Modified influenza b hemagglutinin polypeptides and nucleic acids and uses thereof

Abstract

The present application relates to modified influenza B hemagglutinin polypeptides and nucleic acids, e.g., messenger ribonucleic acids (mRNAs), encoding the same, as well as compositions comprising the same, vaccines comprising the same, and methods of using the same, e.g., for preventing and / or treating a disease or condition caused by an influenza B virus.

Description

Cross-references to related applications

[0001] This application claims priority to European application No. 23306481.5, filed on 6 September 2023, the entire contents of which are hereby incorporated by reference in their entirety. Submission of sequence list

[0002] The sequence list relating to this application is submitted electronically as an XML file, and its entirety is hereby incorporated by reference in this specification. The XML file containing the sequence list is named P51681WO_SL.xlm and is 17,434 bytes in size. Technical Field

[0003] This application relates to modified influenza B hemagglutinin polypeptides and nucleic acids encoding them, such as messenger ribonucleic acid (mRNA), as well as compositions containing them, vaccines containing them, and methods of using them, for example for the prevention and / or treatment of diseases or conditions caused by influenza B virus. Background Technology

[0004] Influenza viruses cause severe morbidity and mortality in humans worldwide, resulting in more than half a million deaths annually. Before the advent of SARS-CoV-2, influenza was a leading cause of respiratory illness. It is estimated that influenza B accounts for approximately 25% of all influenza cases globally. Vaccination is the most common preventative measure. Currently approved influenza vaccines are inactivated vaccines (containing whole viral particles or viral particles treated with lipid-dissolving agents (“split” vaccines), purified glycoproteins expressed in cell cultures (“subunit vaccines”), or live attenuated virus vaccines (which are typically produced in cell cultures or eggs). Other types of vaccines, such as nucleic acid-based or viral vector-based vaccines, are also under development. Since COVID-19, messenger RNA (mRNA) has emerged as a novel and highly effective nucleic acid for vaccine development.

[0005] Influenza hemagglutinin (HA) is a typical class I fusion protein and a major component of current influenza vaccines. HA is a metastable trimeric glycoprotein that undergoes a conformational change from its so-called pre-fusion state to its post-fusion state. However, generating class I fusion proteins (such as HA) through recombinant protein expression is challenging because they are often inherently unstable, expressed at low levels, and fail to form properly folded trimers. Recent clinical trial data also indicate that mRNA vaccines encoding influenza B HA elicit suboptimal neutralizing responses, which may affect their efficacy as vaccines.

[0006] Therefore, there is an urgent need to develop vaccines, whether recombinant vaccine vectors or nucleic acid vaccines, that can deliver influenza B HA with improved expression, quality and stability. Summary of the Invention

[0007] This article discloses a modified influenza B hemagglutinin (HA) peptide with features such as increased immunogenicity, improved stability of the pre-fusion conformation, improved expression, reduced sialic acid binding, and / or reduced antigenicity against non-neutralizing antibodies. These features would enable the induction of a higher neutralizing response, which could translate into better vaccine efficacy or lower reactivity when used at lower doses. Therefore, in one respect, this article provides an artificial messenger ribonucleic acid (mRNA) encoding an influenza B HA polypeptide, wherein the influenza B HA polypeptide comprises: a) at least one proline substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one proline substitution is located at amino acid positions 363, 366, 371, 372, 376, 380, 383, 390, 391, 393, 395, 397, 399, 421, 430, 431, 434, 436 and / or 490, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) The cysteine ​​substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least two cysteine ​​substitutions are located at amino acid positions 20 and 387, 35 and 408, 36 and 415, 37 and 411, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 232 and 433, 23 3 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 380 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 396 and 514, 401 and 475, 430 and 437, 430 and 438 and / or 430 and 439, as per reference SEQ a) Indexed by the amino acid sequence of SEQ ID NO: 1; c) At least one cavity-filling amino acid substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one cavity-filling amino acid substitution is located at amino acid positions 460, 467 and / or 474, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; d) One or more interface-stabilizing amino acid substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the one or more interface-stabilizing amino acid substitutions are located at amino acid positions 18, 121, 188, 226, 228, 408, 435 and / or 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1;e) One or more pH sensor knockout amino acid substitutions relative to the corresponding wild-type influenza B HA peptide, wherein the one or more pH sensor knockout amino acid substitutions are located at amino acid positions 226, 228, 237, 239, 383, 388, 391, 401, 405, 408, 435, 460, 474 and / or 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; f) At least one amino acid substitution relative to the corresponding wild-type influenza B HA peptide, wherein the at least one amino acid substitution produces or disrupts the N-linked glycosylation motif in the influenza B HA peptide and is located at amino acid positions 28, 60, 62, 141, 143, 186, 187, 214, 216, 223, 224, 336 and / or 349, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; and / or g) At least one amino acid substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one amino acid substitution is located at amino acid positions 157, 177, 218 and / or 257, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0008] In some embodiments, the influenza B HA peptide comprises two proline substitutions relative to the corresponding wild-type influenza B HA peptide, wherein the two proline substitutions are located at amino acid positions 430 and 436, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza B HA peptide comprises five amino acid substitutions relative to the corresponding wild-type influenza B HA peptide, wherein the five amino acid substitutions are located at amino acid positions 383, 401, 405, 408, and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza B HA peptide comprises amino acid substitutions A430P and N436P, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza B HA peptide comprises amino acid substitutions H383M, S401V, A405V, K408M, and H475M, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza B HA peptide is derived from influenza B / Victoria virus. In some embodiments, the influenza B / Victoria virus is B / Austria / 1359417 / 2021. In some embodiments, the influenza B HA peptide comprises an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5. In some embodiments, the influenza B HA peptide comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5. In some embodiments, the artificial mRNA comprises a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6. In some embodiments, the artificial mRNA comprises the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

[0009] In some embodiments, the artificial mRNA disclosed herein 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 phosphate thioester bond. In some embodiments, the at least one chemically modified nucleotide comprises pseudouridine, 2'-fluororibonucleotide, or 2'-methoxyribonucleotide, optionally wherein the pseudouridine is N1-methylpseudouridine.

[0010] In some embodiments, this document also provides a composition comprising the artificial mRNA disclosed herein encapsulated in lipid nanoparticles (LNPs). In some embodiments, the LNP comprises cationic lipids. In some embodiments, the cationic lipid comprises OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), or IM-001. In some embodiments, the LNP further comprises polyethylene glycol-conjugated (PEGylated) lipids, cholesterol-based lipids, and accessory lipids. In some embodiments, the PEGylated lipids comprise or are 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). In some embodiments, the cholesterol-based lipids comprise or are cholesterol. In some embodiments, the cofactor lipids comprise or are dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE). The cationic lipids are present in a molar ratio of about 35% to about 55%, the PEGylated lipids in a molar ratio of about 0.25% to about 2.75%, the cholesterol-based lipids in a molar ratio of about 20% to about 45%, and the cofactor lipids in a molar ratio of about 5% to about 35%, wherein all molar ratios are relative to the total lipid content of the LNP. In some embodiments, the cationic lipids are present in a molar ratio of about 40%, the PEGylated lipids in a molar ratio of about 1.5%, the cholesterol-based lipids in a molar ratio of about 28.5%, and the cofactor lipids in a molar ratio of about 30%, wherein all molar ratios are relative to the total lipid content of the LNP. In some embodiments, the artificial mRNA encodes the influenza B HA polypeptide of SEQ ID NO: 3, and the LNP comprises about 40% GL-HEPES-E3-E12-DS-4-E10, about 1.5% DMG-PEG2000, about 28.5% cholesterol, and about 30% DOPE in a molar ratio. In some embodiments, the artificial mRNA encodes the influenza B HA polypeptide of SEQ ID NO: 5, and the LNP comprises about 40% GL-HEPES-E3-E12-DS-4-E10, about 1.5% DMG-PEG2000, about 28.5% cholesterol, and about 30% DOPE in a molar ratio. In some embodiments, the composition is an immunogenic composition.

[0011] In a further aspect, this document provides an influenza B HA polypeptide comprising one or more amino acid substitutions relative to a corresponding wild-type influenza B HA polypeptide, wherein the one or more amino acid substitutions comprise: a) two proline substitutions at amino acid positions 430 and 436, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; or b) amino acid substitutions at amino acid positions 383, 401, 405, 408, and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza B HA polypeptide comprises amino acid substitutions A430P and N436P, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza B HA polypeptide comprises amino acid substitutions H383M, S401V, A405V, K408M, and H475M, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza B HA polypeptide is derived from influenza B / Victoria virus. In some embodiments, the influenza B / Victoria virus is B / Austria / 1359417 / 2021. In some embodiments, the influenza B HA peptide comprises an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the influenza B HA peptide comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5.

[0012] In some embodiments, this document also provides a trimeric influenza B HA peptide complex comprising three copies of any influenza B HA peptide disclosed herein. In some embodiments, the trimeric influenza B HA peptide complex disclosed herein exhibits stronger immunogenicity compared to a trimeric influenza B HA peptide complex prepared from a corresponding wild-type influenza B HA peptide without amino acid substitutions. In some embodiments, the trimeric influenza B HA peptide complex disclosed herein exhibits immunogenicity comparable to that of a trimeric influenza B HA peptide complex prepared from a corresponding wild-type influenza B HA peptide without amino acid substitutions. In some embodiments, a hemagglutination inhibition assay is used to measure immunogenicity.

[0013] Further, this disclosure provides an artificial nucleic acid encoding any of the influenza B HA peptides disclosed herein. In some embodiments, the artificial nucleic acid comprises at least one chemically modified nucleotide and / or phosphate thioester bond. In some embodiments, this 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, this disclosure provides a host cell comprising the vector.

[0014] In a further aspect, this disclosure provides a composition comprising any one of the influenza B HA peptide disclosed herein, a trimer influenza B HA peptide complex, an artificial nucleic acid, or a carrier provided herein. In some embodiments, the composition is an immunogenic composition.

[0015] A vaccine is also provided 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 an mRNA vaccine, and the vaccine further comprises mRNA encoding an influenza H3 HA peptide and mRNA encoding an influenza H1 HA peptide. In some embodiments, the vaccine is an mRNA vaccine, and the vaccine further comprises mRNA encoding an influenza H3 HA peptide, mRNA encoding an influenza H1 HA peptide, mRNA encoding an influenza N2 neuraminidase (NA) peptide, mRNA encoding an influenza N1 NA peptide, and mRNA encoding an influenza NA peptide from the influenza B / Victoria lineage. In some embodiments, the vaccine is a recombinant vaccine, and the vaccine further comprises an influenza H3 HA peptide and an influenza H1 HA peptide. In some embodiments, the vaccine is a recombinant vaccine, and the vaccine further comprises an influenza H3 HA peptide, an influenza H1 HA peptide, an influenza N2 NA peptide, an influenza N1 NA peptide, and an influenza NA peptide from the influenza B / Victoria lineage.

[0016] On the other hand, this document also provides a method for immunizing a subject, or a method for alleviating one or more symptoms of influenza B virus infection, comprising administering any of the vaccines disclosed herein to a subject in need. In some embodiments, the disclosed method prevents a subject from contracting influenza B virus, reduces the likelihood of a subject contracting influenza B virus, or reduces the likelihood of a subject developing severe illness due to influenza B virus infection. In some embodiments, the subject is a human being, for example, aged 6 months or older, less than 18 years of age, at least 6 months and less than 18 years of age, at least 18 years and less than 65 years of age, at least 6 months and less than 5 years of age, at least 5 years 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.

[0017] In a further aspect, this document provides an in vitro method for preparing any of the trimeric influenza beta-HA peptide complexes disclosed herein, the method comprising culturing any of the host cells disclosed herein in a cell culture medium and expressing the trimeric influenza beta-HA peptide complex. In some embodiments, the method further comprises the step of purifying the trimeric influenza beta-HA peptide complex from the cell culture medium. Attached Figure Description

[0018] 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.

[0019] Figure 1 The structure of the influenza B hemagglutinin (HA) trimer in its pre-fusion conformation was depicted. RBS: receptor binding site; FP: fusion peptide; RR1: refolded region 1; CH: central helix; RR2: refolded region 2.

[0020] Figure 2A-2B The study focused on pH sensor knockout and stem interface stabilization. Figure 2A ) and header interface stabilization ( Figure 2B Representative modification designs were used. The amino acid positions were indexed by referring to the amino acid sequence of SEQ ID NO: 1.

[0021] Figure 3 An example gating scheme for analyzing flow cytometry data of HA expressed on the upper surface of 293FT cells is described.

[0022] Figure 4 The surface expression levels of the representative modified influenza B / Austria / 1359417 / 2021 HA peptide described in Example 1, measured by flow cytometry using monoclonal antibodies CR8071 (degenerate esterase (VE) specific), R95-1D05 (receptor binding site (RBS) specific), and CR9114 (stem region specific), as measured in 293FT cells, were depicted. Construct names are listed on the x-axis. 1_Aus: Baseline control; 2_Aus and 5-Aus to 16_Aus: Representative modified influenza B / Austria / 1359417 / 2021 HA designs disclosed herein. MFI: Median fluorescence intensity.

[0023] Figure 5 The surface expression levels of the representative modified influenza B / Phuket / 3073 / 2013 HA peptide described in Example 1, measured by flow cytometry using the monoclonal antibodies CR8071 (VE-specific), R95-1D05 (RBS-specific), and CR9114 (stem region-specific), were assessed in 293FT cells. Construct names are listed on the x-axis. 17_Phu: Baseline control; 18_Phu and 21_Phu through 32_Phu: Representative modified influenza B / Phuket / 3073 / 2013 HA designs disclosed herein. MFI: Median fluorescence intensity.

[0024] Figure 6Example substitutions localized on the pre-fusion influenza B HA (protein database ID 4m44) are depicted. Two in the trimer HA are shown as bands, and the other as the molecular surface. In magnified views representing each stabilization strategy, the side chains of the stabilizing mutations are depicted as globular and rod-like structures. The amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0025] Figure 7 The surface expression levels of additional representative novel Austrian HA peptides described in Example 2 on 293FT cells, measured by flow cytometry using the monoclonal antibodies CR8071 (VE-specific), R95-1D05 (RBS-specific), and CR9114 (stem region-specific), were depicted. Construct names are listed on the x-axis. 1_Aus: Baseline control; 33_Aus to 43_Aus, 45_Aus to 52_Aus, and 54_Aus to 59_Aus: Representative modified influenza B / Austria / 1359417 / 2021 HA designs disclosed herein. MFI: Median fluorescence intensity.

[0026] Figure 8 The surface expression levels of the additional representative modified influenza B / Phuket / 3073 / 2013 HA peptides described in Example 2, measured by flow cytometry using the monoclonal antibodies CR8071 (VE-specific), R95-1D05 (RBS-specific), and CR9114 (stem region-specific), were depicted on 293FT cells. Construct names are listed on the x-axis. 17_Phu: Baseline control; 60_Phu to 70_Phu, 72_Phu to 79_Phu, and 81_Phu to 86_Phu: Representative modified influenza B / Phuket / 3073 / 2013 HA designs disclosed herein. MFI: Median fluorescence intensity.

[0027] Figure 9 The surface expression levels of the additional representative modified influenza B / Phuket / 3073 / 2013 HA peptide described in Example 3, measured by flow cytometry using the monoclonal antibodies R95-1D05 (RBS-specific) and CR9114 (stem region-specific), were depicted on 293FT cells. Construct names are listed on the x-axis. Phu_17 or 17_Phu: baseline control. MFI: median fluorescence intensity.

