza Hl HA polypeptide, an mRNA encoding an influenza N1 NA polypeptide, an mRNA encoding an HA polypeptide from the influenza B / Victoria lineage, and an mRNA encoding an NA polypeptide from the influenza B / Victoria lineage, wherein at least one of the influenza H3 HA polypeptide and the influenza Hl HA polypeptide is a modified influenza A HA polypeptide according to the present disclosure.

[0194] In some embodiments, the immunogenic composition or vaccine comprises an mRNA encoding an influenza H3 HA polypeptide, an mRNA encoding an influenza Hl HA polypeptide, an mRNA encoding an HA polypeptide from the influenza B / Victoria lineage, and an mRNA encoding an HA polypeptide from the influenza B / Yamagata lineage, wherein at least one of the influenza H3 HA polypeptide and the influenza Hl HA polypeptide is a modified influenza A HA polypeptide according to the present disclosure.

[0195] In some embodiments, the immunogenic composition or vaccine comprises an mRNA encoding an influenza H3 HA polypeptide, an mRNA encoding an influenza N2 NA polypeptide, an mRNA encoding an influenza Hl HA polypeptide, an mRNA encoding an influenza N1 NA polypeptide, an mRNA encoding an HA polypeptide from the influenza B / Victoria lineage, an mRNA encoding an NA polypeptide from the influenza B / Victoria lineage, an mRNA encoding an HA polypeptide from the influenza B / Yamagata lineage, and an mRNA encoding an NA polypeptide from the influenza B / Yamagata lineage, wherein at least one of the influenza H3 HA polypeptide and the influenza Hl HA polypeptide is a modified influenza A HA polypeptide according to the present disclosure.

[0196] In some embodiments, the immunogenic composition or vaccine comprises an influenza H3 HA polypeptide, an influenza Hl HA polypeptide, and an influenza B HA polypeptide from the influenza B / Victoria lineage, wherein at least one of the influenza H3 HAAttorney Docket No. 0171.0129-PCT polypeptide and the influenza Hl HA polypeptide is a modified influenza A HA polypeptide according to the present disclosure.

[0197] In some embodiments, the immunogenic composition or vaccine comprises a modified influenza A HA polypeptide from a Hl influenza virus, a wild-type influenza H3 HA polypeptide, and a modified influenza HA polypeptide from an influenza B / Victoria lineage. Exemplary modified influenza HA polypeptides from an influenza B / Victoria lineage are described in, for example, PCT publication No. WO 2025 / 051975, incorporated herein by reference in its entirety.

[0198] In some embodiments, the immunogenic composition or vaccine comprises an influenza H3 HA polypeptide, an influenza N2 NA polypeptide, an influenza Hl HA polypeptide, an influenza N1 NA polypeptide, an HA polypeptide from the influenza B / Victoria lineage, and an NA polypeptide from the influenza B / Victoria lineage, wherein at least one of the influenza H3 HA polypeptide and the influenza Hl HA polypeptide is a modified influenza A HA polypeptide according to the present disclosure.

[0199] In some embodiments, the immunogenic composition or vaccine comprises an influenza H3 HA polypeptide, an influenza Hl HA polypeptide, an HA polypeptide from the influenza B / Victoria lineage, and an HA polypeptide from the influenza B / Yamagata lineage, wherein at least one of the influenza H3 HA polypeptide and the influenza Hl HA polypeptide is a modified influenza A HA polypeptide according to the present disclosure.

[0200] In some embodiments, the immunogenic composition or vaccine comprises an influenza H3 HA polypeptide, an influenza N2 NA polypeptide, an influenza Hl HA polypeptide, an influenza N1 NA polypeptide, an HA polypeptide from the influenza B / Victoria lineage, an NA polypeptide from the influenza B / Victoria lineage, an HA polypeptide from the influenza B / Yamagata lineage, and an NA polypeptide from the influenza B / Yamagata lineage, wherein at least one of the influenza H3 HA polypeptide and the influenza Hl HA polypeptide is a modified influenza A HA polypeptide according to the present disclosure.

[0201] 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, 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, including all values and subranges therebetween. In certainAttorney Docket No. 0171.0129-PCT 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 of the modified influenza A HA polypeptide.

[0202] 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 may constitute 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).

[0203] In some embodiments, the composition of the disclosure is an immunogenic composition or vaccine capable of eliciting an immune response against influenza A viruses in a subject.

[0204] In some embodiments, the immunogenic compositions or vaccines 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 influenza A 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 or vaccine. 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. Lipid Nanoparticle

[0205] 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 lipidsAttorney Docket No. 0171.0129-PCT(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.

[0206] 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. Cationic Lipid

[0207] 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.Attorney Docket No. 0171.0129-PCTAttorney Docket No. 0171.0129-PCTAttorney Docket No. 0171.0129-PCTAttorney Docket No. 0171.0129-PCT

[0208] The cationic lipid may be selected from the group comprising [ckkE10] / [OF-02], [(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl]4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA); 1,2- dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); di((Z)-non-2-en-l-yl)9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319); 9-heptadecanyl 8-{(2-hydroxyethyl)[6- oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); [(4-hydroxybutyl)azanediyl]di(hexane- 6,l-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-oxoethyl]pentanamide (DOGS); [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17- [(2R)-6-methylheptan-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) (C 12-200); 3 ,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (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, 1 -diyl))bis (azanetriyl))tetrakis(ethane-2, l-diyl)(9Z,9'Z,9''Z,9"'Z, 12Z, 12'Z, 12''Z, 12"'Z)-tetrakis(octadeca- 9,12-dienoate) (OF-Deg-Lin); TT3; Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene- 1,3,5- tricarboxamide; Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino] butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl 8-((2- hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); IM-001; and combinations thereof.

[0209] 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.Attorney Docket No. 0171.0129-PCT

[0210] 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

[0211] 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).

[0212] 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(methoxypolyethylene 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-polyethylene 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.

[0213] 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- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000.C. Cholesterol-Based Lipid

[0214] 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.Attorney Docket No. 0171.0129-PCT5,744,335), imidazole cholesterol ester (“ICE”; WO2011 / 068810), sitosterol (22,23- dihydrostigmasterol), 0-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 ((30,25R)-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

[0215] 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).

[0216] 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-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl- phosphatidy ethanolamine (SOPE), or a combination thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.Attorney Docket No. 0171.0129-PCT

[0217] 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

[0218] 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.

[0219] 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%),Attorney Docket No. 0171.0129-PCT wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0220] 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%.

[0221] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG- PEG2000).

[0222] In various embodiments, the cholesterol-based lipid is cholesterol.

[0223] In some embodiments, the helper lipid is l,2-dioleoyl-SN-glycero-3- pho sphoethanolamine (D OPE) .

[0224] 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%.

[0225] 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%.

[0226] 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%.

[0227] 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%.

[0228] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 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%.

[0229] 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%.

[0230] 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%.Attorney Docket No. 0171.0129-PCT

[0231] 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%.

[0232] 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%.

[0233] 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%.

[0234] 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%.

[0235] In certain embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 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%.

[0236] 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%; l,2-distearoyl-5n- 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%.

[0237] 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%; l,2-distearoyl-5n-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%.

[0238] 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%; l,2-distearoyl-5n-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%.