[0028] Figure 10The surface expression levels of the additional representative modified influenza B / Austria / 1359417 / 2021 HA peptide described in Example 3, measured by flow cytometry using the monoclonal antibodies R95-1D05 (RBS-specific) and CR9114 (stem region-specific), were depicted on 293FT cells. Construct names are listed on the x-axis. 1_Aus: Baseline control. MFI: Median fluorescence intensity.

[0029] Figure 11A-11D The summary results obtained from Examples 1-3 are depicted. Figure 11A The modified B / PHUKET / 3073 / 2013HA stem showed at least a 2-fold increase in the ratio of RBS-binding antibodies. Figure 11B The modified B / PHUKET / 3073 / 2013 HA and B / Austria / 1359417 / 2021 HA stems showed an increase of at least 2-fold in the ratio of RBS-binding antibodies. Figure 11C In strains B / PHUKET / 3073 / 2013 and B / Austria / 1359417 / 2021, common modification designs increased the ratio of stem-to-RBS binding antibodies by at least 2-fold. Figure 11D Summary results classified by modification type for each of the B / PHUKET / 3073 / 2013 and B / Austria / 1359417 / 2021 strains.

[0030] Figure 12A-12B The modeling structure of influenza B HA trimer was depicted, showing the positions of amino acid substitutions in two representative modified influenza B HA peptides. Figure 12A : Construct 16_Aus containing H381M_H473M_S399V_K406M_A403V replacements (left: pre-fusion conformation; right: post-fusion conformation); Figure 12B The construct 40_Aus contains A428P_N434P substitutions (left: pre-fusion conformation; right: post-fusion conformation). Amino acid positions are indexed by referring to the amino acid sequence of SEQ ID NO: 2.

[0031] Figure 13The in vitro characterization of two representative modified influenza B HA peptides (“40_Aus” and “16_Aus”) compared to their corresponding wild-type HA peptides (WT) is depicted using the percentage of total 293FT cells that were positive for binding to monoclonal antibodies as measured by flow cytometry. These figures depict the percentage of viable cells bound to each individual monoclonal antibody targeting HA (R95_1D05: RBD binding, CR9114: stem binding, and CR8071: VE binding) out of 30,000 cells. Construct names are listed on the x-axis, and the percentage of positive cells bound to each antibody is listed on the y-axis.

[0032] Figure 14 The in vitro expression of two representative modified influenza B HA peptides (“40_Aus” and “16_Aus”) compared with their corresponding wild-type HA peptides (WT) was depicted in mRNA-transfected HeLa cells.

[0033] Figure 15 The fusion (ability to mediate cell-cell fusion) of two representative modified influenza B HA peptides (“B / Austria_40” and “B / Aus 16”) was depicted compared with the corresponding wild-type HA peptide (“B / Austria WT”) and a negative control containing substitutions and fusion defects at the HA1-HA2 cleavage sites (“B / Aus HA0”).

[0034] Figure 16 The structural characterization of a representative modified influenza B peptide was depicted using negative staining electron microscopy (nsEM). Left: nsEM image of the wild-type HA peptide from B / Austria / 1359417 / 2021 after low pH exposure; Right: nsEM image of the HA peptide from a representative modified influenza B peptide (40_Aus design) after low pH exposure.

[0035] Figures 17A-17B The immunogenicity of a representative modified influenza B HA peptide delivered in mice via LNP-formulated mRNA compared to the wild-type HA peptide from B / Austria / 1359417 / 2021 is depicted. See Tables 4 and 5 for the construct designs listed at the top of the figures.

[0036] Figures 18A-18B Immunogenicity of a representative modified influenza B HA peptide (40_Aus design) as a purified recombinant soluble protein delivery was depicted compared to the wild-type HA peptide from B / Austria / 1359417 / 2021. Figure 18A Hint titer of cell cultured virus; Figure 18B HAI titer of oocyte-cultured virus.

[0037] Figures 19A-19B The in vitro expression of two representative modified influenza B HA peptide designs in different groups of influenza B / Victoria virus strains was described. Figure 19A :40_Aus design; Figure 19B :16_Aus design.

[0038] Figure 20A and 20B The in vitro characterization of the modified influenza B HA peptides generated based on a non-exhaustive combinatorial approach is described. See Table 8 for the construct designs listed at the top of the figures. Figure 20A : Binding of RBD-specific antibody (R95-1D05) and stem region-specific antibody (CR9114); Figure 20B The ratio of stem region-specific (CR9114) binding to RBD-specific (R95-1D05) binding.

[0039] Figure 21 The in vitro expression of modified influenza B HA peptides generated based on a non-exhaustive combinatorial approach was depicted. See Table 8 for the construct designs listed at the top of the figures. Detailed Implementation

[0040] Various exemplary embodiments will now be described in detail with reference to the accompanying drawings and discussed in the following detailed description. It should be understood that the following detailed description is provided to enable the reader to have a more complete understanding of certain embodiments, features, and details of this disclosure and should not be construed as limiting the scope of this disclosure.

[0041] To facilitate understanding of this disclosure, certain terms are defined below. Further definitions of the following and other terms may be provided in the specification. If a term's definition set forth below differs from those in an application or patent incorporated by reference, the definition set forth in this application shall be used to interpret the meaning of that term. definition

[0042] Unless the context clearly specifies otherwise, the singular forms “a / an” and “the” as used in this specification and the appended claims include plural indicators. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or those that will become obvious to a person skilled in the art upon reading this disclosure.

[0043] The use of sequential terms such as “first,” “second,” and “third” to modify claim elements in claims does not imply any priority, order of precedence, or sequence of one claim element relative to another, or the chronological order of performing method actions. Rather, it serves only as a label to distinguish one claim element with a specific name from another element with the same name (but for the purpose of using sequential terms) to differentiate claim elements.

[0044] The terms “about” or “approximately” are used herein to mean a typical tolerance range in the art. For example, “about” can be understood as approximately 2 standard deviations from the average. According to some embodiments, when referring to measurable values ​​such as quantities, “about” means to cover 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 a specified value, because these variations apply to performing the disclosed methods and / or manufacturing and using the disclosed compositions. When “about” appears before a series of values ​​or ranges, it should be understood that “about” may modify each value in that series or range.

[0045] According to this disclosure, an "amino acid" can be any of twenty naturally occurring (or "standard") amino acids or their variants, such as D-proline (the D-enantiomer of proline), or any variant not naturally occurring in proteins, such as leucine. Standard amino acids can be grouped 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; for example, cysteine ​​can form covalent disulfide bonds (or disulfide bridges) with other cysteine ​​residues, proline forms rings with the polypeptide backbone, and glycine is more flexible than other amino acids. Table 1 shows the abbreviations and properties of standard amino acids. Table 1. Standard amino acids, abbreviations, and properties.

[0046] As used herein in the specification and claims, the term “and / or” should be understood to mean “any one or both” of the elements so combined, that is, elements that exist in combination in some cases and separately in others. Other elements may optionally be present, whether related to or unrelated to those explicitly identified by the “and / or” clause, unless explicitly stated otherwise. Thus, as a non-limiting example, a reference to “A and / or B” used in conjunction with open-ended language such as “comprising” may, in one embodiment, refer to A without B (optionally including elements other than B); in another embodiment, refer to B only without A (optionally including elements other than A); in yet another embodiment, refer to A and B (optionally including other elements); and so on.

[0047] As used herein, the term "antibody" refers to an immunoglobulin molecule with a specific amino acid sequence produced by B lymphocytes. In some embodiments, antibodies are induced in humans or other animals by a specific antigen (immunogen). An antibody is characterized by a demonstrably specific reaction with an antigen, with the antibody and antigen each defined according to the other. The terms "inducing an antibody response," "inducing a neutralizing antibody response," "inducing an immunogenic response," or grammatical equivalents refer to the ability of an antigen or other molecule to induce antibody production. In some embodiments, the term "antibody" refers to any recombinant antibody used for in vitro assays (e.g., HA screening assays), including one or more polypeptides encoded substantially by an immunoglobulin gene or a fragment of an immunoglobulin gene. Such antibodies may exist as complete immunoglobulins or as fragments of the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Exemplary antibody fragments include, but are not limited to, F(ab)'2, Fab', and single-chain Fv (scFv).

[0048] As used herein, the term "antigen" refers to a factor that elicits an immune response; and / or (ii) a factor that binds to T cell receptors when exposed to or administered to an organism (e.g., when presented by an MHC molecule) or binds to antibodies (e.g., produced by B cells). In some embodiments, an antigen elicits a humoral response in an organism (e.g., including the production of antigen-specific antibodies); alternatively or additionally, in some embodiments, an antigen elicits a cellular response in an organism (e.g., involving T cells whose receptors specifically interact with the antigen). Those skilled in the art will understand that a particular antigen may elicit an immune response in one or more members of a target organism (e.g., mouse, ferret, rabbit, primate, human), but not in all members of the target organism species. In some embodiments, the 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%, and 99% (inclusive of all values ​​and subranges therebetween) of the target organism species. In some embodiments, the antigen binds to antibodies and / or T-cell receptors and may or may not induce a specific physiological response in the organism. In some embodiments, for example, the antigen may bind to antibodies and / or T-cell receptors in vitro, regardless of whether such interaction occurs in vivo. In some embodiments, the antigen reacts with products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The antigen includes the modified influenza B HA peptide described herein.

[0049] As used herein, "artificial nucleic acid molecule" and the like can generally be understood as a nucleic acid (e.g., DNA or RNA) that is not naturally occurring. Therefore, "artificial messenger ribonucleic acid (mRNA)" refers to mRNA that is not naturally occurring. In other words, an artificial nucleic acid molecule can be understood as a non-natural nucleic acid molecule or mRNA molecule. Such a nucleic acid or mRNA molecule may be non-natural due to its individual sequence (not naturally occurring) and / or due to other modifications (e.g., structural modifications of nucleotides that are not naturally occurring). Artificial nucleic acid molecules can be DNA molecules, RNA molecules (e.g., mRNA), or hybrid molecules containing both DNA and RNA portions. Typically, artificial nucleic acid molecules can be designed and / or generated using genetic engineering methods to correspond to a desired artificial nucleotide sequence (heterologous sequence). Furthermore, the term "artificial nucleic acid molecule" and the like (e.g., "artificial mRNA") is not limited to meaning "single molecule" but is generally understood to refer to a collection containing the same molecules. Therefore, it may involve multiple identical molecules contained in an aliquot sample.

[0050] As used herein, the phrase “indexed by reference to the amino acid sequence of SEQ ID NO: 1” refers to standardized biological sequence alignment, which allows comparison of a query sequence (e.g., a modified HA peptide sequence to which one or more modifications described herein have been or will be applied) with a subject sequence (e.g., a wild-type influenza HA peptide sequence, such as the HA peptide sequence of B / Phuket / 3073 / 2013 (SEQ ID NO: 1)) to identify amino acid residues in the target sequence corresponding to the same positions in the subject sequence. Typically, the target and query sequences share characteristic portions or features but differ slightly in length and / or sequence identity. For example, residue numbers in a specific target sequence or targeted modification can be identified and described based on the amino acid sequence of B / Phuket / 3073 / 2013. The sequence is aligned to the full-length HA protein sequence of B / Phuket / 3073 / 2013 (SEQ ID NO: 1), including the signal peptide, transmembrane, and cytoplasmic tail domains. The N-terminal methionine of the signal peptide is residue 1. Therefore, the phrase "amino acid position x indexed by reference to the amino acid sequence of SEQ ID NO: 1" is used herein to specify the position / identity of an amino acid residue in a polypeptide of interest (e.g., a modified influenza B HA polypeptide) by referring to the corresponding amino acid at position x in the HA polypeptide sequence of B / Phuket / 3073 / 2013 (SEQ ID NO: 1). Similarly, when referring to SEQ ID NO: 2, the phrase "indexed by reference to the amino acid sequence of SEQ ID NO: 2" is used herein to specify the position / identity of an amino acid residue in a polypeptide of interest (e.g., a modified influenza B HA polypeptide) by referring to the corresponding amino acid position in the HA polypeptide sequence of B / Austria / 1359417 / 2021 (SEQ ID NO: 2).

[0051] The terms “at least,” “less than,” “greater than,” or “up to” preceding a numerical value or series of numerical values ​​(e.g., “at least two”) are understood to include the numerical value adjacent to the term “at least,” “less than,” or “greater than,” as well as any subsequent numerical values ​​or integers that can logically be included, as shown from the context. When the terms “at least,” “less than,” “greater than,” or “up to” appear before a series of numerical values ​​or a range, it should be understood that “at least,” “less than,” “greater than,” or “up to” can modify each numerical value in that series or range.

[0052] As used herein, the term "bioactivity" refers to an observable biological effect or outcome achieved by a pharmaceutical agent or entity of interest. For example, in some embodiments, a specific binding interaction is bioactivity. In some embodiments, the regulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is bioactivity. In some embodiments, the presence or extent of bioactivity is assessed by detecting direct or indirect products produced by a biological pathway or event of interest. In some embodiments, the bioactivity of an HA peptide refers to the ability of the HA peptide to induce neutralizing antibodies. In these cases, the term "bioactivity" may be used interchangeably with "immunogenic activity."

[0053] As used herein, a "codon-optimized" nucleic acid sequence refers to a modified nucleic acid sequence that improves and optimizes the expression of the encoded protein for a specific expression system. A codon-optimized nucleic acid sequence encodes the same protein as the unoptimized parent sequence on which the codon-optimized nucleic acid sequence is based. For example, a nucleic acid sequence can be codon-optimized for expression in mammalian cells (e.g., CHO cells, human cells, mouse cells, etc.), bacterial cells (e.g., E. coli), insect cells, yeast cells, or plant cells.

[0054] As used herein, the term "epitope" includes any portion that is specifically recognized, wholly or partially, by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope consists of a plurality of amino acid residues in an antigen. In some embodiments, when the antigen adopts an associated three-dimensional conformation, the amino acid residues are surface-exposed. In some embodiments, when the antigen adopts such a conformation, the amino acid residues are physically close to or continuous with each other in space. In some embodiments, when the antigen adopts an alternative conformation (e.g., linearization; e.g., a nonlinear epitope), at least some amino acids are physically separated from each other.

[0055] As used herein, the term "head region" refers to a segment of the influenza B HA polypeptide, which is comprised of approximately amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Morphologically, the head region can be defined as a globular domain of HA.

[0056] As used herein, the term "hemagglutinin polypeptide" or "HA polypeptide" refers to a polypeptide whose amino acid sequence includes at least one characteristic sequence of influenza A or B HA. A variety of HA sequences from influenza isolates are known in the art; in fact, the National Center for Biotechnology Information (NCBI) maintains a database (ncbi.nlm.nih.gov / genomes / FLU / ) containing over 40,000 HA sequences (for both influenza A and B viruses). Referring to this database, those skilled in the art can readily identify characteristic sequences of common HA polypeptides and / or specific HA polypeptides (e.g., influenza B HA or influenza A HA, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 polypeptides; or HA mediating infection in a specific host; such as humans, birds, seals, etc.).

[0057] As used in this article, "H1" refers to influenza virus subtype 1 hemagglutinin (HA). Influenza A viruses are divided into Group 1 and Group 2. Groups 1 and 2 are further divided into subtypes, which are classified according to the sequences of two proteins on the viral surface: HA and neuraminidase (NA). Currently, 18 recognized HA subtypes exist (H1-H18). Therefore, H1 differs from other HA subtypes (including H2-H18).

[0058] As used in this article, "H3" refers to influenza virus subtype 3 HA. Therefore, H3 is different from other HA subtypes (including H1, H2, and H4-H18).

[0059] As used in this article, "N1" refers to influenza virus subtype 1 NA. Influenza A viruses are divided into Group 1 and Group 2. Groups 1 and 2 are further divided into subtypes, which are classified according to the sequences of two proteins, HA and NA, on the viral surface. Currently, 11 recognized NA subtypes exist (N1-N11). Therefore, N1 differs from other NA subtypes (including N2-N11).

[0060] As used in this article, “N2” refers to influenza virus subtype 2 neuraminidase (NA). Therefore, N2 is different from other NA subtypes (including N1 and N3-N11).

[0061] The term "host" is used herein to refer to a system (e.g., a cell, organism, etc.) in which the polypeptide of interest is present. In some embodiments, a host is a system susceptible to infection by a specific infectious agent. In some embodiments, a host is a system expressing a specific polypeptide of interest.

[0062] As used herein, the term "host cell" refers to a cell into which foreign DNA has been introduced (recombinantly or otherwise). For example, a host cell can be used to generate the modified influenza B HA peptide described herein using standard recombinant techniques. Those skilled in the art will understand upon reading this disclosure that these terms refer not only to the specific subject cell but also to the progeny of such cells. Such progeny may actually differ from the parent cell because certain modifications can occur in the progeny due to mutations or environmental influences, but are still included within the scope of the term "host cell" as used herein. In some embodiments, the host cell includes any prokaryotic and eukaryotic cell adapted to express foreign DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include those of prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains of *Escherichia coli*, *Bacillus*, *Streptomyces*, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., *Saccharomyces cerevisiae*, *Schizochytrium spp.*, *Pichia pastoris*, *Pichia methanata*, etc.), plant cells, microalgae (including eukaryotic algae such as *Chlamydomonas*, *Chlorella*, *Microchlorophyll*, *Schizochytrium*, diatoms such as *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, *Spodoptera litura*, etc.), non-human animal cells, human cells, or cell fusions (e.g., hybridomas or tetrasomes). In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells are eukaryotic cells and are selected from the following cell types: CHO (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293EBNA, 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 cells, C127 cells, SP2 / 0, NS-0, MMT060562, testicular Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the above-mentioned cells. In some embodiments, the cells contain one or more viral genes, for example, retinal cells expressing viral genes (e.g., PER.C6™ cells).

[0063] As used herein, the terms “in some embodiments,” “in some embodiments,” “in other embodiments,” “in some other embodiments,” etc., refer to embodiments of all aspects of this disclosure unless the context clearly indicates otherwise.

[0064] As used in this article, "mRNA vaccine" refers to a vaccine that uses messenger RNA (mRNA) to produce an immune response.

[0065] The term "mutation" refers to the deletion, addition, or substitution of amino acid residues in the amino acid sequence of a modified protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide.

[0066] As used herein, the term "N-linked glycosylation motif" refers to an amino acid sequence on the surface of a polypeptide (e.g., a protein) that accommodates the attachment of a glycan. An N-linked glycosylation motif contains a contiguous sequence NxS / Ty, where N is asparagine, x and y are any residues other than proline (P), and S / T are serine or threonine residues. Glycans are either polysaccharides or oligosaccharides. The term "glycan" can also refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or proteoglycan.

[0067] As used herein, the term "prevent, preventing, or prevention" means preventing or avoiding the manifestation of a disease, delaying the onset of one or more symptoms of a particular disease, condition, or illness (e.g., infection with a virus, such as influenza virus), and / or reducing the frequency and / or severity of such one or more symptoms. In some embodiments, prevention is evaluated on a population basis such that if a statistically significant reduction in the development, frequency, and / or intensity of one or more symptoms of a particular disease, condition, or illness is observed in a population susceptible to such a disease, condition, or illness, the agent is considered to "prevent" the disease, condition, or illness.

[0068] As used herein, the term "preventive effective amount" refers to an amount sufficient to prevent the manifestation of a disease, delay the onset of one or more symptoms of a particular disease, condition, or illness (e.g., infection with a virus, such as influenza virus), and / or reduce the frequency and / or severity of such one or more symptoms.

[0069] The term "sequence identity," as known in the art, refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparison. In the art, "identity" also refers to the degree of sequence correlation between polypeptide or polynucleotide sequences, as determined by matching such sequence strings. Sequence identity can be readily calculated using known methods, including but not limited to those described in: *Computational Molecular Biology*, edited by Lesk, AM, Oxford University Press, New York, 1988; *Biocomputing: Informatics and Genome Projects*, edited by Smith, DW, Academic Press, New York, 1993; *Computer Analysis of Sequence Data, Part I*, edited by Griffin, AM and Griffin, HG, Humana Publishing, New Jersey, 1994; *Sequence Analysis in Molecular Biology*, von Heinje, G., Academic Press, 1987; and *Sequence Analysis Primer*, edited by Gribskov, M. and Devereux, J., M Stockton Publishing, New York, 1991; and Carillo, H., and Lipman, D., Siam J. Applied Math., 48:1073 (1988). Typical methods for determining identity are designed to give the maximum match between test sequences. Methods for determining sequence identity and similarity are compiled in publicly available computer programs. Typical computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, SF et al., J. Molec. Biol. 215:403-410 (1990)).The BLAST X procedure is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, ed. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)). The well-known Smith-Waterman algorithm can also be used to determine identity. In some embodiments, sequence identity is determined using the BLAST procedure with default parameters.

[0070] As used herein, the term "receptor binding site" or "RBS" encompasses continuous or discontinuous amino acid residues in the head region of an influenza HA peptide, including amino acids involved in the direct binding of sialic acid to target cell receptor proteins. The amino acid residues constituting the "receptor binding site" or "RBS" of an influenza HA peptide can be described from the crystal structure of the HA peptide complexed with a sialic acid analog and the identification of amino acid residues that are substantially similar to the analog, or can be described with reference to the HA peptide sequence from a specific viral strain (e.g., B / Victoria / 02 / 1987, B / Yamagata / 16 / 1988). Therefore, in some embodiments, the "receptor binding site" or "RBS" of a modified HA peptide as described herein can be determined using a reference HA peptide sequence. In some embodiments, the "receptor binding site" or "RBS" of a modified HA peptide as described herein can be determined using the crystal structure of the HA peptide sequence. Exemplary reference crystal structures of HA peptides include the crystal structure of influenza virus B / Yamanashi / 166 / 1998 complexed with the avian-like receptor LSTA (PDBID 4M40). Therefore, in some embodiments, the RBS can be defined as a region comprising all amino acid residues within 5 angstroms of the LSTA molecule in the crystal structure of the HA of influenza B / Yamanashi / 166 / 1998 complexed with LSTA (PDB ID 4M40). In some embodiments, the RBS can be defined as a region of a modified influenza B HA polypeptide consisting of residues at amino acid positions 110, 151-157, 165, 173-175, 208-218, 248, and 254-259, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0071] As used in this article, a “recombinant vaccine” refers to a vaccine that uses genetic engineering to produce antigens from pathogens (such as influenza viruses) using harmless organisms (such as yeast or bacteria).

[0072] As used herein, the term "stem region" refers to a discontinuous region of the influenza B HA polypeptide containing approximately amino acid residues 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Morphologically, the stem region can be defined as an elongated domain emerging from the globular head.

[0073] As used herein, the term "subject" means any member of the animal kingdom. In some embodiments, "subject" refers to a human being. In some embodiments, "subject" refers to a non-human animal. In some embodiments, a subject includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, a non-human subject is a mammal (e.g., rodents, mice, rats, rabbits, ferrets, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, a subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In some embodiments, a subject is an adult, an adolescent, or an infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be used interchangeably with the term "subject."

[0074] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. One type of vector is the "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. Another type of vector is the viral vector, in which an additional DNA segment can be linked to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) can integrate into the host cell's genome after introduction and thus replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0075] As used in this article, the term "wildtype" generally refers to the normal form of a protein or nucleic acid found in nature. For example, wild-type HA peptides are found in natural isolates of influenza viruses. Many different wild-type HA sequences can be found in the NCBI Influenza Virus Sequence Database (ncbi.nlm.nih.gov / genomes / FLU / ). Structure of influenza B HA polypeptide

[0076] Influenza B HA is a trimeric glycoprotein anchored to the viral capsid and has two main functions. During entry, HA first mediates viral attachment to the target cell surface through interaction with sialic acid receptors. Then, after viral endocytosis, it triggers fusion of the viral and endosomal membranes to release its genome into the cytoplasm of the target cell. The influenza B HA contains a large extracellular domain of approximately 500 amino acids, which is cleaved by host-derived enzymes to generate two polypeptides still linked by disulfide bonds. The majority of the N-terminal fragment (approximately 320-330 amino acids), called HA1, forms the distal globular domain, also known as the head region, which contains the receptor-binding site (RBS) and most of the determinants recognized by viral neutralizing antibodies. The smaller C-terminal portion (approximately 180 amino acids), called HA2, consists of a fusion peptide, N-terminal refold region 1 (RR1), a central helix, and C-terminal refold region 2 (RR2), forming a stem-like structure, also known as the stem region, which anchors the head region to the cell or viral membrane. The three monomeric HA subunits combine through symmetric operations to form a trimer biomolecule of HA. Figure 1 The schematic structure of the influenza B HA trimer is shown in the figure.

[0077] Like other class I fusion proteins, influenza B HA transitions from a high-energy, metastable pre-fusion state to a post-fusion conformation, a transition triggered by low pH. Unintentionally constrained by any theory, the triggering of pH-based HA conformational changes is generally considered to be due to the protonation of amino acids acting as pH sensors. However, the residues or combinations of residues that function as pH sensors are not fully elucidated. Since membrane fusion events occur within a pH range of approximately 5–6, the most likely residues to function as pH sensors are histidine, aspartic acid, and / or glutamate, which possess pK values ​​within the appropriate pH range. a .

[0078] Most HA neutralizing antibodies bind to loops surrounding the RBS and interfere with receptor binding and attachment. Because these loops are highly variable, most antibodies targeting these regions are strain-specific. Functional and structural analyses of recently developed fully human monoclonal antibodies against influenza A HA with broad cross-neutralizing potency revealed that these antibodies do not target receptor binding and attachment, but rather interfere with membrane fusion processes and target 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, WO 2010 / 130636 and WO 2013 / 007770, all of which are incorporated herein by reference.

[0079] Amino acid sequences of numerous influenza B HA peptides from different influenza B viruses of the B / Yamagata and B / Victoria lineages, as well as nucleic acid sequences encoding such peptides, are known in the art and readily available in influenza virus databases maintained, for example, by the National Center for Biotechnology Information (NCBI) (ncbi.nlm.nih.gov / genomes / FLU / ). For instance, the amino acid sequences of wild-type HA peptides from representative influenza B viruses of the B / Yamagata and B / Victoria lineages are listed in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. HA peptide of influenza B / Phuket / 3073 / 2013 (B / Yamagata lineage) MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAP GGPYRLGTSGSCPNATSKIGFFATMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIVVDYMMQKPGKTGTIVYQRGVLLPQKVWCASG RSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEI LELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMLAIFIVYMVSRDNVSCSICL (SEQ ID NO: 1). HA peptide of influenza B / Austria / 1359417 / 2021 (B / Victoria lineage): MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHVRLSTHNVINTEDA PGGPYEIGTSGSCLNITNGKGFFATMAWAVPKNKTATNPLTIEVPYICTEEEDQITVWGFHSDDETQMARLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGK SKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEI LELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL (SEQ ID NO: 2).

[0080] The HA peptide of influenza B virus exhibits significant sequence conservation in two existing lineages. For example, the HA peptide (SEQ ID NO: 1) of B / Phuket / 3073 / 2013 (B / Yamagata lineage) and the HA peptide (SEQ ID NO: 2) of B / Austria / 1359417 / 2021 (B / Victoria lineage) share 92% sequence identity. Within the same lineage, the sequence identity is even greater. Given the substantial conservation of the influenza B HA sequence, those skilled in the art can readily compare the amino acid positions between different influenza B HA sequences to identify the corresponding amino acid positions between different influenza B virus strains.

[0081] Therefore, for the purposes of this disclosure (unless the context otherwise requires), the amino acid positions in the modified influenza B HA peptide are given with reference to the amino acid sequence of the full-length wild-type HA peptide of B / Phuket / 3073 / 2013 as shown in SEQ ID NO: 1. However, it should be noted, and those skilled in the art will understand, that different influenza B HA sequences may have different numbering systems, for example, if additional amino acid residues are added or removed compared to SEQ ID NO: 1. Therefore, it should be understood that when a particular amino acid residue is referred to by its number, the description is not limited to the amino acid precisely located at that numbered position when counting from the beginning of the given amino acid sequence, but rather any and all equivalent / corresponding amino acid residues in the influenza B HA peptide sequence are expected, even if the residue is not at the same precise numbered position, for example, if the given influenza B HA peptide sequence is shorter or longer than SEQ ID NO: 1, or has insertions or deletions compared to SEQ ID NO: 1. Modified influenza B HA peptide

[0082] This disclosure provides modified influenza B HA peptides, wherein one or more mutations have been introduced into the amino acid sequence relative to the corresponding wild-type influenza B HA peptide. In some embodiments, the modified influenza B HA peptides disclosed herein possess certain beneficial characteristics compared to the corresponding wild-type influenza B HA peptides, such as increased immunogenicity, improved stability of the pre-fusion conformation, improved expression, reduced sialic acid binding, and / or reduced antigenicity against non-neutralizing antibodies. In some embodiments, the modified influenza B HA peptides disclosed herein present epitopes for recognition by broadly protective antibodies, and can therefore be used to create epitope-based universal vaccines to induce protection against a wide range of influenza B virus strains. Artificial nucleic acid molecules encoding the modified influenza B HA peptides disclosed herein are also provided.

[0083] Compared to the amino acid sequence of the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide of this disclosure contains one or more amino acid mutations in the head region and / or stem region. The amino acid mutations introduced in the modified influenza B HA peptide of this disclosure include amino acid substitutions, deletions, or additions. In some embodiments, the one or more amino acid mutations may be independent of substitutions, insertions, deletions, and truncations. In some embodiments, the only mutation introduced in the amino acid sequence of the modified influenza B HA peptide of this disclosure is an amino acid substitution relative to the corresponding wild-type influenza B HA peptide, and may include conserved and / or non-conserved substitutions.

[0084] Conservative substitutions can be made, for example, based on the similarity of the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the amino acid residues involved. For example, as shown in Table 1, 20 naturally occurring amino acids can be divided into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, “conservative substitution” is defined as the exchange of an amino acid by another amino acid listed in the same group as the six standard amino acid groups shown above. For example, the exchange of Asp by Glu in a modified polypeptide retains a negative charge. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt the α-helix. As used in this article, “non-conservative substitution” is defined as the exchange of an amino acid with another amino acid listed in a different group of the six standard amino acid groups shown above.

[0085] In some embodiments, substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolidone, N-formylmethionine β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, engineered amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and general amino acid analogs). In some embodiments, amino acid substitutions at specific amino acid positions are selected based on factors including, but not limited to, the likelihood of steric hindrance, charge attraction, charge repulsion, common properties of amino acid side chains, secondary and / or tertiary structural considerations, and / or frequency of use in the respective host cells. Those skilled in the art will understand which factors should be considered when designing amino acid substitutions for the modified influenza B HA peptides disclosed herein.