[0239] In certain embodiments, the LNP comprises: IM-001 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%.Attorney Docket No. 0171.0129-PCT

[0240] 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.

[0241] Accordingly, in some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding any of the influenza A 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.

[0242] 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 the molar 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.

[0243] In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) encoding the influenza A HA polypeptide of SEQ ID NO: 3Attorney Docket No. 0171.0129-PCT 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) encoding the influenza A HA polypeptide of SEQ ID NO: 5 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) encoding the influenza A HA polypeptide of SEQ ID NO: 7 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) encoding the influenza A HA polypeptide of SEQ ID NO: 9 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%.

[0244] In some embodiments, provided herein is a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 4 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: 6 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: 8 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: 10 encapsulated in a LNP, wherein the LNPAttorney Docket No. 0171.0129-PCT 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%.II. Processes for Making LNP Vaccines

[0245] 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.

[0246] 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.

[0247] 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 whichAttorney Docket No. 0171.0129-PCT 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.

[0248] 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.

[0249] 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.

[0250] 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 greaterAttorney Docket No. 0171.0129-PCT 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.

[0251] 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 Lett. (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.

[0252] 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.

[0253] 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 polydispersity 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.

[0254] 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.,Attorney Docket No. 0171.0129-PCT 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).

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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 about 0.20, 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.

[0259] 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 encapsulationAttorney Docket No. 0171.0129-PCT efficiency of at least 90% (e.g., at least 91%, 92%, 93%, 94%, or 95%, including all values and subranges therebetween).

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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 acidAttorney Docket No. 0171.0129-PCT 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.Adjuvants

[0265] In some embodiments, the immunogenic composition or vaccine of the present disclosure comprises an adjuvant. In other embodiments, the immunogenic composition or vaccine of the present disclosure does not contain an adjuvant. Similarly, in some embodiments, the immunogenic composition or vaccine of the present disclosure can be administered with an adjuvant to boost the immune response. In other embodiments, the immunogenic composition or vaccines can be administered without an adjuvant. As used herein, the term “adjuvant” refers to a substance or combination of substances that may be used to enhance an immune response to an antigen component of a vaccine or immunogenic composition. Adjuvants can include a suspension of minerals (alum, aluminum salts, including, for example, aluminum hydroxide / oxyhydroxide (A1OOH), aluminum phosphate (AIPO4), aluminum hydroxyphosphate sulfate (AAHS) and / or potassium aluminum sulfate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example, see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biological molecules, such as lipids and costimulatory molecules. Exemplary biological adjuvants include, but are not limited to, AS04 (Didierlaurent et al., J. Immunol., 2009, 183:6186-6197), IL-2, RANTES, GM-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.Attorney Docket No. 0171.0129-PCT

[0266] In certain embodiments, the adjuvant is a squalene-based adjuvant comprising an oil-in- water adjuvant emulsion comprising at least: squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and a hydrophobic nonionic surfactant. In certain embodiments, the emulsion is thermoreversible, optionally wherein about 90% of the population by volume of the oil drops has a size less than about 200 nm.

[0267] In certain embodiments, the polyoxyethylene alkyl ether is of formula CH3-(CH2)X-(O- CH2-CH2)n-OH, in which n is an integer from 10 to 60, and x is an integer from 11 to 17. In certain embodiments, the polyoxyethylene alkyl ether surfactant is polyoxyethylene(12) cetostearyl ether.

[0268] In certain embodiments, about 90% of the population by volume of the oil drops has a size less than about 160 nm. In certain embodiments, about 90% of the population by volume of the oil drops has a size less than about 150 nm. In certain embodiments, about 50% of the population by volume of the oil drops has a size less than about 100 nm. In certain embodiments, about 50% of the population by volume of the oil drops has a size less than about 90 nm.

[0269] In certain embodiments, the adjuvant further comprises at least one alditol, including, but not limited to, glycerol, erythritol, xylitol, sorbitol and mannitol.

[0270] In some embodiments the hydrophilic / lipophilic balance (HLB) of the hydrophilic nonionic surfactant is greater than or equal to about 10. In certain embodiments, the HLB of the hydrophobic nonionic surfactant is less than about 9. In certain embodiments, the HLB of the hydrophilic nonionic surfactant is greater than or equal to about 10 and the HLB of the hydrophobic nonionic surfactant is less than about 9.

[0271] In certain embodiments, the hydrophobic nonionic surfactant is a sorbitan ester, such as sorbitan monooleate, or a mannide ester surfactant. In certain embodiments, the amount of squalene is between about 5% and about 45%. In certain embodiments, the amount of polyoxyethylene alkyl ether surfactant is between about 0.9% and about 9%. In certain embodiments, the amount of hydrophobic nonionic surfactant is between about 0.7% and about 7%. In certain embodiments, the adjuvant comprises: i) about 32.5% of squalene, ii) about 6.18% of polyoxyethylene(12) cetostearyl ether, iii) about 4.82% of sorbitan monooleate, and iv) about 6% of mannitol.

[0272] In certain embodiments, the adjuvant further comprises an alky Ipolyglyco side and / or a cryoprotective agent, such as a sugar, in particular dodecylmalto side and / or sucrose.Attorney Docket No. 0171.0129-PCT

[0273] In certain embodiments, the adjuvant comprises AF03, as described in Klucker et al., J. Pharm. Sci., 2012, 101(12):4490-4500, which is hereby incorporated by reference in its entirety. In certain embodiments, the adjuvant comprises a liposome-based adjuvant, such as SPA14. SPA14 is a liposome-based adjuvant (ASOl-like) containing a toll-like receptor 4 (TLR4) agonist (E6020) and saponin (QS21).

[0274] In some embodiments, the immunogenic composition or vaccine composition does not comprise an adjuvant. In certain embodiments, the one or more mRNA molecules encapsulated in a LNP may serve to adjuvate one or more of the modified influenza A HA polypeptides in the composition. See e.g., Shirai et al., Vaccines, 2020, 8(433): 1-18. In other embodiments, the vaccine or immunogenic composition further comprises an adjuvant.Administration

[0275] 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.

[0276] 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.

[0277] 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 PharmaceuticalAttorney Docket No. 0171.0129-PCTSciences, 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. Patent No. 4,270,537, U.S. Patent No. 5,015,235, U.S. Patent No. 5,141,496, U.S. Patent No. 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 WO1999 / 34850, incorporated herein by reference, and functional equivalents thereof.

[0278] 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. Patent No. 5,599,302, U.S. Patent No. 5,334,144, U.S. Patent No. 5,993,412, U.S. Patent No. 5,649,912, U.S. Patent No. 5,569,189, U.S. Patent No. 5,704,911, U.S. Patent No. 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.

[0279] 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 thoseAttorney Docket No. 0171.0129-PCT 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.

[0280] 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.

[0281] 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

[0282] 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 vaccine or immunogenic 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 immunogenic compositions or vaccines described herein.

[0283] 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, anAttorney Docket No. 0171.0129-PCT 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.

[0284] Accordingly, in some embodiments, the disclosure provides a method of immunizing a subject comprising administering to the subject in need thereof any of the vaccines or immunogenic compositions 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 vaccines or immunogenic compositions 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 vaccine or immunogenic 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.

[0285] 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 method or 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 whoAttorney Docket No. 0171.0129-PCT 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.