[0086] Therefore, this document provides a modified influenza B HA peptide comprising a head region and a stem region, wherein the modified influenza B HA peptide comprises one or more modifications selected from proline mutations, disulfide bridge-forming mutations, interface stabilization mutations, pH sensor inactivation mutations, glycan-engineered mutations in the head region, glycan-engineered mutations in the stem region, and / or sialic acid binding interference mutations, as detailed below. In some embodiments, the modified influenza B HA peptide disclosed herein comprises at least one of the aforementioned two 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 inactivation mutation. In some embodiments, the modified influenza B HA peptide disclosed herein comprises at least one of the aforementioned three modifications, such as at least one proline mutation, at least one disulfide bridge-forming mutation, and at least one pH sensor inactivation mutation. In some embodiments, the modified influenza B HA peptide disclosed herein comprises at least one of the aforementioned four modifications, such as at least one proline mutation, at least one disulfide bridge-forming mutation, at least one interface stabilization mutation, and at least one pH sensor inactivation mutation. In some embodiments, the modified influenza B HA peptide disclosed herein comprises at least one of the aforementioned five modifications, such as at least one proline mutation, at least one disulfide bridge formation mutation, at least one interface stabilization mutation, at least one pH sensor inactivation mutation, and at least one glycan-engineered mutation in the head region. In some embodiments, the modified influenza B HA peptide disclosed herein comprises at least one of the aforementioned six modifications, such as at least one proline mutation, at least one disulfide bridge formation mutation, at least one interface stabilization mutation, at least one pH sensor inactivation mutation, at least one glycan-engineered mutation in the head region, and at least one sialic acid binding interference mutation. In some embodiments, the modified influenza B HA peptide disclosed herein comprises at least one of each of the aforementioned seven modifications.

[0087] In some embodiments, one or more of the above modifications stabilize the modified influenza B HA peptide disclosed herein in a pre-fusion conformation. Monoclonal antibodies targeting well-defined structural epitopes of the influenza B HA peptide, such as stem region-specific antibodies (e.g., CR9114), degenerate esterase (VE)-specific antibodies (e.g., CR8071), and receptor-binding site (RBS)-specific antibodies (e.g., R95-1D05), can be used to study the conformation of the modified influenza B HA peptide disclosed herein using flow cytometry. Binding of an RBS-specific antibody (e.g., R95-1D05) indicates total surface expression and integrity of the sialic acid-binding pocket on the surface-expressed HA peptide. Binding of VE-specific antibodies (e.g., CR8071) and stem region-specific antibodies (e.g., CR9114) indicates that the surface-expressed HA peptide is in a pre-fusion conformation. The binding ratio of stem region-specific antibodies (e.g., CR9114) to RBS-specific antibodies (e.g., R95-1D05) (e.g., CR9114 MFI / R95-1D05 MFI) indicates the ratio of pre-fusion-locked HA peptides presented on the cell surface to total HA peptides; a higher ratio indicates a higher percentage of surface-expressed HA peptides in a pre-fusion-locked conformation. Therefore, in some embodiments, the stabilization of the pre-fusion conformation is measured by the increased binding of the modified influenza B HA peptide to the stem region-specific antibody (e.g., CR9114) compared to the corresponding wild-type influenza B HA peptide. In some embodiments, the stabilization of the pre-fusion conformation is measured by the increased (e.g., at least two-fold) binding ratio of the stem region-specific antibody (e.g., CR9114) to the RBS-specific antibody (e.g., R95-1D05) compared to the corresponding wild-type influenza B HA peptide. In some embodiments, the stabilization of the pre-fusion conformation is measured by the increased binding of the modified influenza B HA peptide to a stem region-specific antibody (e.g., CR9114) and the increased binding ratio (e.g., two-fold or higher) of the stem region-specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) compared to the corresponding wild-type influenza B HA peptide.

[0088] Morphologically, the head region can be defined as a spherical structural domain of the HA, while the stem region can be defined as an elongated structural domain emerging from the spherical head. Figure 1The present disclosure provides a schematic structure of the modified influenza B HA polypeptide. In some embodiments, the head region of the modified influenza B HA polypeptide according to the present disclosure can be defined as the segment of the modified influenza B HA polypeptide at approximately amino acid positions 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the stem region, as a discontinuous region of the HA polypeptide, can be defined as containing approximately amino acid residues 16-56 and 308-547 of the modified influenza B HA polypeptide, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0089] Wild-type influenza B HA peptides contain a signal peptide (e.g., amino acids 1-15 corresponding to SEQ ID NO: 1) at their N-terminus, which directs the transport of the HA peptide during production and is typically not present in the final peptide, for example, used in vaccines. Therefore, in some embodiments, the modified influenza B HA peptides disclosed herein do not contain a signal peptide. In other embodiments, the modified influenza B HA peptides disclosed herein contain a signal peptide naturally derived from one or more modified wild-type influenza B HA peptides. In some embodiments, the modified influenza B HA peptides disclosed herein contain a signal peptide heterologous to the wild-type influenza B HA peptide. In some embodiments, such heterologous signal peptides are influenza HA signal peptides derived from influenza A HA peptides. In some embodiments, such heterologous signal peptides are influenza HA signal peptides derived from influenza B HA peptides different from wild-type influenza B HA peptides. In some embodiments, the heterologous signal peptides are from non-influenza sources. Any signal peptide known in the art that can direct the transport of the HA peptide during production can be used. For example, for the recombinant production of the modified influenza B HA polypeptide disclosed herein in insect cells, any signal peptide from mammals and viruses can be used to guide protein secretion in insect cells.

[0090] The introduction of one or more modified influenza B HA peptides according to this disclosure may be derived from any wild-type influenza B virus known in the art or to be discovered in the future, including but not limited to HA peptides of B / Yamagata influenza virus, such as B / Phuket / 3073 / 2013, and HA peptides of B / Victoria influenza virus, such as B / Austria / 1359417 / 2021. Other influenza B / Victoria viruses may include, but are not limited to, B / Washington / 02 / 2019, B / Lisboa / 37 / 2019, B / India / Punniv564 / 2021, B / Hunanjishou / 1678 / 2021, B / Yekaterinburg / 3292V / 2020, B / Kenya / 180 / 2021, B / Qinghaigonghe / 1259 / 2021, B / Shanghaihuangpu / 1825 / 2021, B / Bangladesh / 2002 / 2019, B / Colorado / 06 / 2017, and B / Brisbane / 60 / 2008. In some embodiments, the modified influenza B HA peptide disclosed herein is derived from the influenza B / Yamagata virus. In some embodiments, the modified influenza B HA peptide disclosed herein is derived from the influenza B / Phuket / 3073 / 2013 strain. In some embodiments, the modified influenza B HA peptide disclosed herein is derived from influenza B / Victoria virus. In some embodiments, the modified influenza B HA peptide disclosed herein is derived from influenza B / Austria / 1359417 / 2021 strain. In some embodiments, the modified influenza B HA peptide disclosed herein is derived from standard care strains. It should be understood that any influenza B strain not specifically mentioned herein may be a source for introducing one or more modified HA peptides according to this disclosure. proline mutation

[0091] In some embodiments, the modified influenza B HA peptides provided herein contain at least one proline mutation (e.g., substitution) in the stem region relative to the corresponding wild-type influenza B HA peptide (including amino acid residues 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1). The introduction of at least one proline mutation is designed to disrupt at least one helical structure, such as an α-helix, present in the stem region of the fusion-conformed influenza B HA peptide. Without intending to be bound by any theory, proline is the only naturally occurring amino acid in which the side chain is bonded to the nitrogen of the main chain to form a five-membered pyrrolidine ring. This pyrrolidine ring restricts the rotation of the N-Cα bond, reducing the main chain conformational entropy of the protein in its unfolded form relative to other naturally occurring amino acids. Therefore, without intending to be bound by any theory, the introduction of the proline mutation can increase protein stability by reducing the entropy difference between the unfolded and folded forms.

[0092] The stem region of the influenza B HA polypeptide consists of a fusion peptide, an N-terminal refold region 1 (RR1), a central helix, and a C-terminal refold region 2 (RR2). In some embodiments, at least one proline mutation is introduced into the fusion peptide of the stem region. In some embodiments, at least one proline mutation is introduced into the N-terminal refold region 1 of the stem region. In some embodiments, at least one proline mutation is introduced into the central helix of the stem region. In some embodiments, at least one proline mutation is introduced into the C-terminal refold region 2 of the stem region.

[0093] In some embodiments, the modified influenza B HA peptide disclosed herein contains one or more proline mutations at amino acid positions 363, 366, 371, 372, 376, 380, 383, 390, 391, 393, 395, 397, 399, 421, 430, 431, 434, 436, and / or 490, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide disclosed herein contains one or more proline mutations at amino acid positions 372, 397, 399, 421, 430, 431, 434, and / or 436, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Specifically, relative to the corresponding wild-type influenza B HA peptide, the influenza B HA peptide may contain one or more proline substitutions at amino acid positions 363, 366, 371, 372, 376, 380, 383, 390, 391, 393, 395, 397, 399, 421, 430, 431, 434, 436 and / or 490, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. For example, relative to the corresponding wild-type influenza B HA peptide, the influenza B HA peptide may contain one or more proline substitutions at amino acid positions 372, 397, 399, 421, 430, 431, 434 and / or 436, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the influenza B HA peptide contains two proline substitutions at amino acid positions 430 and 436, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0094] In some embodiments, relative to the corresponding wild-type influenza B HA polypeptide, the modified influenza B HA polypeptide disclosed herein comprises one or more proline substitutions selected from F363P, A366P, L371P, E372P, E376P, A380P, H383P, A390P, H391P, V393P, V395P, A397P, L399P, V421P, A430P, M431P, L434P, N436P and / or S490P, wherein the amino acid positions are indexed by referring to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to the corresponding wild-type influenza B HA polypeptide, the modified influenza B HA polypeptide disclosed herein comprises one or more proline substitutions selected from E372P, A397P, L399P, V421P, A430P, M431P, L434P and / or N436P, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0095] For example, relative to the corresponding wild-type influenza B HA peptide, an influenza B HA peptide containing proline substitutions (e.g., A430P and N436P) at amino acid positions 430 and 436 was found to be stable in the pre-fusion conformation, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. Therefore, in a particular embodiment, the influenza B HA peptide disclosed herein comprises proline substitutions A430P and N436P relative to the corresponding wild-type influenza B HA peptide, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. Disulfide bridge formation mutation

[0096] In some embodiments, the modified influenza B HA peptide provided herein contains at least two cysteine ​​mutations at appropriate amino acid positions, such that the introduction of at least two cysteine ​​mutations forms disulfide bridges (or disulfide bonds) in the modified influenza B HA peptide. Such mutations are referred to herein as “disulfide bridge-forming mutations.” Without wishing to be bound by any theory, the introduced disulfide bridges appear to stabilize the conformational state of the modified influenza B HA peptide, such as the pre-fusion conformation. To stabilize the pre-fusion conformation of the modified influenza B HA peptide, the amino acid correspondences mutated to cysteine ​​are chosen to be very close in the pre-fusion conformation but far apart in the post-fusion conformation. For example, such amino acid pairs can be identified by visually examining the crystal structure of the pre-fusion conformation of the influenza B HA peptide, such as the crystal structure of influenza virus B / Yamanashi / 166 / 1998 (PDB ID4M40), or by using computational protein design software for further quantitative selection, such as BioLuminate™ (Schrodinger LLC, NY, 2015), Discovery Studio™ (Accelrys, San Diego, 2015), MOE™ (Chemical Computing Group Inc., Montreal, 2015), and Rosetta™ (University of Washington, Seattle, 2015).

[0097] In some embodiments, the modified influenza B HA peptide provided herein comprises at least two cysteine ​​mutations that are appropriately positioned such that the at least two cysteine ​​mutations form disulfide bridges connecting HA1 and HA2 of the modified influenza B HA peptide to stabilize the pre-fusion conformation and reduce or eliminate the post-fusion conformation. In some embodiments, the modified influenza B HA peptide provided herein comprises at least two cysteine ​​mutations that are appropriately positioned such that the at least two cysteine ​​mutations form disulfide bridges between the loop region of HA1 and the helix of HA2 in the modified influenza B HA peptide. In some embodiments, the modified influenza B HA peptide provided herein comprises at least two cysteine ​​mutations that are appropriately positioned such that the at least two cysteine ​​mutations form disulfide bridges in the stem region of the modified influenza B HA peptide.

[0098] In some embodiments, the modified influenza B HA peptide disclosed herein contains at least two cysteine ​​mutations at amino acid positions 20 and 387, 35 and 408, 36 and 415, 37 and 411, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 232 and 433, 233 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 380 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 396 and 514, 401 and 475, 430 and 437, 430 and 438, and / or 430 and 439, as described by reference to SEQ ID NO: The amino acid sequence of SEQ ID NO: 1 is indexed. In some embodiments, the modified influenza B HA polypeptide disclosed herein contains at least two cysteine ​​mutations at amino acid positions 20 and 387, 36 and 415, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 233 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 401 and 475, 430 and 437, 430 and 438, and / or 430 and 439, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide disclosed herein contains at least two cysteine ​​mutations at amino acid positions 383 and 401, and / or amino acid positions 401 and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0099] Specifically, relative to the corresponding wild-type influenza B HA peptide, the influenza B HA peptide has the following amino acid positions: 20 and 387, 35 and 408, 36 and 415, 37 and 411, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 232 and 433, 233 and 434, 239 and 276, 346. At least two disulfide bridges forming cysteine ​​substitutions may be included at positions 465, 367 and 478, 378 and 397, 380 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 396 and 514, 401 and 475, 430 and 437, 430 and 438, and / or 430 and 439, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. For example, relative to the corresponding wild-type influenza B HA peptide, the influenza B HA peptide may contain at least two disulfide bridges forming cysteine ​​substitutions at amino acid positions 20 and 387, 36 and 415, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 233 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 401 and 475, 430 and 437, 430 and 438, and / or 430 and 439, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In a particular embodiment, relative to the corresponding wild-type influenza B HA peptide, the influenza B HA peptide contains at least two disulfide bridges forming cysteine ​​substitutions at amino acid positions 383 and 401, and / or amino acid positions 401 and 475, as indexed by reference to the amino acid sequence of SEQ ID NO:1.

[0100] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein comprises a subset selected from I20C and S387C, T35C and K408C, A36C and S415C, G37C and K411C, L125C and M431C, L127C and M431C, N185C and K223C, P186C and P224C, P186C and V239C, T188C and Q241C, N232C and E433C, G233C and L434C, V239C and T276C. The amino acid positions are substituted with at least two cysteine ​​residues of A346C and N465C, I367C and A478C, M378C and A397C, A380C and A397C, H383C and S401C, S387C and L510C, A394C and Q507C, A394C and L510C, A396C and L510C, A396C and A514C, S401C and H475C, A430C and E437C, A430C and I438C, and / or A430C and L439C, wherein the amino acid positions are indexed by referring to the amino acid sequence of SEQ ID NO: 1. In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein comprises a subset selected from I20C and S387C, A36C and S415C, L125C and M431C, L127C and M431C, N185C and K223C, P186C and P224C, P186C and V239C, T188C and Q241C, G233C and L434C, V239C and T276C, A346C, and others. At least two cysteine ​​substitutions of C and N465C, I367C and A478C, M378C and A397C, H383C and S401C, S387C and L510C, A394C and Q507C, A394C and L510C, A396C and L510C, S401C and H475C, A430C and E437C, A430C and I438C, and / or A430C and L439C, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0101] For example, relative to the corresponding wild-type influenza B HA peptide, an influenza B HA peptide containing disulfide bridges forming cysteine ​​substitutions at amino acid positions 383 and 401 (e.g., H383C and S401C) and 401 and 475 (e.g., S401C and H475C) was found to be stable in the pre-fusion conformation, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. Therefore, in a particular embodiment, relative to the corresponding wild-type influenza B HA peptide, the influenza B HA peptide contains disulfide bridges forming cysteine ​​substitutions at amino acid positions 383 and 401, or amino acid positions 401 and 475, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. Interface stability mutation

[0102] In some embodiments, the modified influenza B HA peptide provided herein, relative to the corresponding wild-type influenza B HA peptide, contains one or more amino acid mutations (e.g., one or more amino acid substitutions) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1) and / or the stem region (containing amino acid residues 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein such one or more amino acid mutations stabilize the modified influenza B HA peptide in a pre-fusion conformation by means of interface stabilization. These mutations are also referred to herein as “interface-stabilizing mutations.” Without wishing to be bound by any theory, interface stabilization can be achieved in various ways, such as cavity filling and the formation of polar interactions with adjacent residues (e.g., the formation of hydrogen bonds or salt bridges). Thus, in some embodiments, the one or more amino acid mutations that stabilize the modified influenza B HA peptide in a pre-fusion conformation by means of interface stabilization include at least one cavity-filling mutation in the stem region. In some embodiments, the modified influenza B HA peptide is stabilized by interface stabilization in a pre-fusion conformation by one or more amino acid mutations, including one or more amino acid mutations in the head and / or stem regions, to form polar interactions with adjacent amino acid residues. In some embodiments, the polar interactions comprise hydrogen bonds. In some embodiments, the polar interactions comprise salt bridges.

[0103] The term "cavity-filling mutation" refers to a mutation that causes amino acid residues in a parent peptide, such as wild-type influenza B HA peptides (e.g., HA peptides of B / PHUKET / 3073 / 2013 or B / Austria / 1359417 / 2021), to be replaced by amino acids intended to fill the internal cavities (i.e., interstitial spaces) present in the folded structure of the parent peptide. Without being bound by any theory, these cavity-filling mutations may contribute to stabilizing the pH-sensitive interface between the head and stem regions, thereby stabilizing the pre-fusion conformation of the modified influenza B HA peptide. For example, cavities in the pre-fusion conformation of wild-type influenza B HA peptides can be identified by visually examining the crystal structure of the pre-fusion conformation of the influenza B HA peptide, such as the crystal structure of influenza virus B / Yamanashi / 166 / 1998 (PDB ID 4M40), or by using computational protein design software for further quantitative selection, such as BioLuminate™ (Schrodinger LLC, NY, 2015), Discovery Studio™ (Accelrys, San Diego, 2015), MOE™ (Chemical Computation Group, 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 embedded in the pre-fusion conformation but exposed to the solvent in the post-fusion 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 amino acids with basic side chains at neutral pH (e.g., arginine (R), lysine (K), and histidine (H)). For example, a lysine (K) mutation can be introduced into the stem region to fill the cavity between the central helices and form a polar interaction with adjacent residues.

[0104] In some embodiments, the modified influenza B HA peptide of this disclosure contains at least one interface-stabilizing (e.g., cavity-filling) mutation (e.g., substitution) at amino acid position 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, relative to the corresponding wild-type influenza B HA peptide. In some embodiments, the modified influenza B HA peptide of this disclosure contains at least one interface-stabilizing (e.g., cavity-filling) mutation by substituting histidine (H), phenylalanine (F), or lysine (K) for the amino acid at position 460 (as indexed by reference to the amino acid sequence of SEQ ID NO: 1). In some embodiments, the modified influenza B HA peptide of this disclosure contains at least one interface-stabilizing (e.g., cavity-filling) mutation, which includes substituting lysine (K) for the amino acid at position 460 (as indexed by reference to the amino acid sequence of SEQ ID NO: 1).

[0105] In some embodiments, the amino acid at position 460 of the modified influenza B HA peptide (indexed as per the amino acid sequence of SEQ ID NO: 1) is not substituted with methionine (M), leucine (L), tryptophan (W), tyrosine (Y), or arginine (R). In some embodiments, the amino acid at position 460 of the modified influenza B HA peptide (indexed as per the amino acid sequence of SEQ ID NO: 1) is not substituted with arginine (R).

[0106] In some embodiments, the modified influenza B HA peptide of this disclosure contains at least one interface-stabilizing (e.g., cavity-filling) mutation (e.g., substitution) at amino acid position 467, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, relative to the corresponding wild-type influenza B HA peptide. In some embodiments, the modified influenza B HA peptide of this disclosure contains at least one interface-stabilizing (e.g., cavity-filling) mutation by substituting the amino acid at position 467 (as indexed by reference to the amino acid sequence of SEQ ID NO: 1) with histidine (H), phenylalanine (F), glutamine (Q), or tyrosine (Y). In some embodiments, the modified influenza B HA peptide of this disclosure contains at least one interface-stabilizing (e.g., cavity-filling) mutation by substituting the amino acid at position 467 (as indexed by reference to the amino acid sequence of SEQ ID NO: 1) with phenylalanine (F) or tyrosine (Y).

[0107] In some embodiments, the modified influenza B HA peptide of this disclosure contains at least one interface-stabilizing (e.g., cavity-filling) mutation (e.g., substitution) at amino acid position 474, as indexed by reference to the amino acid sequence of SEQ ID NO: 1, relative to the corresponding wild-type influenza B HA peptide. In some embodiments, the modified influenza B HA peptide of this disclosure contains at least one interface-stabilizing (e.g., cavity-filling) mutation by substituting an amino acid at position 474 (as indexed by reference to the amino acid sequence of SEQ ID NO: 1) with phenylalanine (F), asparagine (N), glutamine (Q), or tyrosine (Y). In some embodiments, the modified influenza B HA peptide of this disclosure contains at least one interface-stabilizing (e.g., cavity-filling) mutation by substituting an amino acid at position 474 (as indexed by reference to the amino acid sequence of SEQ ID NO: 1) with glutamine (Q).

[0108] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein comprises at least one interfacial stabilizing (e.g., cavity-filling) substitution selected from A460K, G467F, G467Q, G467Y and E474Q, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0109] In some embodiments, one or more amino acid mutations (e.g., substitutions) that stabilize the modified influenza B HA peptide in its pre-fusion conformation by interface stabilization relative to the corresponding wild-type influenza B HA peptide include one or more substitutions at amino acid positions 18, 121, 188, 226, 228, 408, 435, and / or 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more amino acid mutations that stabilize the modified influenza B HA peptide in its pre-fusion conformation by interface stabilization include one or more substitutions selected from D18W, Q121K, T188N, K226M, T228V, K408R, H435L, and A460K, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. pH sensor inactivation mutation

[0110] In some embodiments, the modified influenza B HA peptides provided herein contain one or more amino acid mutations (e.g., one or more amino acid substitutions) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1) and / or the stem region (containing amino acid residues 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), relative to the corresponding wild-type influenza B HA peptide, wherein the introduction of such one or more amino acid mutations inactivates one or more pH sensors in the head region and / or stem region. In this disclosure, these mutations are also referred to as “pH sensor inactivation mutations” or “pH sensor knockout mutations.” The transition of the influenza B HA peptide from a high-energy metastable pre-fusion state to its post-fusion conformation is triggered by low pH. Therefore, without being bound by any theory, inactivating one or more pH sensors of the influenza B HA peptide may contribute to stabilizing the pre-fusion conformation of the modified influenza B HA peptide. Any amino acid residues or combinations of residues in influenza B HA peptides known in the art or to be identified in the future that serve as pH sensors may be substituted to inactivate these pH sensors. For example, pH sensors in the stem region and / or head interface can be deactivated by replacing histidine (H) with hydrophobic residues such as leucine (L) or methionine (M).

[0111] In some embodiments, one or more amino acid mutations (e.g., one or more amino acid substitutions) that inactivate one or more pH sensors in the head and / or stem regions of the modified influenza B HA peptide disclosed herein are located at amino acid positions 226, 228, 237, 383, 388, 391, 401, 405, 408, 435, 460, 474 and / or 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more amino acid mutations (e.g., one or more amino acid substitutions) that inactivate one or more pH sensors in the head and / or stem regions of the modified influenza B HA peptide disclosed herein are located at amino acid positions 226, 228, 237, 383, 388, 401, 405, 408, 435, 460 and / or 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more amino acid mutations (e.g., one or more amino acid substitutions) that inactivate one or more pH sensors in the head and / or stem regions of the modified influenza B HA peptide disclosed herein are located at amino acid positions 226, 228, 237, 383, 388, 401, 408, 435, 460 and / or 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0112] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein contains one or more amino acid mutations (e.g., one or more amino acid substitutions) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the introduction of such one or more amino acid mutations inactivates one or more pH sensors in the head region. In these embodiments, the mutation may be a substitution at amino acid positions 226, 228, and / or 237, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Thus, in some embodiments, the modified influenza B HA peptide disclosed herein contains a pH sensor inactivation mutation in the head region, which includes a substitution at amino acid position 226, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 226 is substituted with glutamine (Q) or methionine (M). In some embodiments, the amino acid at position 226 is substituted with methionine (M). In some embodiments, the modified influenza B HA peptide disclosed herein comprises a pH sensor inactivation mutation comprising a substitution at amino acid position 228, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 228 is substituted with valine (V). In some embodiments, the modified influenza B HA peptide disclosed herein comprises a pH sensor inactivation mutation comprising a substitution at amino acid position 237, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 237 is substituted with isoleucine (I) or leucine (L). In some embodiments, the amino acid at position 237 is substituted with leucine (L).

[0113] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein contains at least one pH sensor inactivation mutation (e.g., at least one amino acid substitution) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), which contains at least two substitutions at amino acid positions 226 and 237, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acids at positions 226 and 237 are substituted with glutamine (Q) and isoleucine (I), respectively. In some embodiments, the amino acids at positions 226 and 237 are substituted with glutamine (Q) and leucine (L), respectively. In some embodiments, the amino acids at positions 226 and 237 are substituted with methionine (M) and isoleucine (I), respectively. In some embodiments, the amino acids at positions 226 and 237 are substituted with methionine (M) and leucine (L), respectively.

[0114] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein contains at least one pH sensor inactivation mutation (e.g., at least one amino acid substitution) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), which comprises at least two substitutions at amino acid positions 226 and 228, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acids at positions 226 and 228 are substituted with methionine (M) and valine (V), respectively.

[0115] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein contains one or more amino acid mutations (e.g., one or more amino acid substitutions) in the stem region (containing amino acid residues 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the introduction of such one or more amino acid mutations inactivates one or more pH sensors in the stem region. In these embodiments, the mutation may be a substitution at amino acid positions 383, 388, 391, 401, 405, 408, 435, 460, 474 and / or 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Thus, in some embodiments, the modified influenza B HA peptide disclosed herein contains a pH sensor inactivation mutation in the stem region, which comprises a substitution at amino acid position 383, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 383 is substituted with isoleucine (I), leucine (L), methionine (M), or proline (P). In some embodiments, the amino acid at position 383 is substituted with leucine (L) or methionine (M). In some embodiments, the modified influenza B HA peptide disclosed herein includes a pH sensor inactivation mutation in the stem region, which includes a substitution at amino acid position 388, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 388 is substituted with tryptophan (W). In some embodiments, the modified influenza B HA peptide disclosed herein includes a pH sensor inactivation mutation in the stem region, which includes a substitution at amino acid position 391, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 391 is substituted with proline (P). In some embodiments, the modified influenza B HA peptide disclosed herein includes a pH sensor inactivation mutation in the stem region, which includes a substitution at amino acid position 401, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 401 is substituted with isoleucine (I) or valine (V). In some embodiments, the amino acid at position 401 is substituted with valine (V). In some embodiments, the modified influenza B HA peptide disclosed herein contains a pH sensor inactivation mutation in the stem region, the mutation comprising a substitution at amino acid position 405, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 405 is substituted with Ile or valine (V).In some embodiments, the modified influenza B HA peptide disclosed herein includes a pH sensor inactivation mutation in the stem region, the mutation comprising a substitution at amino acid position 408, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 408 is substituted with methionine (M). In some embodiments, the modified influenza B HA peptide disclosed herein includes a pH sensor inactivation mutation in the stem region, the mutation comprising a substitution at amino acid position 435, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 435 is substituted with glutamic acid (E), tyrosine (Y), or phenylalanine (F). In some embodiments, the modified influenza B HA peptide disclosed herein includes a pH sensor inactivation mutation in the stem region, the mutation comprising a substitution at amino acid position 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 460 is substituted with lysine (K). In some embodiments, the modified influenza B HA peptide disclosed herein includes a pH sensor inactivation mutation in the stem region, the mutation comprising a substitution at amino acid position 474, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 474 is substituted with glutamine (Q). In some embodiments, the modified influenza B HA peptide disclosed herein includes a pH sensor inactivation mutation in the stem region, the mutation comprising a substitution at amino acid position 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 475 is substituted with isoleucine (I), leucine (L), methionine (M), tyrosine (Y), or tryptophan (W). In some embodiments, the amino acid at position 475 is substituted with tyrosine (Y) or tryptophan (W).

[0116] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein includes at least one pH sensor inactivation mutation in the stem region, the at least one pH sensor inactivation mutation comprising at least two substitutions at amino acid positions 383 and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acids at positions 383 and 475 are substituted with isoleucine (I). In some embodiments, the amino acids at positions 383 and 475 are substituted with leucine (L). In some embodiments, the amino acids at positions 383 and 475 are substituted with methionine (M). In some embodiments, the amino acids at positions 383 and 475 are substituted with isoleucine (I) and leucine (L), respectively. In some embodiments, the amino acids at positions 383 and 475 are substituted with isoleucine (I) and methionine (M), respectively. In some embodiments, the amino acids at positions 383 and 475 are substituted with leucine (L) and isoleucine (I), respectively. In some embodiments, the amino acids at positions 383 and 475 are replaced by leucine (L) and methionine (M), respectively. In some embodiments, the amino acids at positions 383 and 475 are replaced by methionine (M) and isoleucine (I), respectively. In some embodiments, the amino acids at positions 383 and 475 are replaced by methionine (M) and leucine (L), respectively.

[0117] In some embodiments, the modified influenza B HA peptide disclosed herein includes at least one pH sensor inactivation mutation in the stem region, the at least one pH sensor inactivation mutation comprising at least two substitutions at amino acid positions 383 and 401, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acids at positions 383 and 401 are substituted with methionine (M) and valine (V), respectively. In some embodiments, the amino acids at positions 383 and 401 are substituted with leucine (L) and valine (V), respectively.

[0118] In some embodiments, the modified influenza B HA peptide disclosed herein contains at least one pH sensor inactivation mutation in the stem region, the at least one pH sensor inactivation mutation comprising at least two substitutions at amino acid positions 383 and 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acids at positions 383 and 460 are substituted with leucine (L) and lysine (K).

[0119] In some embodiments, the modified influenza B HA peptide provided herein comprises one or more pH sensor inactivation substitutions selected from K226Q, K226M, T228V, H237I, H237L, H383I, H383L, H383M, H383P, H388W, H391P, S401I, S401V, A405I, A405V, K408M, H435E, H435F, A460K, E474Q, H475I, H475L, H475Y, H475W, and H475M, with the amino acid positions indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptides provided herein, relative to the corresponding wild-type influenza B HA peptides, comprise one or more pH sensor inactivation substitutions selected from K226M, T228V, H237L, H383M, H383L, H388W, S401V, K408M, H435E, H435F, H435Y, A460K, H475Y, and H475W, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more pH sensor inactivation mutations in the modified influenza B HA peptides provided herein comprise amino acid substitutions for K226M and H237L, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more pH sensor inactivation mutations in the modified influenza B HA peptides provided herein comprise amino acid substitutions for K226M and T228V, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more pH sensor inactivation mutations in the modified influenza B HA peptide provided herein include amino acid substitutions of H383M and S401V, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more pH sensor inactivation mutations in the modified influenza B HA peptide provided herein include amino acid substitutions of H383L and S401V, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, one or more pH sensor inactivation mutations in the modified influenza B HA peptide provided herein include amino acid substitutions of H383L and A460K, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0120] In some embodiments, the modified influenza B HA peptide provided herein comprises pH sensor inactivation substitutions at amino acid positions 383, 401, 405, 408, and 475, relative to the corresponding wild-type influenza B HA peptide, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide provided herein comprises pH sensor inactivation substitutions H383M, S401V, A405V, K408M, and H475M, relative to the corresponding wild-type influenza B HA peptide, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. Glycan engineered mutation

[0121] The modified influenza B HA peptide disclosed herein may also contain one or more mutations that result in the introduction or disruption of one or more N-linked glycosylation motifs. These mutations are referred to herein as “glycan-engineered mutations”.