[0286] 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 vaccines or immunogenic compositions 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 vaccines or immunogenic compositions described herein.

[0287] The present disclosure provides any of the vaccine or immunogenic 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.

[0288] 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.

[0289] In some embodiments, the vaccine or immunogenic composition, 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 vaccines or immunogenic compositions described herein may be administered prophylactically to prevent the influenza A virus infection or ameliorate the symptoms of a potential influenza A virus infection.

[0290] 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 vaccine or immunogenic composition is administered to a subject suffering from an influenza A virus infection, or the subject is displaying one or more symptoms commonly associated with anAttorney Docket No. 0171.0129-PCT influenza A virus infection. In some embodiments, the subject is known or believed to have been exposed to an influenza A virus infection.

[0291] 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 or immunogenic compositions 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.

[0292] In some embodiments, the vaccines or immunogenic compositions 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), 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).

[0293] In some embodiments, the vaccines or immunogenic compositions 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 of age). 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 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).Attorney Docket No. 0171.0129-PCT

[0294] The methods and uses of the vaccines or immunogenic compositions 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 vaccines or immunogenic compositions described herein include prime-boost vaccination strategies. Prime-boost vaccination comprises administering a priming vaccine or immunogenic composition and then, after a period of time has passed, administering to the subject a boosting vaccine or immunogenic composition. The immune response is “primed” upon administration of the priming vaccine or immunogenic composition and is “boosted” upon administration of the boosting vaccine or immunogenic composition. The priming vaccine or immunogenic composition can include a vaccine or immunogenic composition as described herein and an optional adjuvant. Likewise, the boosting vaccine or immunogenic composition can include a vaccine or immunogenic composition as described herein and an optional adjuvant. The priming vaccine or immunogenic composition can be, but need not be, the same as the boosting vaccine or immunogenic composition. Administration of the boosting vaccine or immunogenic composition is generally weeks or months after administration of the priming vaccine or immunogenic composition, 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 primeboost vaccination is a naive subject, typically a naive infant or child.

[0295] The vaccine or immunogenic composition can be administered using any suitable route of administration, including, for example, parenteral delivery, as discussed above. In some embodiments, the vaccine or immunogenic composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.Other Applications

[0296] The modified influenza A HA polypeptides disclosed herein may have other applications, such as use in an in vitro method for preparation of a trimeric influenza A HA polypeptide complex. Accordingly, provided herein is an in vitro method of preparing a trimeric influenza A HA polypeptide complex, the method comprising expressing an artificial nucleic acid molecule encoding any of the modified influenza A HA polypeptides disclosed herein in a host cell to prepare the trimeric influenza A HA polypeptide complex. In some embodiments, the artificial nucleic acid molecule encoding the modified influenza A HA polypeptide is part of a vector. In other embodiments, the expression of the modified influenza A HA polypeptide is by culturing the host cell in cell culture medium. Thus, in some embodiments, provided herein is anAttorney Docket No. 0171.0129-PCT in vitro method of preparing a trimeric influenza A HA polypeptide complex, the method comprising culturing the host cell in a cell culture medium, and expressing the trimeric influenza A HA polypeptide complex. In some embodiments, the in vitro method disclosed herein further comprises a step of purifying the trimeric influenza A HA polypeptide complex from the cell culture medium.

[0297] In certain embodiments, the trimeric influenza A HA polypeptide complex prepared according to the in vitro method disclosed herein is more stable in prefusion conformation as compared to a trimeric influenza A HA polypeptide complex prepared from a control influenza A HA polypeptide without the one or more modifications. In some embodiments, stability in prefusion conformation is measured by an increased binding of the trimeric influenza A HA polypeptide complex to a stem region- specific antibody (e.g., CR9114) as compared to a trimeric influenza A HA polypeptide complex prepared from a control influenza A 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) as compared to a trimeric influenza A HA polypeptide complex prepared from a control influenza A HA polypeptide without the one or more modifications. In some embodiments, stability in prefusion conformation is measured by an increased binding of the trimeric influenza A 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 influenza A HA polypeptide complex prepared from a control influenza A HA polypeptide without the one or more modifications.

[0298] In certain embodiments, the trimeric influenza A HA polypeptide complex prepared according to the in vitro method disclosed herein is more immunogenic as compared to a trimeric influenza A HA polypeptide complex prepared from a control influenza A 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).Attorney Docket No. 0171.0129-PCTFirst Set of Representative Embodiments of the Present Disclosure

[0299] Embodiment 1. An artificial messenger ribonucleic acid (mRNA) encoding a modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide comprises: a) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 36 and 398 and / or 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1; or b) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 235 and 262 and / or 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0300] Embodiment 2. The artificial mRNA of Embodiment 1, wherein the modified influenza A HA polypeptide comprises amino acid substitutions: a) V36C and S398C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) G219C and R234C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; c) S235C and N262C as indexed by reference to the amino acid sequence of SEQ ID NO: 2; or d) 1411C and D424C as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0301] Embodiment 3. The artificial mRNA of Embodiment 1 or 2, wherein the modified influenza A HA polypeptide is from a H1N1 influenza virus or a H3N2 influenza virus.

[0302] Embodiment 4. The artificial mRNA of Embodiment 3, wherein the H1N1 influenza virus is A / West Virginia / 30 / 2022, or wherein the H3N2 influenza virus is A / Darwin / 06 / 2021.

[0303] Embodiment 5. The artificial mRNA of any one of Embodiments 1-4, wherein the modified influenza A HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

[0304] Embodiment 6. The artificial mRNA of Embodiment 5, wherein the modified influenza A HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

[0305] Embodiment 7. The artificial mRNA of any one of Embodiments 1-6, comprising a nucleic acid sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

[0306] Embodiment 8. The artificial mRNA of Embodiment 7, comprising the nucleic acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.Attorney Docket No. 0171.0129-PCT

[0307] Embodiment 9. The artificial mRNA of any one of Embodiments 1-8, comprising a 5'- cap structure and / or a 3'-poly(A) sequence.

[0308] Embodiment 10. The artificial mRNA of any one of Embodiments 1-9, comprising at least one chemically modified nucleotide and / or a phosphorothioate bond.

[0309] 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.

[0310] Embodiment 12. A composition comprising the artificial mRNA of any one of Embodiments 1-11 encapsulated in a lipid nanoparticle (LNP).

[0311] Embodiment 13. The composition of Embodiment 12, wherein the LNP comprises a cationic lipid.

[0312] Embodiment 14. The composition of Embodiment 13, 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.

[0313] Embodiment 15. The composition of Embodiment 13 or 14, wherein the LNP further comprises a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

[0314] Embodiment 16. The composition of Embodiment 15, 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).

[0315] Embodiment 17. The composition of Embodiment 15 or 16, 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.

[0316] Embodiment 18. The composition of Embodiment 17, 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 helperAttorney Docket No. 0171.0129-PCT 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.

[0317] Embodiment 19. The composition of Embodiment 18, wherein the artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 3, 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%.

[0318] Embodiment 20. The composition of Embodiment 18, wherein the artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 5, 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%.

[0319] Embodiment 21. The composition of Embodiment 18, wherein the artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 7, 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%.

[0320] Embodiment 22. The composition of Embodiment 18, wherein the artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 9, 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%.

[0321] Embodiment 23. The composition of any one of Embodiments 12-22, wherein the composition is an immunogenic composition.

[0322] Embodiment 24. A modified influenza A HA polypeptide comprising one or more amino acid substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the one or more amino acid substitutions comprises: a) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 36 and 398 and / or 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1; or b) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 235 and 262 and / or 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0323] Embodiment 25. The modified influenza A HA polypeptide of Embodiment 24, wherein the modified influenza A HA polypeptide comprises amino acid substitutions: a) V36CAttorney Docket No. 0171.0129-PCT and S398C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) G219C and R234C as indexed by reference to the amino acid sequence of SEQ ID NO: 1 ; c) S235C and N262C as indexed by reference to the amino acid sequence of SEQ ID NO: 2; or d) 1411C and D424C as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0324] Embodiment 26. The modified influenza A HA polypeptide of Embodiment 24 or 25, wherein the modified influenza A HA polypeptide is from a H1N1 influenza virus or a H3N2 influenza virus.

[0325] Embodiment 27. The modified influenza A HA polypeptide of Embodiment 26, wherein the H1N1 influenza virus is A / West Virginia / 30 / 2022, or wherein the H3N2 influenza virus is A / Darwin / 06 / 2021.

[0326] Embodiment 28. The modified influenza A HA polypeptide of any one of Embodiments 24-27, wherein the modified influenza A HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

[0327] Embodiment 29. The modified influenza A HA polypeptide of Embodiment 28, wherein the modified influenza A HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

[0328] Embodiment 30. A trimeric influenza A HA polypeptide complex, comprising three copies of the modified influenza A HA polypeptide of any one of Embodiments 24-29.

[0329] Embodiment 31. An artificial nucleic acid encoding the modified influenza A HA polypeptide of any one of Embodiments 24-29.

[0330] Embodiment 32. The artificial nucleic acid of Embodiment 31, wherein the artificial nucleic acid comprises at least one chemically modified nucleotide and / or a phosphorothioate bond.

[0331] Embodiment 33. A vector comprising the artificial nucleic acid of Embodiment 31 or 32.

[0332] Embodiment 34. The vector of Embodiment 33, wherein the vector is a messenger RNA (mRNA) production vector.

[0333] Embodiment 35. A host cell comprising the vector of Embodiment 33 or 34.

[0334] Embodiment 36. A composition comprising the modified influenza A HA polypeptide of any one of Embodiments 24-29, the trimeric influenza A HA polypeptide complex ofAttorney Docket No. 0171.0129-PCTEmbodiment 30, the artificial nucleic acid of Embodiment 31 or 32, or the vector of Embodiment 33 or 34.

[0335] Embodiment 37. The composition of Embodiment 36, wherein the composition is an immunogenic composition.

[0336] Embodiment 38. A vaccine comprising the composition of Embodiment 23 or 37, and a pharmaceutically acceptable carrier.

[0337] Embodiment 39. The vaccine of Embodiment 38, further comprising an adjuvant.

[0338] Embodiment 40. The vaccine of Embodiment 38 or 39, wherein the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza H3 HA polypeptide and an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage.

[0339] Embodiment 41. The vaccine of Embodiment 38 or 39, wherein the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza H3 HA polypeptide, an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage, an mRNA encoding an influenza N2 neuraminidase (NA) polypeptide, an mRNA encoding an influenza N1 NA polypeptide, and an mRNA encoding an influenza NA polypeptide from the Influenza B / Victoria lineage.

[0340] Embodiment 42. The vaccine of Embodiment 38 or 39, wherein the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza Hl HA polypeptide and an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage.

[0341] Embodiment 43. The vaccine of Embodiment 38 or 39, wherein the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza Hl HA polypeptide, an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage, an mRNA encoding an influenza N2 NA polypeptide, an mRNA encoding an influenza N 1 NA polypeptide, and an mRNA encoding an influenza NA polypeptide from the Influenza B / Victoria lineage.Attorney Docket No. 0171.0129-PCT

[0342] Embodiment 44. The vaccine of Embodiment 38 or 39, wherein the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an influenza H3 HA polypeptide and an influenza HA polypeptide from an influenza B / Victoria lineage.

[0343] Embodiment 45. The vaccine of Embodiment 38 or 39, wherein the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an influenza H3 HA polypeptide, an influenza HA polypeptide from an influenza B / Victoria lineage, an influenza N2 NA polypeptide, an influenza N1 NA polypeptide, and an influenza NA polypeptide from the Influenza B / Victoria lineage.

[0344] Embodiment 46. The vaccine of Embodiment 38 or 39, wherein the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an influenza Hl HA polypeptide and an influenza HA polypeptide from an influenza B / Victoria lineage.

[0345] Embodiment 47. The vaccine of Embodiment 38 or 39, wherein the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an influenza Hl HA polypeptide, an influenza HA polypeptide from an influenza B / Victoria lineage, an influenza N2 NA polypeptide, an influenza N1 NA polypeptide, and an influenza NA polypeptide from the Influenza B / Victoria lineage.

[0346] Embodiment 48. A method of immunizing a subject, the method comprising administering to the subject in need thereof the vaccine of any one of Embodiments 38-47.

[0347] Embodiment 49. The method of Embodiment 48, 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.

[0348] Embodiment 50. The method of Embodiment 48 or 49, wherein the subject is a human.

[0349] Embodiment 51. The method of Embodiment 50, 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, atAttorney Docket No. 0171.0129-PCT 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.

[0350] Embodiment 52. The method of any one of Embodiments 48-51, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0351] Embodiment 53. 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 Embodiments 38-47.

[0352] Embodiment 54. An in vitro method of preparing a trimeric influenza A HA polypeptide complex, the method comprising culturing the host cell of Embodiment 35 in a cell culture medium, and expressing the trimeric influenza A HA polypeptide complex.

[0353] Embodiment 55. The in vitro method of Embodiment 54, 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

[0354] Embodiment 2.1: An artificial nucleic acid, e.g., an artificial messenger ribonucleic acid (mRNA), encoding a modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide comprises: a) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 36 and 398 and / or amino acid positions 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1; or b) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 235 and 262 and / or amino acid positions 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0355] Embodiment 2.2: The artificial nucleic acid of Embodiment 2.1, wherein the modified influenza A HA polypeptide comprises amino acid substitutions: a) V36C and S398C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) G219C and R234C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; c) S235C and N262C as indexed by reference to the amino acid sequence of SEQ ID NO: 2; or d) 1411C and D424C as indexed by reference to the amino acid sequence of SEQ ID NO: 2.Attorney Docket No. 0171.0129-PCT

[0356] Embodiment 2.3: The artificial nucleic acid of Embodiment 2.1 or 2.2, wherein the modified influenza A HA polypeptide is from a H1N1 influenza virus or a H3N2 influenza virus, optionally wherein the H1N1 influenza virus is A / West Virginia / 30 / 2022, or wherein the H3N2 influenza virus is A / Darwin / 06 / 2021.

[0357] Embodiment 2.4: The artificial nucleic acid of any one of Embodiments 2.1-2.3, wherein the modified influenza A HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to, or being identical to, the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, or wherein the artificial nucleic acid comprises a nucleic acid sequence having at least about 90% sequence identity to, or being identical to, the nucleic acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

[0358] Embodiment 2.5: The artificial nucleic acid, e.g. the artificial mRNA, of any one of Embodiments 2.1-2.4, comprising: i) a 5'-cap structure and / or a 3'-poly(A) sequence; ii) at least one chemically modified nucleotide; and / or iii) 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 Nl- methylp seudouridine .