[0122] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein contains at least one amino acid mutation (e.g., at least one amino acid substitution) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the at least one amino acid mutation introduces at least one N-linked glycosylation motif in the head region. In some embodiments, the at least one N-linked glycosylation motif introduced in the head region is located in or adjacent to the RBS of the head region. The RBS of the head region can be defined as a region of the modified influenza B HA peptide consisting of residues at amino acid positions 110, 151-157, 165, 173-175, 208-218, 248, and 254-259, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Alternatively, the RBS can be defined as the region comprising all amino acid residues of the modified influenza B HA peptide that correspond to all amino acid residues within 5 Å of the LSTa molecule in the crystal structure of the HA of influenza virus B / Yamanashi / 166 / 1998 complexed with LSTa (PDB ID 4M40). Not wishing to be bound by any theory, one or more additional N-linked glycosylation motifs may be added to the head region, particularly in or near the RBS, to mask non-neutralizing epitopes present in the region and facilitate neutralization of the modified influenza B HA peptide. In some embodiments, the N-linked glycosylation motif comprises a concordant sequence NxS / Ty, where N is asparagine, S / T is a serine or threonine residue, and x and y are any residues other than proline (P).

[0123] In some embodiments, at least one N-linked glycosylation motif introduced in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1) of the modified influenza B HA peptide disclosed herein, relative to the corresponding wild-type influenza B HA peptide, is generated by introducing one or more substitutions at amino acid positions 60, 62, 141, 143, 186, 187, 214, 216, 223 and / or 224, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 60 is substituted with threonine (T). In some embodiments, the amino acid at position 62 is substituted with threonine (T). In some embodiments, the amino acid at position 141 is substituted with asparagine (N). In some embodiments, the amino acid at position 143 is substituted with threonine (T). In some embodiments, the amino acid at position 186 is substituted with phenylalanine (F). In some embodiments, the amino acid at position 187 is substituted with threonine (T). In some embodiments, the amino acid at position 214 is substituted with asparagine (N). In some embodiments, the amino acid at position 216 is replaced by threonine (T). In some embodiments, the amino acid at position 223 is replaced by asparagine (N). In some embodiments, the amino acid at position 224 is replaced by phenylalanine (F).

[0124] In some embodiments, at least one N-linked glycosylation motif introduced into the head region of the modified influenza B HA polypeptide disclosed herein is generated by substitution of an amino acid selected from K60T, K62T, D141N, E143T, P186F, L187T, Q214N, K216T, K223N, and P224F. In some embodiments, at least one N-linked glycosylation motif introduced into the head region of the modified influenza B HA polypeptide disclosed herein is generated by substitution of an amino acid selected from K62T, P186F, L187T, Q214N, K216T, K223N, and P224F.

[0125] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein further includes at least one amino acid mutation (e.g., at least one amino acid substitution) in the stem region (containing amino acid residues 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the at least one amino acid mutation introduces or disrupts an N-linked glycosylation motif in the stem region. Without wishing to be bound by any theory, engineering one or more N-linked glycosylation motifs in the stem region may help stabilize the modified influenza B HA peptide in its pre-fusion conformation. In some embodiments, the N-linked glycosylation motif contains a concordant sequence of NxS / Ty, where N is asparagine, S / T is a serine or threonine residue, and x and y are any residues other than proline (P).

[0126] In some embodiments, at least one N-linked glycosylation motif introduced or disrupted in the stem region is generated by introducing at least one substitution at amino acid positions 28, 336, and / or 349, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 28 is substituted with threonine (T). In some embodiments, the amino acid at position 336 is substituted with threonine (T). In some embodiments, the amino acid at position 349 is substituted with alanine (A) or valine (V).

[0127] In some embodiments, at least one N-linked glycosylation motif introduced or disrupted in the stem region of the modified influenza B HA polypeptide disclosed herein is generated by substitution of an amino acid selected from P28T, K60T, P336T, T349A, and T349V, wherein the amino acid position is indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0128] In some embodiments, the modified influenza B HA peptide disclosed herein comprises at least one glycan-engineered mutation in the head region and at least one glycan-engineered mutation in the stem region. In some embodiments, the at least one glycan-engineered mutation in the head region comprises at least one substitution at amino acid position 60, and the at least one glycan-engineered mutation in the stem region comprises at least one substitution at amino acid position 28, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one glycan-engineered mutation in the head region comprises the amino acid substitution K60T, and the at least one glycan-engineered mutation in the stem region comprises the amino acid substitution P28T. Sialic acid binding interference mutation

[0129] In some embodiments, the modified influenza B HA peptide provided herein, relative to the corresponding wild-type influenza B HA peptide, contains at least one amino acid mutation (e.g., at least one amino acid substitution) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the at least one amino acid mutation results in reduced sialic acid binding of the modified influenza B HA peptide compared to a control influenza B HA peptide lacking at least one amino acid mutation that reduces sialic acid binding of the modified influenza B HA peptide. Such mutations are also referred to herein as “sialic acid binding interference mutations.” In some embodiments, the at least one amino acid mutation that reduces sialic acid binding of the modified influenza B HA peptide does not impair the integrity of the epitope targeted by the RBS neutralizing antibody. In some embodiments, the at least one amino acid mutation (e.g., substitution) that reduces sialic acid binding of the modified influenza B HA peptide is at amino acid positions 157, 177, 218, and / or 257, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid at position 157 is substituted with alanine (A). In some embodiments, the amino acid at position 177 is replaced by aspartic acid (D). In some embodiments, the amino acid at position 218 is replaced by alanine (A). In some embodiments, the amino acid at position 257 is replaced by leucine (L).

[0130] In some embodiments, at least one sialic acid-binding interference mutation in the modified influenza B HA peptide disclosed herein comprises an amino acid substitution S257L, wherein the amino acid position is indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0131] In some embodiments, the modified influenza B HA peptide provided herein comprises one or more sialic acid binding interference substitutions selected from S157A, V177D, L218A, and S257L. Mutant Combinations

[0132] In some embodiments, relative to the corresponding wild-type influenza B HA peptide, the modified influenza B HA peptide disclosed herein comprises two or more (e.g., three, four, five, six, or seven) modifications (e.g., amino acid substitutions) selected from: a) at least one proline mutation (e.g., substitution) in the stem region (containing amino acid residues 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the at least one proline mutation disrupts at least one helical structure in the stem region of the modified influenza B HA peptide in the fusion conformation; b) at least two cysteine ​​mutations (e.g., substitutions), wherein the at least two cysteine ​​mutations form disulfide bridges in the modified influenza B HA peptide; c) the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1) and / or the stem region (containing amino acid residues 16-56 and 308-547, as indexed by reference to SEQ ID NO: 1) d) One or more amino acid mutations (e.g., substitutions) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1) and / or the stem region (containing amino acid residues 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the one or more amino acid mutations deactivate one or more pH sensors in the head region and / or the stem region; e) At least one amino acid mutation (e.g., substitution) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the at least one amino acid mutation introduces at least one N-linked glycosylation motif in the head region; f) The stem region (containing amino acid residues 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1) At least one amino acid mutation (e.g., substitution) in the stem region (indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the at least one amino acid mutation introduces or disrupts an N-linked glycosylation motif in the stem region; and at least one amino acid mutation (e.g., substitution) in the head region (containing amino acid residues 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1), wherein the at least one amino acid mutation results in reduced sialic acid binding of the modified influenza B HA polypeptide compared to a control influenza B HA polypeptide lacking at least one amino acid mutation that reduces sialic acid binding of the modified influenza B HA polypeptide.

[0133] For example, one or more pH sensor inactivation mutations (e.g., substitutions) can be combined with one or more interface stabilizing mutations (e.g., substitutions), or one or more glycan-engineered amino acid mutations (e.g., substitutions) can be combined with one or more amino acid mutations (e.g., substitutions) that stabilize the modified influenza B HA peptide in its pre-fusion conformation or disrupt its post-fusion conformation. Therefore, in some embodiments, one or more modifications selected from options a)-d) above can be combined with one or more modifications from options e)-g) above. Alternatively or additionally, one or more modifications that disrupt the post-fusion conformation can be combined with one or more modifications that stabilize the pre-fusion conformation of the modified influenza B HA peptide.

[0134] 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 inactivation 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 glycan-engineering mutations (e.g., substitutions) disclosed herein in the head region and / or stem region. In some embodiments, one or more proline mutations (e.g., substitutions) disclosed herein are combined with one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0135] 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 inactivation 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 glycan-engineering mutations (e.g., substitutions) disclosed herein in the head region and / or stem region. In some embodiments, one or more disulfide bridge-forming mutations (e.g., substitutions) disclosed herein are combined with one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0136] In some embodiments, one or more interface-stabilizing mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivation 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 glycan-engineering mutations (e.g., substitutions) disclosed herein in the head region and / or stem region. In some embodiments, one or more interface-stabilizing mutations (e.g., substitutions) disclosed herein are combined with one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0137] In some embodiments, one or more pH sensor inactivation mutations (e.g., substitutions) disclosed herein are combined with one or more glycan-engineered mutations (e.g., substitutions) disclosed herein in the head region and / or stem region. In some embodiments, one or more pH sensor inactivation mutations (e.g., substitutions) disclosed herein are combined with one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0138] In some embodiments, one or more glycan-engineered mutations (e.g., substitutions) in the head region and / or stem region disclosed herein are combined with one or more sialic acid-binding interference mutations (e.g., substitutions) disclosed herein.

[0139] More than two different types of modifications can also be combined. 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) 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) and one or more pH sensor inactivation 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 glycan-engineering mutations (e.g., substitutions) disclosed herein in the head region and / or stem region. 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 sialic acid binding interference 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 inactivation 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 glycan-engineered mutations (e.g., substitutions) disclosed herein in the head and / or stem regions 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 interference 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 inactivation mutations (e.g., substitutions) disclosed herein and one or more glycan-engineered 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 inactivation mutations (e.g., substitutions) disclosed herein and one or more sialic acid binding interference 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 glycan-engineered mutations (e.g., substitutions) in the head region and / or stem region disclosed herein and one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0140] In some embodiments, one or more disulfide bridge-forming mutations (e.g., substitution) disclosed herein are combined with one or more interface-stabilizing mutations (e.g., substitution) disclosed herein and one or more pH sensor inactivation mutations (e.g., substitution) disclosed herein. In some embodiments, one or more disulfide bridge-forming mutations (e.g., substitution) disclosed herein are combined with one or more interface-stabilizing mutations (e.g., substitution) disclosed herein and one or more glycan-engineering mutations (e.g., substitution) disclosed herein in the head and / or stem regions. In some embodiments, one or more disulfide bridge-forming mutations (e.g., substitution) disclosed herein are combined with one or more interface-stabilizing mutations (e.g., substitution) disclosed herein and one or more sialic acid binding interference mutations (e.g., substitution) disclosed herein. In some embodiments, one or more disulfide bridge-forming mutations (e.g., substitution) disclosed herein are combined with one or more pH sensor inactivation mutations (e.g., substitution) disclosed herein and one or more glycan-engineering mutations (e.g., substitution) disclosed herein in the head and / or stem regions. In some embodiments, one or more disulfide bridge-forming mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivation mutations (e.g., substitutions) disclosed herein and one or more sialic acid binding interference 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 glycan-engineered mutations (e.g., substitutions) disclosed herein in the head region and / or stem region and one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0141] In some embodiments, one or more interface-stabilizing mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivation mutations (e.g., substitutions) disclosed herein and one or more glycan-engineered mutations (e.g., substitutions) disclosed herein in the head and / or stem regions. In some embodiments, one or more interface-stabilizing mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivation mutations (e.g., substitutions) disclosed herein and one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein are combined with one or more glycan-engineered mutations (e.g., substitutions) disclosed herein in the head and / or stem regions and one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0142] In some embodiments, one or more pH sensor inactivation mutations (e.g., substitutions) disclosed herein are combined with one or more glycan-engineered mutations (e.g., substitutions) in the head region and / or stem region disclosed herein and one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0143] 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 inactivation 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, and one or more glycan-engineering mutations (e.g., substitutions) disclosed herein in the head and / or stem regions. 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 sialic acid binding interference 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 inactivation mutations (e.g., substitutions) disclosed herein, and one or more glycan-engineered mutations (e.g., substitutions) disclosed herein in the head and / or stem regions. 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 inactivation mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interference 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 glycan-engineered mutations (e.g., substitutions) disclosed herein in the head and / or stem regions, and one or more sialic acid binding interference 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 glycan-engineering mutations (e.g., substitutions) disclosed herein in the head and / or stem regions. 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 interference 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 inactivation mutations (e.g., substitutions) disclosed herein, one or more glycan engineered mutations (e.g., substitutions) in the head region and / or stem region disclosed herein, and one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0144] In some embodiments, one or more disulfide bridge-forming mutations (e.g., substitution) disclosed herein are combined with one or more interface-stabilizing mutations (e.g., substitution) disclosed herein, one or more pH sensor inactivation mutations (e.g., substitution) disclosed herein, and one or more glycan-engineering mutations (e.g., substitution) disclosed herein in the head region and / or stem region. In some embodiments, one or more disulfide bridge-forming mutations (e.g., substitution) disclosed herein are combined with one or more interface-stabilizing mutations (e.g., substitution) disclosed herein, one or more pH sensor inactivation mutations (e.g., substitution) disclosed herein, and one or more sialic acid binding interference mutations (e.g., substitution) disclosed herein. In some embodiments, one or more disulfide bridge-forming mutations (e.g., substitution) disclosed herein are combined with one or more pH sensor inactivation mutations (e.g., substitution) disclosed herein, one or more glycan-engineering mutations (e.g., substitution) disclosed herein in the head region and / or stem region, and one or more sialic acid binding interference mutations (e.g., substitution) disclosed herein.

[0145] In some embodiments, one or more interface stabilizing mutations (e.g., substitutions) disclosed herein are combined with one or more pH sensor inactivation mutations (e.g., substitutions) disclosed herein, one or more glycan engineered mutations (e.g., substitutions) in the head region and / or stem region disclosed herein, and one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0146] 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 inactivation mutations (e.g., substitutions) disclosed herein, and one or more glycan-engineered mutations (e.g., substitutions) disclosed herein in the head and / or stem regions. 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 inactivation mutations (e.g., substitutions) disclosed herein, and one or more sialic acid binding interference 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 inactivation mutations (e.g., substitutions) disclosed herein, one or more glycan-engineered mutations (e.g., substitutions) disclosed herein in the head and / or stem regions, and one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0147] 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 inactivation mutations (e.g., substitutions) disclosed herein, one or more glycan-engineered mutations (e.g., substitutions) disclosed herein in the head region and / or stem region, and one or more sialic acid binding interference 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 inactivation mutations (e.g., substitutions) disclosed herein, one or more glycan-engineered mutations (e.g., substitutions) disclosed herein in the head region and / or stem region, and one or more sialic acid binding interference mutations (e.g., substitutions) disclosed herein.