[0359] Embodiment 2.6: A composition comprising the artificial nucleic acid of any one of Embodiments 2.1-2.5, optionally encapsulated in a lipid nanoparticle (LNP), optionally wherein the composition is an immunogenic composition.

[0360] Embodiment 2.7: The composition of Embodiment 2.6, wherein the LNP comprises: i) a cationic lipid; and optionally ii) a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid; optionally wherein: a) 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; b) the PEGylated lipid comprises or is 1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000); and / or c) the cholesterol- based lipid comprises or is cholesterol; and / or d) the helper lipid comprises or is dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), and optionally wherein: a) the cationic lipid is present at a molar ratio between about 35% and about 55%, such as 40%; b) the PEGylated lipid is present at a molar ratio between about 0.25% and about 2.75%, such as 1.5%; c) the cholesterol-basedAttorney Docket No. 0171.0129-PCT lipid is present at a molar ratio between about 20% and about 45%, such as 28.5%; and d) the helper lipid is present at a molar ratio between about 5% and about 35%, such as 30%, wherein all of the molar ratios are relative to the total lipid content of the LNP.

[0361] Embodiment 2.8: A modified influenza A HA polypeptide comprising one or more amino acid substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the one or more amino acid substitutions comprises: a) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 36 and 398 and / or amino acid positions 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1; or b) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 235 and 262 and / or amino acid positions 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0362] Embodiment 2.9: The modified influenza A HA polypeptide of Embodiment 2.8, wherein the modified influenza A HA polypeptide comprises amino acid substitutions: a) V36C and S398C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) G219C and R234C as indexed by reference to the amino acid sequence of SEQ ID NO: 1 ; c) S235C and N262C as indexed by reference to the amino acid sequence of SEQ ID NO: 2; or d) 1411C and D424C as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

[0363] Embodiment 2.10: The modified influenza A HA polypeptide of Embodiment 2.8 or 2.9, wherein the modified influenza A HA polypeptide is from a H1N1 influenza virus or a H3N2 influenza virus, optionally wherein the H1N1 influenza virus is A / West Virginia / 30 / 2022, or wherein the H3N2 influenza virus is A / Darwin / 06 / 2021.

[0364] Embodiment 2.11: The modified influenza A HA polypeptide of any one of Embodiments 2.8-2.10, wherein the modified influenza A HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to, or being identical to, the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

[0365] Embodiment 2.12: A trimeric influenza A HA polypeptide complex, comprising three copies of the modified influenza A HA polypeptide of any one of Embodiments 2.8-2.11.

[0366] Embodiment 2.13: A composition comprising the modified influenza A HA polypeptide of any one of Embodiments 2.8-2.11, or the trimeric influenza A HA polypeptideAttorney Docket No. 0171.0129-PCT complex of Embodiment 2.12, optionally wherein the composition is an immunogenic composition.

[0367] Embodiment 2.14: A vaccine comprising the composition of Embodiment 2.6, 2.7, or 2.13, a pharmaceutically acceptable carrier, and optionally an adjuvant..

[0368] Embodiment 2.15: The vaccine of Embodiment 2.14, wherein the vaccine is: i) a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza H3 HA polypeptide and an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage; ii) a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza H3 HA polypeptide, an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage, an mRNA encoding an influenza N2 neuraminidase (NA) polypeptide, an mRNA encoding an influenza N1 NA polypeptide, and an mRNA encoding an influenza NA polypeptide from the Influenza B / Victoria lineage; iii) a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza Hl HA polypeptide and an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage; iv) a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza Hl HA polypeptide, an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage, an mRNA encoding an influenza N2 NA polypeptide, an mRNA encoding an influenza N 1 NA polypeptide, and an mRNA encoding an influenza NA polypeptide from the Influenza B / Victoria lineage; v) a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an influenza H3 HA polypeptide and an influenza HA polypeptide from an influenza B / Victoria lineage; vi) a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an influenza H3 HA polypeptide, an influenza HA polypeptide from an influenza B / Victoria lineage, an influenza N2 NAAttorney Docket No. 0171.0129-PCT polypeptide, an influenza N 1 NA polypeptide, and an influenza NA polypeptide from the Influenza B / Victoria lineage; vii) a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an influenza Hl HA polypeptide and an influenza HA polypeptide from an influenza B / Victoria lineage; or viii) a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an influenza Hl HA polypeptide, an influenza HA polypeptide from an influenza B / Victoria lineage, an influenza N2 NA polypeptide, an influenza N 1 NA polypeptide, and an influenza NA polypeptide from the Influenza B / Victoria lineage.EXAMPLES

[0369] 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 Modified Influenza A Hemagglutinin (HA) Polypeptides

[0370] This example illustrates the design and generation of various modified influenza A HA polypeptides with one or more introduced amino acid mutations that contribute to the stabilization of the modified influenza A HA polypeptides in the prefusion conformation. The structure of an influenza A HA trimer in the prefusion conformation is shown in FIG. 1. The working hypothesis was that stabilizing the HA in the prefusion-closed conformation would elicit an increased neutralizing response and reducing HA binding to sialic acid would increase exposure of the RBS neutralizing epitopes to the immune system. The design strategies include introduction of cavity filling substitutions, disulfide bridge forming substitutions, pH sensor knock-out substitutions, helix break proline substitutions, sialic acid binding site ablation substitutions, and combinations thereof.

[0371] Amino acid substitutions were introduced into the wild-type influenza A HA from A / West Virginia / 30 / 2022 (a H1N1 subtype, SEQ ID NO: 1) and the wild-type influenza A HA from A / Darwin / 06 / 2021 (a H3N2 subtype, SEQ ID NO: 2) as summarized in Tables 3 and 4.Attorney Docket No. 0171.0129-PCTTable 3. Structural designs and rationale of modified influenza A HA polypeptides based on wildtype influenza A HA from A / West Virginia / 30 / 2022 (modified influenza Hl HA polypeptides; the numbering of the amino acid positions is as indexed by reference to the amino acid sequence of SEQ ID NO: 1).Attorney Docket No. 0171.0129-PCTTable 4. Structural designs and rationale of modified influenza A HA polypeptides based on wildtype influenza A HA from A / Darwin / 06 / 2021 (modified influenza H3 HA polypeptides; the numbering of the amino acid positions is as indexed by reference to the amino acid sequence of SEQ ID NO: 2).Attorney Docket No. 0171.0129-PCTAttorney Docket No. 0171.0129-PCT

[0372] Aggregated results categorized by types of modification in each A / West Virginia / 30 / 2022 and A / Darwin / 06 / 2021 strain are shown in FIG. 2A-2B. FIG. 2A shows the distribution of the modified HA polypeptides from the Hl strain A / West Virginia / 30 / 2022 that were screened for improvement in expression and prefusion confirmation, while maintaining antigenic likeness. FIG. 2B shows the distribution of the modified HA polypeptides from the H3 strain A / Darwin / 6 / 2021 that were screened for improvement in expression and prefusion confirmation, while maintaining antigenic likeness.Example 2. In vitro Expression of the Modified Influenza Hl HA Polypeptides

[0373] This example describes the in vitro expression of the modified influenza Hl HA polypeptides described in Example 1 in mRNA-transfected HeLa cells.