[0148] In a specific embodiment, the influenza B HA polypeptide provided herein comprises: a) at least one proline substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one proline substitution is located at amino acid positions 363, 366, 371, 372, 376, 380, 383, 390, 391, 393, 395, 397, 399, 421, 430, 431, 434, 436 and / or 490, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) The cysteine ​​substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least two cysteine ​​substitutions are located at amino acid positions 20 and 387, 35 and 408, 36 and 415, 37 and 411, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 232 and 433, 23 3 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 380 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 396 and 514, 401 and 475, 430 and 437, 430 and 438 and / or 430 and 439, as per reference SEQ c) Indexed by the amino acid sequence of SEQ ID NO: 1; d) At least one cavity-filling amino acid substitution relative to the corresponding wild-type influenza B HA peptide, wherein the at least one cavity-filling amino acid substitution is located at amino acid positions 460, 467 and / or 474, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; e) One or more interface-stabilizing amino acid substitutions relative to the corresponding wild-type influenza B HA peptide, wherein the one or more interface-stabilizing amino acid substitutions are located at amino acid positions 18, 121, 188, 226, 228, 408, 435 and / or 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; f) One or more pH sensor knockout amino acid substitutions relative to the corresponding wild-type influenza B HA peptide, wherein the one or more pH sensor knockout amino acid substitutions are located at amino acid positions 226, 228, 237, 239, 383, 388, 391, 401, 405, 408, 435, 460, 474 and / or 475, as indexed by reference to SEQ ID NO: 1. Index the amino acid sequence of NO:1;f) A substitution of at least one amino acid relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one amino acid substitution generates or disrupts the N-linked glycosylation motif in the influenza B HA polypeptide and is located at amino acid positions 28, 60, 62, 141, 143, 186, 187, 214, 216, 223, 224, 336 and / or 349, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; and / or g) A substitution of at least one amino acid relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one amino acid substitution is located at amino acid positions 157, 177, 218 and / or 257, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. Exemplary modified influenza B HA peptide

[0149] In some embodiments, the modified influenza B HA peptides provided herein, relative to the corresponding wild-type influenza B HA peptides, comprise amino acid substitutions at the following positions: 1) Amino acid position 18; 2) Amino acid position 28; 3) Amino acid position 62; 4) Amino acid position 157; 5) Amino acid position 188; 6) Amino acid position 121; 7) Amino acid position 177; 8) Amino acid position 218; 9) Amino acid position 226; 10) Amino acid position 257; 11) Amino acid position 336; 12) Amino acid position 349; 13) Amino acid position 363; 14) Amino acid position 366; 15) Amino acid position 372; 16) Amino acid position 380; 17) Amino acid position 383; 18) Amino acid position 388; 19) Amino acid position 390; 20) Amino acid position 391; 21) Amino acid position 397; 22) Amino acid position 399; 23) Amino acid position 408; 24) Amino acid position 421 25) Amino acid position 430; 26) Amino acid position 435; 27) Amino acid position 436; 28) Amino acid position 460; 29) Amino acid position 467; 30) Amino acid position 474; 31) Amino acid position 475; 32) Amino acid positions 125 and 431; 33) Amino acid positions 214 and 216; 34) Amino acid positions 383 and 475; 35) Amino acid positions 380 and 397; 36) Amino acid positions 396 and 514; 37) Amino acid positions 430 and 436; 38) Amino acid positions 37 and 411; 39) Amino acid positions 467 and 474; 40) Amino acid positions 383 and 401; 41) Amino acid positions 431 and 434; 42) Amino acid positions 232 and 433; 43) Amino acid positions 401 and 475; 44) Amino acid positions 35 and 408; 45) Amino acid positions 430 and 439; 46) Amino acid positions 239 and 276; 47) Amino acid positions 188 and 241; 48) Amino acid positions 36 and 415; 49) Amino acid positions 387 and 510; 50) Amino acid positions 20 and 387; 51) Amino acid positions 186 and 239; 52) Amino acid positions 186 and 224; 53) Amino acid positions 346 and 465; 54) Amino acid positions 185 and 223; 55) Amino acid positions 378 and 397; 56) Amino acid positions 127 and 431; 57) Amino acid positions 233 and 434; 58) Amino acid positions 367 and 478; 59) Amino acid positions 430 and 439; 60) Amino acid positions 430 and 438; 61) Amino acid positions 430 and 437; 62) Amino acid positions 396 and 510; 63) Amino acid positions 394 and 507; 64) Amino acid positions 394 and 510; 65) Amino acid positions 186 and 187; 66) Amino acid positions 62 and 336; 67) Amino acid positions 383, 460, and 475; 68) Amino acid positions 383, 401, and 475; 69) Amino acid positions 28, 60, and 336; 70) Amino acid positions 226, 228, and 237; 71) Amino acid positions 223, 224, and 225; 72) Amino acid positions 226, 237, 383, and 475; 73) Amino acid positions at 383, 401, 405, and 475; 74) Amino acid positions 383, 401, 408, and 475; 75) Amino acid positions 226, 237, 383, 460, and 475; 76) Amino acid positions 226, 228, 237, 383, and 475; 77) Amino acid positions 383, 401, 405, 408, and 475; or 78) Amino acid positions 28, 60, 141, 143, and 336 As indexed by reference to the amino acid sequence of SEQ ID NO: 1. As shown herein, such one or more amino acid substitutions can stabilize the modified influenza B HA peptide in its pre-fusion (closed) conformation, for example, by increasing the binding of the modified influenza B HA peptide to a stem region-specific antibody (e.g., CR9114) compared to the corresponding wild-type influenza B HA peptide. In some embodiments, the stabilization of the pre-fusion conformation of the modified influenza B HA peptide is measured by determining the binding ratio of the stem region-specific antibody (e.g., CR9114) to the RBS-specific antibody (e.g., R95-1D05) compared to the corresponding wild-type influenza B HA peptide. In some embodiments, the modified influenza B HA peptide is at least twice as likely as the corresponding wild-type influenza B HA peptide to bind the stem region-specific antibody (e.g., CR9114) to the RBS-specific antibody (e.g., R95-1D05). In some embodiments, the stabilization of the pre-fusion conformation is measured by determining the binding ratio of the modified influenza B HA peptide to a stem region-specific antibody (e.g., CR9114) and by determining the binding ratio of the stem region-specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) compared to the corresponding wild-type influenza B HA peptide.

[0150] In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions at positions 383 and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, such amino acid substitutions are combined with one or more amino acid substitutions at position 460 (optionally in combination with amino acid substitutions at positions 226 and 227) or at positions 401 and 408 (optionally in combination with amino acid substitutions at position 405), as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0151] In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions at positions 383, 475, and 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions at positions 226, 237, 383, 460, and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions at positions 383, 401, 405, 408, and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0152] In some embodiments, the modified influenza B HA peptide provided herein comprises one or more amino acid substitutions at amino acid positions 349, 383, 397, 401, 421, 430, 436, or 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions at amino acid positions 349, 397, 421, or 430, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions at amino acid positions 1) 430 and 436, 2) 383 and 401, or 3) 401 and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0153] In some embodiments, the modified influenza B HA peptides provided herein, relative to the corresponding wild-type influenza B HA peptides, comprise the following amino acid substitutions: 1) D18W; 2) P28T; 3) K62T; 4) Q121K; 5)S157A; 6) V177D; 7) T188N; 8) L218A; 9) K226M; 10)S257L; 11) P336T; 12)T349A; 13)T349V; 14) F363P; 15) A366P; 16)E372P; 17) A380P; 18)H383P; 19)H388W; 20) A390P; 21)H391P; 22)A397P; 23)L399P; 24) K408R; 25) K408M; 26)V421P; 27) A430P; 28)H435E; 29)H435L; 30)H435Y; 31)H435F; 32)N436P; 33) A460K; 34) A460R; 35)G467F; 36)G467Q; 37)G467Y; 38)E474Q; 39)H475Y; 40)H475W; 41) T35C and K408C; 42) G37C and K411C; 43) N232C and E433C; 44) A380C and A397C; 45) A396C and A514C; 46) H383L and H475L; 47) H383C and S401C; 48) S401C and H475C; 49) A430P and N436P; 50)M431P and L434P; 51) G467Y and E474Q; 52) Q214N and K216T; 53) L125C and M431C; 54) A430C and L439C; 55) V239C and T276C; 56) T188C and Q241C; 57) A36C and S415C; 58) S387C and L510C; 59) I20C and S387C; 60) P186C and V239C; 61) P186C and P224C; 62) A346C and N465C; 63) N185C and K223C; 64) M378C and A397C; 65) L127C and M431C; 66) G233C and L434C; 67) I367C and A478C; 68) A430C and L439C; 69) A430C and I438C; 70) A430C and E437C; 71) A396C and L510C; 72) A394C and Q507C; 73) A394C and _L510C; 74) P186F and L187T; 75) K62T and P336T; 76) P28T, K60T, and P336T; 77) H383L, A460K, and H475L; 78) H383L, S401V and H475L; 79) K226M, T228V, and H237L; 80) K223N, P224F and Q225T; 81) K226Q, H237I, H383I and H475I; 82) K226M, H237L, H383L and H475L; 83) H383L, S401I, A405I and H475L; 84) H383L, S401V, A405V and H475L; 85) H383L, S401V, K408M and H475L; 86) H383M, S401V, K408M and H475M; 87) P28T, K60T, D141N, E143T and P336T; 88) K226M, H237L, H383L, A460K and H475L; 89) K226M, T228V, H237L, H383L and H475L; 90) H383L, S401V, A405V, K408M and H475L; or 91) H383M, S401V, A405V, K408M and H475M, The amino acid positions are indexed by referring to the amino acid sequence of SEQ ID NO: 1.

[0154] In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions H383L and H475L, or H383M and H475M, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, such amino acid substitutions are combined with one or more other amino acid substitutions, such as A460K (optionally combined with K226M and H227L), or S401V and K408M (optionally combined with A405V), as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0155] In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions of H383L, H475L, and A460K, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions of K226M, H237L, H383L, A460K, and H475L, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions of H383M, S401V, A405V, K408M, and H475M, wherein the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0156] In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions of T349V, A397P, V421P, or A430P, as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified influenza B HA peptide provided herein comprises amino acid substitutions of A430P and N436P, H383C and S401C, or S401C and H475C, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0157] Exemplary influenza B HA peptides containing substitutions relative to the wild-type HA peptide are listed in Table 2A (where the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 1) and Table 2B (where the amino acid positions are indexed by reference to the amino acid sequence of SEQ ID NO: 2). Table 2A. Exemplary influenza B HA peptides (amino acid positions are indexed by referring to the amino acid sequence of SEQ ID NO: 1). Table 2BA. Exemplary influenza B HA peptides (amino acid positions are indexed by referring to the amino acid sequence of SEQ ID NO: 2).

[0158] The representative modified influenza B HA polypeptide disclosed herein may have the amino acid sequence shown in SEQ ID NO: 3: MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHVRLSTHNVINTEDA PGGPYEIGTSGSCLNITNGKGFFATMAWAVPKNKTATNPLTIEVPYICTEEEDQITVWGFHSDDETQMARLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGK SKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGPMDELHPEI LELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL (SEQ ID NO: 3).

[0159] Another representative modified influenza B HA polypeptide according to this disclosure may have the amino acid sequence shown in SEQ ID NO: 5: MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHVRLSTHNVINTEDA PGGPYEIGTSGSCLNITNGKGFFATMAWAVPKNKTATNPLTIEVPYICTEEEDQITVWGFHSDDETQMARLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGK SKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWMGYTSHGAHGVAVAADLKVTQEVINMITKNLNSLSELEVKNLQRLSGAMDELHNEI LELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEMLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL (SEQ ID NO: 5).

[0160] Therefore, in some embodiments, the influenza B HA polypeptide disclosed herein comprises an amino acid sequence having at least about 80%, for example, at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (inclusive of all values ​​and subranges therein) sequence identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments, the influenza B HA polypeptide disclosed herein comprises an amino acid sequence having at least about 80%, for example, at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (inclusive of all values ​​and subranges therein) sequence identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the influenza B HA polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the influenza B HA polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 5.

[0161] Similar to wild-type influenza B HA peptides, the modified influenza B HA peptides disclosed herein are capable of forming trimeric HA complexes through symmetric manipulation. Therefore, in some embodiments, a trimeric influenza B HA peptide complex comprising any three copies of any one of the modified influenza B HA peptides according to this disclosure is provided herein. In some embodiments, the trimeric influenza B HA peptide complex of this disclosure exhibits higher pre-fusion conformational stability compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications. Conformational stability can be measured using any method known in the art. In some embodiments, the pre-fusion conformational stability of the trimeric influenza B HA peptide complex of this disclosure is measured by increased binding to a stem-region-specific antibody (e.g., CR9114) compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications. In some embodiments, the stability of the pre-fusion conformation is measured by the increased binding ratio of stem region-specific antibody (e.g., CR9114) to RBS-specific antibody (e.g., R95-1D05) compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications.

[0162] In some embodiments, the trimeric influenza B HA peptide complex disclosed herein exhibits stronger immunogenicity compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications (e.g., a corresponding wild-type influenza B HA peptide without amino acid substitutions). In some embodiments, the trimeric influenza B HA peptide complex disclosed herein exhibits comparable immunogenicity compared to a control influenza B HA peptide without one or more modifications (e.g., a corresponding wild-type influenza B HA peptide without amino acid substitutions). Immunogenicity can be measured using any method known in the art. In some embodiments, a hemagglutination inhibition assay (HAI) is used to measure immunogenicity. Changes in the position of mutation or substitution

[0163] While this document provides a modified influenza B HA peptide with a specific amino acid mutation or substitution at an exemplary amino acid position, this disclosure also contemplates amino acid mutations or substitutions near the specified amino acid position. Therefore, in some embodiments, this disclosure also includes amino acid positions within three residues (e.g., within one or two residues) of the amino acid position specified herein. For example, disclosure of a mutation or substitution of an influenza B HA peptide at amino acid position 336 may also include mutations or substitutions at positions 333, 334, 335, 337, 338, or 339, respectively.

[0164] The exemplary modified influenza B HA peptide described herein illustrates this point, particularly regarding mutations or substitutions that lead to disulfide bridge formation or proline stabilization. For example, amino acid substitutions of cysteine ​​at amino acid positions 428 and 435, 428 and 436, and 428 and 437 all result in disulfide bridge formation. Similarly, amino acid substitutions of cysteine ​​at amino acid positions 392 and 505, 392 and 508, and 394 and 508 all result in disulfide bridge formation. Likewise, amino acid substitutions of proline at amino acid positions 397 or 399 stabilize the exemplary influenza B HA peptide in its pre-fusion conformation. Another example is the substitution of proline at amino acid positions 429 and 432, or alternatively at amino acid positions 428 and 434, which achieves the same or similar effects. Nucleic acid construction and expression

[0165] This disclosure also provides an artificial nucleic acid molecule encoding the disclosed modified influenza B HA peptide. The nucleic acid may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. The modified influenza B HA peptide provided herein can be synthesized as a DNA sequence using standard methods known in the art, followed by cloning and expression in a recombinant host system using a suitable vector. The modified influenza B HA peptide provided herein can also be synthesized as an RNA, such as a messenger RNA (mRNA) sequence. References to the nucleotide sequences shown herein include DNA molecules having the specified sequences and RNA molecules (e.g., mRNA) having the specified sequences, wherein U or a derivative thereof (e.g., pseudouridine) replaces T, unless the context requires otherwise. Other nucleotide derivatives or modified nucleotides may be incorporated into the artificial nucleic acid molecule encoding the disclosed modified influenza B HA peptide. The synthetic DNA or mRNA sequence encoding the disclosed modified influenza B HA peptide can be codon-optimized to improve and optimize the expression of the encoded protein for a specific expression system. Any codon optimization algorithm known in the art can be used to generate codon-optimized nucleic acid sequences.