[0374] In this example, mRNAs encoding the wild-type HA polypeptide (WT) from A / West Virginia / 30 / 2022, or a panel of modified Hl HA polypeptides from the same A / West Virginia / 30 / 2022 strain, were transfected into HeLa cells that were seeded the day prior to transfection and incubated overnight at 37°C / 5% CO2 in a humidified incubator. The following day, mRNAs were transfected using Lipofectamine (following manufacturer’ s protocol) and cells were incubated for 20+0.5 hours at 37°C / 5% CO2 in a humidified incubator. At the experimental endpoint, cells were fixed at room temperature with 4% paraformaldehyde, washed with PBS, permeabilized with digitonin, 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 conjugatedAttorney Docket No. 0171.0129-PCT 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. The background-subtracted image MFI was reported for evaluation of protein expression.

[0375] As shown in FIG. 3, the in vitro expression of the majority of the modified Hl HA polypeptides is very similar to that of WT (shown by the black dashed line), with a few exceptions where low expression as observed, suggesting that not all HA modifications are beneficial for expression.Example 3. In vitro Characterization of the Modified Influenza Hl HA Polypeptides

[0376] This example describes the in vitro characterization of the modified influenza Hl HA polypeptides described in Example 1.

[0377] Monoclonal antibodies to well-defined, structural epitopes were selected against the surface of H1N1 A / WestVirgina / 30 / 2022 HA polypeptide to ensure that the protein was properly folded and presented at the cell surface and retained antigenic likeness to the WT protein. A lateral patch (head) antibody (045-09-2B05), a stem-region- specific antibody (CR9114), and an interprotomer interface binding antibody (FluA20) were used to investigate the conformation of the Hl design candidates on the surface of cells using flow cytometry. The binding of 045-09- 2B05 indicates the total surface expression and proper folding of the HA head. The binding of CR9114 indicates that the surface expressed HA polypeptides present a properly folded stem region and are in the prefusion state. The binding of FluA20 indicates that the HA trimer is breathing and in an open state, whereas the absence of FluA20 binding indicates that the HA trimer is in more of a closed state.

[0378] To assess the surface expression and presentation of the wild-type and the panel of Hl designs, 239FT cells were transfected in triplicate with a single DNA plasmid (pDNA) encoding each individual HA polypeptide. 24 hours 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 045-09-2B05, CR9114, and FluA20 or left in staining buffer alone (Fc control not shown). All reactions were then stained with a secondary antibody against human IgG conjugated to Alexa Fluor 488. Cells were washed in staining bufferAttorney Docket No. 0171.0129-PCT 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). The mean fluorescent intensity (MFI) was normalized to the percent of positive stained cells for each monoclonal antibody binding measured by flow cytometry is shown in FIG. 4A-4C. The graphs depict the percentage of live cells that bound to each individual monoclonal antibody targeting HA from 30,000 cells. Construct names are listed on the x-axis and MFI normalized by percent values of positive cells bound by each antibody on the y-axis. The black dashed line shows the expression level of the WT protein.

[0379] As shown in FIG. 4A, most of the designs show similar binding of the lateral patch monoclonal antibody, which closely correlates with the expression data on FIG. 3. This data suggests that most of the designs are properly folded and show similar levels of expression to the WT. FIG. 4B shows the binding of the stem antibody (which binds the prefusion HA trimer), and most of the designs have similar levels of CR9114 binding, suggesting most of the designs are in the prefusion state. Overall, there is good correlation between low binding of the lateral patch and stem antibody, suggesting that those designs were negatively impacted by the substitutions introduced. Finally, FIG. 4C shows binding of the FluA20 binding antibody, which shows that the Hl design panel can be grouped in three bins, one group that shows similar binding to the WT, one group that shows higher FluA20 binding to the WT (and thus more of an open head conformation), and some with a lower FluA20 binding, suggesting that those designs have more of a closed head conformation.Example 4. In vitro Expression of the Modified Influenza H3 HA Polypeptides

[0380] This example describes the in vitro expression of the modified influenza H3 HA polypeptides described in Example 1 in mRNA-transfected HeLa cells.

[0381] In this example, mRNAs encoding for the wild-type HA polypeptide (WT) from A / Darwin / 6 / 2021, or a panel of modified H3 polypeptides from the same A / Darwin / 6 / 2021 strain, were transfected into HeLa cells. The experiment was carried out as described in Example 2. As shown in FIG. 5, most of the H3 designs showed similar expression to the WT protein.Attorney Docket No. 0171.0129-PCTExample 5. In vitro Characterization of the Modified Influenza H3 HA Polypeptides

[0382] This example describes the in vitro characterization of the modified influenza H3 HA polypeptides described in Example 1.

[0383] Monoclonal antibodies to well-defined, structural epitopes were selected against the surface of H3N2 A / Darwin / 6 / 2021HA polypeptide to ensure that the protein was properly folded and presented at the cell surface and retained antigenic likeness to the WT protein. A head antibody (F0005-126), a stem-region-specific antibody (CR9114), and an interprotomer interface binding antibody (FluA20) were used to investigate the conformation of the H3 design candidates on the surface of cells using flow cytometry. The binding of F0005-126 indicates the total surface expression and proper folding of the HA head. The binding of CR9114 indicates that the surface expressed HA polypeptides present a properly folded stem region and are in the prefusion state. The binding of FluA20 indicates that the HA trimer is breathing and in an open state, whereas the absence of FluA20 binding indicates that the HA trimer is in more of a closed state. The experiment was done as described in Example 3.

[0384] As shown in FIG. 6A, most of the designs show similar binding of the head monoclonal antibody, which correlates with the expression data on FIG. 5. This data suggests that most of the designs are properly folded and show similar levels of expression to the WT. FIG. 6B shows the binding of the stem antibody (which binds the prefusion HA trimer), and most of the designs have similar levels of CR9114 binding, suggesting most of the designs are in the prefusion state. Overall, there is good correlation between low binding of the head and stem antibody, suggesting that those designs were negatively impacted by the substitutions introduced. Finally, FIG. 6C shows binding of the FluA20 binding antibody, which shows that the H3 design panel can be grouped in three bins, one group that shows similar binding to the WT, one group that shows higher FluA20 binding to the WT (and thus more of an open head conformation), and a smaller group with a lower FluA20 binding, suggesting that those designs have more of a closed head conformation.Example 6. Fusogenicity (Ability to Mediate Cell-Cell Fusion) of Modified Influenza A HA Polypeptides

[0385] This example describes the functional assessment of the prefusion- stabilized designs in a fusion assay previously described in Milder et al., Proc Natl Acad Sci USA, 2022,Attorney Docket No. 0171.0129-PCT119(6):e2115379119. The hypothesis is that prefusion stabilized designs will show no, or less fusion, compared to the wild-type protein. Briefly, Vero cells were co-transfected with plasmid DNAs encoding various influenza HA polypeptides (e.g., the wild-type HA polypeptide from A / WestVirgina / 30 / 2022 or A / Darwin / 6 / 2021, the Hl designs or H3 designs described in Example 1) and a reporter GFP plasmid (to assess fused cells) using the Trans-IT transfection reagent in media containing trypsin (to allow for proper processing of HAO into HA1-HA2). After transfection, cells were incubated overnight at 37°C / 5% CO2 in a humidified incubator. Cells were imaged under a fluorescent microscope prior to the low pH incubation to set a baseline control. Cells were then exposed to pH 5.0 for 10 minutes, after which the media was aspirated and replaced with normal cell culture media. After 1 hour incubation at 37°C / 5% CO2 in a humidified incubator, cells were imaged again to determine the formation of syncytia (multinucleated fused cells), which would indicate that the transfected HA polypeptide was able to mediate cell fusion.

[0386] As shown in FIG. 7A, the wild-type HA polypeptide from A / WestVirgina / 30 / 2022 (“Hl WV_WT”) can mediate fusion and syncytia formation. The HAO polypeptide, which has a substitution in the HA1-HA2 cleavage site and thus is fusion deficient (“Hl WV_HA0”), serves as a negative control for the assay and shows no fusion activity. The representative Hl design “Hl WV_512” shows no fusion, comparable to HAO, while the other representative Hl design “Hl WV_466” shows robust fusion as observed in the wild-type counterpart. These results suggest that at least the “Hl WV_512” design described herein is stabilized in the prefusion conformation.

[0387] Similarly, as shown in FIG. 7B, the wild- type HA polypeptide from A / Darwin / 6 / 2021 (“H3 Darwin / 6_WT”) can mediate fusion and syncytia formation. The HAO polypeptide, which has a substitution in the HA1-HA2 cleavage site and thus is fusion deficient (“H3 Darwin / 6_HA0”), serves as a negative control for the assay and shows no fusion activity. The two representative H3 designs, “H3 Darwin / 6_542” and “H3 Darwin / 6_571,” shows no fusion, comparable to HAO, suggesting that these two designs are stabilized in the prefusion conformation.Example 7. Immunogenicity of Influenza A HA Polypeptides Delivered as Lipid Nanoparticle (LNP) Formulated mRNA

[0388] This example describes the immunogenicity of representative modified influenza A HA polypeptides delivered as LNP-formulated mRNA in mice.Attorney Docket No. 0171.0129-PCT

[0389] Immunogenicity was assessed using a prime-boost regimen in naive mice, using a dose of 0.2 pg mRNA / LNP formulated HA per animal. In this study, 6-8-week-old BALB / C mice in groups of 6 were immunized by the intramuscular route with the indicated monovalent formulated HA polypeptide, encompassing different Hl or H3 design, or a negative control consisting of the buffer used for administration without LNP. 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 influenza A / WestVirgina / 30 / 2022 strain for the Hl designs or influenza A / Darwin / 6 / 2021 strain for the H3 designs by the HAI assay and a high content imaging-based micro -neutralization test (HINT).

[0390] The HAI 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 1:5,120 for testing against the B / Michigan / 01 / 2021 strain. The HAI assay was performed in V bottom 96- well plates using 4 HAU of chicken embryonated egg amplified virus and 0.5% turkey red blood cell (TBC) (serum starting dilution = 1:10). The HAI titer was determined as the highest dilution of serum resulting in complete inhibition of hemagglutination and is reported by the dilution titer.

[0391] The HINT assay was performed as follows. Briefly, serial 2-fold dilutions of receptor destroying enzyme (RDE)-treated sera from 1:40 to 1:40,960 were mixed with an equal volume of virus, about 1000 focus forming units (FFU), and incubated for 60 minutes at 37°C / 5% CO2 in a humidified incubator. After incubation, an MDCK SIAT1 cell suspension of 30,000 cells / well was added to the virus-sera mixture and incubated for about 22 hours at 37°C / 5% CO2 in a humidified incubator. The cell monolayers were fixed with methanol and prepared for staining. Wells were then incubated with anti-influenza monoclonal antibody against NP, followed by an Alexa FLUOR® 488-conjugated secondary antibody. Cells were washed and plates scanned on CTL ImmunoSpot Cell Imaging v2. Counts from plate were transferred into SoftMax Pro 6 GxP software in the Sanofi Universal Exporter to calculate neutralization 50 (NT50) titers that achieves 50% foci reduction from sigmoidal curve (using the 4PL Sigmoidal dose-response [variable slope] function).

[0392] As shown in FIG. 8A, of the 5 representative modified influenza Hl HA polypeptides tested, all showed robust microneutralization (HINT) titers after 2 doses (geometrical mean titersAttorney Docket No. 0171.0129-PCT(GMT) of the wild-type polypeptide shown by the dashed line). As shown in FIG. 8B, of the 5 representative modified influenza Hl HA polypeptides tested, all showed statistically similar HAI titers to the wild-type influenza A HA polypeptide from A / WestVirgina / 30 / 2022 (“HA_A_H1N1_WV_WT”; GMT of the wild-type polypeptide shown by the dashed line). No responses were seen with the negative control as expected (“LNP dil” for LNP diluent).

[0393] Similar results were observed with the H3 designs. As shown in FIG. 8C, of the 5 representative modified influenza H3 HA polypeptides tested, all showed robust microneutralization (HINT) titers after 2 doses (GMT of the wild-type polypeptide shown by the dashed line). As shown in FIG. 8D, of the 5 representative modified influenza H3 HA polypeptides tested, all showed statistically similar HAI titers to the wild-type influenza A HA polypeptide from A / Darwin / 6 / 2021 (“HA_A_H3N2_Darwin_WT”; GMT of the wild-type polypeptide shown by the dashed line). No responses were seen with the negative control as expected (“LNP dil” for LNP diluent).

[0394] 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 in the 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.

[0395] 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,Attorney Docket No. 0171.0129-PCT 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. In general, where embodiments or aspects of the present disclosure, is / are referred to as comprising particular elements, features, etc., certain embodiments or aspects consist, or consist essentially of, such elements, features, etc. 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.

[0396] 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. Attorney Docket No. 0171.0129-PCTCLAIMS1. An artificial messenger ribonucleic acid (mRNA) encoding a modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide comprises: a) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 36 and 398 and / or 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1; or b) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 235 and 262 and / or 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO:2.

2. The artificial mRNA of claim 1, wherein the modified influenza A HA polypeptide comprises amino acid substitutions: a) V36C and S398C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) G219C and R234C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; c) S235C and N262C as indexed by reference to the amino acid sequence of SEQ ID NO: 2; or d) 1411C and D424C as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

3. The artificial mRNA of claim 1 or 2, wherein the modified influenza A HA polypeptide is from a H1N1 influenza virus or a H3N2 influenza virus.

4. The artificial mRNA of claim 3, wherein the H1N1 influenza virus is A / West Virginia / 30 / 2022, or wherein the H3N2 influenza virus is A / Darwin / 06 / 2021.Attorney Docket No. 0171.0129-PCT5. The artificial mRNA of any one of claims 1-4, wherein the modified influenza A HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

6. The artificial mRNA of claim 5, wherein the modified influenza A HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

7. The artificial mRNA of any one of claims 1-6, comprising a nucleic acid sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

8. The artificial mRNA of claim 7, comprising the nucleic acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

9. The artificial mRNA of any one of claims 1-8, comprising a 5'-cap structure, a 5'- untranslated region, a 3'-untranslated region, and / or a 3'-poly(A) sequence.

10. The artificial mRNA of any one of claims 1-9, comprising at least one chemically modified nucleotide and / or a phosphorothioate bond.

11. The artificial mRNA of claim 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.

12. A composition comprising the artificial mRNA of any one of claims 1-11 encapsulated in a lipid nanoparticle (LNP).

13. The composition of claim 12, wherein the LNP comprises a cationic lipid.Attorney Docket No. 0171.0129-PCT14. The composition of claim 13, 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.

15. The composition of claim 13 or 14, wherein the LNP further comprises a polyethylene glycol conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

16. The composition of claim 15, wherein: a) the PEGylated lipid comprises or is l,2-dimyristoyl-rac-glycero-3- methoxypoly ethylene 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).

17. The composition of claim 15 or 16, 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.

18. The composition of Embodiment 17, 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.Attorney Docket No. 0171.0129-PCT19. The composition of claim 18, wherein the artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 3, 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%.

20. The composition of claim 18, wherein the artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 5, 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%.

21. The composition of claim 18, wherein the artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 7, 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%.

22. The composition of claim 18, wherein the artificial mRNA encodes the influenza A HA polypeptide of SEQ ID NO: 9, 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%.

23. The composition of any one of claims 12-22, wherein the composition is an immunogenic composition.

24. A modified influenza A HA polypeptide comprising one or more amino acid substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the one or more amino acid substitutions comprises: a) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 36 and 398 and / or 219 and 234 as indexed by reference to the amino acid sequence of SEQ ID NO: 1; orAttorney Docket No. 0171.0129-PCT b) at least two cysteine substitutions relative to a corresponding wild-type influenza A HA polypeptide, wherein the at least two cysteine substitutions are at amino acid positions 235 and 262 and / or 411 and 424 as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

25. The modified influenza A HA polypeptide of claim 24, wherein the modified influenza A HA polypeptide comprises amino acid substitutions: a) V36C and S398C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) G219C and R234C as indexed by reference to the amino acid sequence of SEQ ID NO: 1; c) S235C and N262C as indexed by reference to the amino acid sequence of SEQ ID NO: 2; or d) 1411C and D424C as indexed by reference to the amino acid sequence of SEQ ID NO: 2.

26. The modified influenza A HA polypeptide of claim 24 or 25, wherein the modified influenza A HA polypeptide is from a H1N1 influenza virus or a H3N2 influenza virus.

27. The modified influenza A HA polypeptide of claim 26, wherein the H1N1 influenza virus is A / West Virginia / 30 / 2022, or wherein the H3N2 influenza virus is A / Darwin / 06 / 2021.

28. The modified influenza A HA polypeptide of any one of claims 24-27, wherein the modified influenza A HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.

29. The modified influenza A HA polypeptide of claim 28, wherein the modified influenza A HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.Attorney Docket No. 0171.0129-PCT30. A trimeric influenza A HA polypeptide complex, comprising three copies of the modified influenza A HA polypeptide of any one of claims 24-29.

31. An artificial nucleic acid encoding the modified influenza A HA polypeptide of any one of claims 24-29.

32. The artificial nucleic acid of claim 31, wherein the artificial nucleic acid comprises at least one chemically modified nucleotide and / or a phosphorothioate bond.

33. A vector comprising the artificial nucleic acid of claim 31 or 32.

34. The vector of claim 33, wherein the vector is a messenger RNA (mRNA) production vector.

35. A host cell comprising the vector of claim 33 or 34.

36. A composition comprising the modified influenza A HA polypeptide of any one of claims 24-29, the trimeric influenza A HA polypeptide complex of claim 30, the artificial nucleic acid of claim 31 or 32, or the vector of claim 33 or 34.

37. The composition of claim 36, wherein the composition is an immunogenic composition.

38. A vaccine comprising the composition of claim 23 or 37, and a pharmaceutically acceptable carrier.

39. The vaccine of claim 38, further comprising an adjuvant.

40. The vaccine of claim 38 or 39, wherein the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza H3 HA polypeptide and an mRNA encoding an influenza HA polypeptide132Attorney Docket No. 0171.0129-PCT from an influenza B / Victoria lineage, optionally wherein the influenza H3 HA polypeptide is a wild-type influenza H3 HA polypeptide and the influenza HA polypeptide from an influenza B / Victoria lineage is a modified influenza HA polypeptide from an influenza B / Victoria lineage.

41. The vaccine of claim 38 or 39, wherein the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza H3 HA polypeptide, an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage, an mRNA encoding an influenza N2 neuraminidase (NA) polypeptide, an mRNA encoding an influenza N 1 NA polypeptide, and an mRNA encoding an influenza NA polypeptide from the Influenza B / Victoria lineage.

42. The vaccine of claim 38 or 39, wherein the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza Hl HA polypeptide and an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage.

43. The vaccine of claim 38 or 39, wherein the vaccine is a mRNA vaccine comprising an mRNA encoding the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an mRNA encoding an influenza Hl HA polypeptide, an mRNA encoding an influenza HA polypeptide from an influenza B / Victoria lineage, an mRNA encoding an influenza N2 NA polypeptide, an mRNA encoding an influenza N 1 NA polypeptide, and an mRNA encoding an influenza NA polypeptide from the Influenza B / Victoria lineage.

44. The vaccine of claim 38 or 39, wherein the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an influenza H3 HA polypeptide and an influenza HA polypeptide from an influenza B / Victoria lineage.133Attorney Docket No. 0171.0129-PCT45. The vaccine of claim 38 or 39, wherein the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a Hl influenza virus, and wherein the vaccine further comprises an influenza H3 HA polypeptide, an influenza HA polypeptide from an influenza B / Victoria lineage, an influenza N2 NA polypeptide, an influenza N1 NA polypeptide, and an influenza NA polypeptide from the Influenza B / Victoria lineage.

46. The vaccine of claim 38 or 39, wherein the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an influenza Hl HA polypeptide and an influenza HA polypeptide from an influenza B / Victoria lineage.

47. The vaccine of claim 38 or 39, wherein the vaccine is a recombinant vaccine comprising the modified influenza A HA polypeptide, wherein the modified influenza A HA polypeptide is from a H3 influenza virus, and wherein the vaccine further comprises an influenza Hl HA polypeptide, an influenza HA polypeptide from an influenza B / Victoria lineage, an influenza N2 NA polypeptide, an influenza N1 NA polypeptide, and an influenza NA polypeptide from the Influenza B / Victoria lineage.

48. A method of immunizing a subject, the method comprising administering to the subject in need thereof the vaccine of any one of claims 38-47.

49. The method of claim 48, 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.

50. The method of claim 48 or 49, wherein the subject is a human.

51. The method of claim 50, 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 than134Attorney Docket No. 0171.0129-PCT65 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.

52. The method of any one of claims 48-51, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally .

53. 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 38-47.

54. An in vitro method of preparing a trimeric influenza A HA polypeptide complex, the method comprising culturing the host cell of claim 35 in a cell culture medium, and expressing the trimeric influenza A HA polypeptide complex.

55. The in vitro method of claim 54, further comprising a step of purifying the trimeric influenza A HA polypeptide complex from the cell culture medium.135

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