[0166] The representative codon-optimized mRNA sequence encoding the influenza B HA peptide of SEQ ID NO: 3 is shown in SEQ ID NO: 4:

[0167] The representative codon-optimized mRNA sequence encoding the influenza B HA peptide of SEQ ID NO: 5 is shown in SEQ ID NO: 6:

[0168] In some embodiments, the artificial nucleic acid molecule (e.g., mRNA) encoding the influenza B HA polypeptide disclosed herein comprises 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% (inclusive of all values ​​and subranges) sequence identity with the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the artificial nucleic acid molecule (e.g., mRNA) encoding the influenza B HA polypeptide disclosed herein comprises 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% (inclusive of all values ​​and subranges) sequence identity with the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the artificial nucleic acid molecule (e.g., mRNA) encoding the influenza B HA polypeptide disclosed herein comprises or is composed of the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the artificial nucleic acid molecule (e.g., mRNA) encoding the influenza B HA polypeptide disclosed herein comprises or is composed of the nucleic acid sequence of SEQ ID NO: 6.

[0169] For expressing the modified influenza B HA peptide disclosed herein, suitable recombinant host cells include, but are not limited to, insect cells, mammalian cells, avian cells, bacterial cells, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (clonal isolates derived from the parental Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi293 cells, typically transformed with cleaved type 5 adenovirus DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, but are not limited to, 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 vector systems, are well known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are available, for example, from Ingenie, Inc. (San Diego, CA) in kit form. Avian cell expression systems are also known to those skilled in the art and are described, for example, in U.S. Patent 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 known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al., editor, 1989), Butterworth, London.

[0170] In some embodiments, the cell contains one or more viral genes, such as retinal cells expressing viral genes (e.g., PER.C6™ cells). In some embodiments, the host cell is an SF9 cell of the fall armyworm (Spodopterafrugiperda). See U.S. Patent No. 6,103,526, which is incorporated herein by reference in its entirety. In some embodiments, the host cell is an SF9 cell of the fall armyworm that has been infected with a baculovirus vector (e.g., Autographa californica nucleopolyhedrovirus). In some embodiments, the host cell is a CHO cell.

[0171] A variety of suitable vectors for expressing recombinant proteins in insect or mammalian cells are well known and conventional in the art. Suitable vectors may contain a number of components, including but not limited to one or more of the following: an origin of replication; a selective marker gene; one or more expression control elements, such as transcriptional control elements (e.g., promoters, enhancers, terminators) and / or one or more translation signals; and a signal sequence or leader sequence for targeting a secretory pathway in a selected host cell (e.g., of mammalian origin or from heterologous mammalian or non-mammalian species). For example, for expression in insect cells, a suitable baculovirus expression vector, such as pFastBac (Ingenieur), is used to generate recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express the recombinant protein. For expression in mammalian cells, vectors that drive construct expression in desired mammalian host cells (e.g., CHO cells) can be used.

[0172] Modified influenza B HA peptides can be purified using any suitable method. For example, methods for purifying recombinant influenza HA peptides are known in the art. See, for example, Wang et al., Vaccine, 2006, 24(12):2176-2185. Suitable methods for purifying the desired protein are well known in the art and include precipitation and various types of chromatography, such as hydrophobic interactions, ion exchange, affinity, chelation, and size exclusion. Suitable purification protocols can be created using two or more of these or other suitable methods. If desired, modified influenza B HA peptides may include purification-enhancing “tags,” such as epitope tags or histidine (HIS) tags. Such tagged peptides can be readily purified, for example, from conditioned media by chelation chromatography or affinity chromatography.

[0173] The purified peptide can be analyzed using 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 such as helices and β-sheets. ELISA, Octet, and FACS can be used to investigate the binding of the modified influenza B HA peptide disclosed herein to a wide range of 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). Therefore, the modified influenza B HA peptide according to this disclosure can be selected with the desired conformation (e.g., a stable pre-fusion conformation).

[0174] Therefore, in some embodiments, this document provides artificial nucleic acids encoding any of the modified influenza B HA peptides described herein. The artificial nucleic acids disclosed herein may be in the form of DNA or RNA, such as messenger RNA (mRNA). In some embodiments, the artificial nucleic acids disclosed herein are DNA molecules. In some embodiments, the artificial nucleic acids disclosed herein are RNA molecules. In some embodiments, the artificial nucleic acids disclosed herein are mRNA molecules.

[0175] This document also provides vectors containing the artificial nucleic acid molecules (e.g., mRNA) disclosed herein. RNA sequences encoding target proteins (e.g., mRNA encoding influenza HA protein) can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Vectors of particular interest may include expression vectors, replication vectors, probe-generating vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0176] In some embodiments, the vector can be used to express mRNA in host cells. In various embodiments, the vector can be used as a template for in vitro transcription (IVT). The construction of the most preferably 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.

[0177] In some embodiments, the vectors disclosed herein may 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.

[0178] Various RNA polymerase promoters are known. In some embodiments, the promoter may be the T7 RNA polymerase promoter. Other useful promoters may include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Common nucleotide sequences for the T7, T3, and SP6 promoters are known.

[0179] This article also discloses host cells (e.g., mammalian cells, such as human cells) including the vectors or RNA compositions disclosed herein.

[0180] A variety of different methods can be used to introduce polynucleotides into target cells, including but not limited to electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Massachusetts) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipid transfection, polymer encapsulation, peptide-mediated transfection, and biological projectile particle delivery systems (such as “gene guns”) (see, for example, Nishikawa et al. (2001). Hum Gene Ther.). [Human Gene Therapy] 12(8): 861-70 or TransIT-RNA Transfection Kit (Mirus, Madison, WI).

[0181] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane capsule).

[0182] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors disclosed herein, various measurements can be performed to confirm the presence of mRNA sequences in host cells. RNA

[0183] In some embodiments, the vaccine or immunogenic composition disclosed herein may comprise one or more self-amplifying RNAs encoding the influenza B HA polypeptide disclosed herein. Antigen expression from conventional mRNA is proportional to the number of mRNA molecules from the vaccine or immunogenic composition successfully delivered to the subject. However, self-amplifying RNA contains genetically engineered replicons derived from self-replicating viruses, and therefore can be added to the vaccine or immunogenic composition at lower doses than conventional mRNA while achieving comparable results.

[0184] In some embodiments, the RNA is messenger RNA (mRNA) containing an open reading frame (ORF) encoding the influenza B HA polypeptide disclosed herein. In some embodiments, the RNA (e.g., mRNA) further comprises at least one 5' UTR, 3' UTR, multiple (A) tails, and / or a 5' cap. A.5' cap

[0185] The 5' cap on mRNA can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Several types of 5' caps are known. The 7-methylguanosine cap (also known as "m7G" or "Cap-0") contains a guanosine nucleotide linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond.

[0186] Typically, a 5' cap is added as follows: First, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphate groups; then, guanosine triphosphate (GTP) is added to the terminal phosphate ester via guanylate transferase, creating a 5'5'5 triphosphate bond; then, the 7-nitrogen of guanine is 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 US Publication Nos. US 2016 / 0032356 and US Publication No. US 2018 / 0125989, which are incorporated herein by reference.

[0187] 5'-capping of polynucleotides can be performed concurrently with in vitro transcription reactions using the following chemical RNA cap analogs, according to the manufacturer's protocol, to generate 5'-guanosine cap structures: 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5'))(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies). 5'-Capping of modified RNA can be performed post-transcriptionally using a vaccinia virus capping enzyme to produce a cap 0 structure: m7G(5')ppp(5')G. A cap 1 structure can be produced using both a vaccinia virus capping enzyme and a 2'-O methyltransferase to produce: m7G(5')ppp(5')G-2'-O-methyl. A cap 2 structure can be generated from the cap 1 structure, followed by 2'-O-methylation of the penultimate nucleotide at the 5' end using a 2'-O methyltransferase. A cap 3 structure can be generated from the cap 2 structure, followed by 2'-O-methylation of the penultimate nucleotide at the 5' end using a 2'-O methyltransferase.

[0188] In some embodiments, the mRNA disclosed herein comprises a 5' cap selected from the group consisting of: 3'-O-Me-m7G(5')ppp(5')G (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.

[0189] In some embodiments, the mRNA disclosed herein comprises the following 5' cap: B. Untranslated Region (UTR)

[0190] In some embodiments, the mRNA disclosed herein includes a 5' and / or a 3' untranslated region (UTR). In the mRNA, the 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues until the transcription termination signal.

[0191] In some embodiments, the mRNA disclosed herein may include a 5' UTR containing one or more elements that affect the stability or translation of the mRNA. In some embodiments, the 5' UTR may have a length of about 10 to 5,000 nucleotides. In some embodiments, the 5' UTR may have a length of about 50 to 500 nucleotides. In some embodiments, the 5' UTR has a length of at least about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, or about 650 nucleotides. The lengths are approximately 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1,000 nucleotides, 1,500 nucleotides, 2,000 nucleotides, 2,500 nucleotides, 3,000 nucleotides, 3,500 nucleotides, 4,000 nucleotides, 4,500 nucleotides, or 5,000 nucleotides.

[0192] In some embodiments, the mRNA disclosed herein may include a 3' UTR, which includes one or more of the following: a polyadenylation signal, a protein binding site affecting the positional stability of the mRNA in the cell, or one or more binding sites of the miRNA. In some embodiments, the 3' UTR may have a length of 50 to 5,000 nucleotides or longer. In some embodiments, the 3' UTR may have a length of 50 to 1,000 nucleotides or longer. In some embodiments, the length of the 3' UTR is at least about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 450 nucleotides, about 500 nucleotides, about 550 nucleotides, about 600 nucleotides, about 650 nucleotides, about 700 nucleotides, or about 750 nucleotides. 0 nucleotides, approximately 800 nucleotides in length, approximately 850 nucleotides in length, approximately 900 nucleotides in length, approximately 950 nucleotides in length, approximately 1,000 nucleotides in length, approximately 1,500 nucleotides in length, approximately 2,000 nucleotides in length, approximately 2,500 nucleotides in length, approximately 3,000 nucleotides in length, approximately 3,500 nucleotides in length, approximately 4,000 nucleotides in length, approximately 4,500 nucleotides in length, or approximately 5,000 nucleotides in length.

[0193] In some embodiments, the mRNA disclosed herein may contain a 5' or 3' UTR derived from a gene different from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0194] In some embodiments, the 5' and / or 3' UTR sequences may be derived from stable mRNA (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycling enzymes) to increase mRNA stability. For example, the 5' UTR sequence may include a portion of the CMV, i.e., the early 1 (IE1) gene or a fragment thereof, to improve mRNA nuclease resistance and / or improve mRNA half-life. Including a sequence encoding human growth hormone (hGH) or a fragment thereof at the 3' end or untranslated region of the mRNA is also considered. Typically, these modifications improve mRNA stability and / or pharmacokinetic properties (e.g., half-life) relative to their unmodified counterparts and include modifications, for example, to improve mRNA resistance to in vivo nuclease digestion.

[0195] Exemplary 5' UTR includes a sequence derived from the CMV, i.e., the early 1 (IE1) gene (US Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference) or the sequence GGGAUCCUACC (SEQ ID NO: 7) (US Publication No. 2016 / 0151409, which is incorporated herein by reference).

[0196] 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) bundle. Furthermore, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In some embodiments, the 5' UTR derived from the 5' UTR of a TOP gene lacks the 5' TOP motif (oligopyrimidine bundle) (e.g., U.S. Publications 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0197] In some embodiments, 5' UTR is derived from the ribosomal protein large 32 (L32) gene (US Publication No. 2017 / 0029847, ibid.).

[0198] In some embodiments, the 5' UTR is derived from the 5' UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (US Publication No. 2016 / 0166710, ibid.).

[0199] In some embodiments, the 5' UTR is derived from the 5' UTR of the ATP5A1 gene (US Publication No. 2016 / 0166710, ibid.).

[0200] In some embodiments, the internal ribosome entry site (IRES) is used instead of the 5' UTR.

[0201] In some embodiments, the 5' UTR contains the nucleic acid sequence of GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO:8).

[0202] In some embodiments, the 3' UTR contains the nucleic acid sequence CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO:9).

[0203] The 5'UTR and 3'UTR are described in further detail in International Publication No. WO 2012 / 075040 (incorporated hereby by reference). C. Polyadenylation tail

[0204] As used herein, the terms "multiple (A) sequence," "multiple (A) tail," and "multiple (A) region" refer to the adenosine nucleotide sequence at the 3' position of an mRNA molecule. A multiple (A) tail can confer stability to mRNA and protect it from exonuclease degradation. A multiple (A) tail can enhance translation. In some embodiments, the multiple (A) tail is substantially homopolymeric. For example, a 100-adenosine nucleotide multiple (A) tail can substantially have a length of 100 nucleotides. In some embodiments, the multiple (A) tail can be interrupted by at least one nucleotide different from the adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a 100-adenosine nucleotide multiple (A) tail can have a length exceeding 100 nucleotides (including 100 adenosine nucleotides and at least one nucleotide different from the adenosine nucleotide or a segment of nucleotides). In some embodiments, the multiple (A) tail comprises the following sequence: AAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 10).

[0205] As used herein, “multiple (A) tails” typically refers to RNA. However, in the context of this disclosure, the term also refers to the corresponding sequence in a DNA molecule (e.g., “multiple (T) sequences”).

[0206] The multiple (A) tail can contain 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 multiple (A) tail can be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.

[0207] In some embodiments where the nucleic acid is RNA, the multi(A) tail of the nucleic acid is obtained from a DNA template during in vitro transcription of RNA. In some embodiments, the multi(A) tail is obtained in vitro using conventional chemical synthesis methods without transcription from a DNA template. In various embodiments, the multi(A) tail is generated by enzymatic polyadenylation of RNA (after in vitro transcription of RNA) using commercially available polyadenylation kits and corresponding protocols, or alternatively by using immobilized multi(A) polymerases (e.g., using the methods and means described in International Publication No. WO 2016 / 174271).

[0208] Nucleic acids can contain multiple (A) tails obtained through enzymatic polyadenylation, with most nucleic acid molecules containing approximately 100 (+ / -20) to approximately 500 (+ / -50) or approximately 250 (+ / -20) adenosine nucleotides.

[0209] In some embodiments, the nucleic acid may include a multi(A) tail derived from the template DNA, and may additionally include at least one additional multi(A) tail generated by enzymatic polyadenylation, for example as described in International Publication No. WO 2016 / 091391.

[0210] In some embodiments, the nucleic acid contains at least one polyadenylation signal.

[0211] In various embodiments, the nucleic acid may contain at least one multiple (C) sequence.

[0212] As used herein, the term "multiple (C) sequence" is intended to be a cytosine nucleotide sequence of up to about 200 cytosine nucleotides. In some embodiments, a multiple (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, a multiple (C) sequence comprises about 30 cytosine nucleotides. D. Chemical modification

[0213] The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may contain 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 may include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) (including, but not limited to, purines (adenine (A) and guanine (G))) or pyrimidines (thymine (T), cytosine (C), and uracil (U))). In some embodiments, the disclosed mRNA can be synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines, such analogs or derivatives as 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), dihydrouracil, 2-thio-uracil, 4-thio-uracil, 5-carboxyl-... Methylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxyuracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, piracetamidine, β-D-mannosyl-piracetamidine, aminophosphate, thiophosphate, peptide nucleotide, methylphosphonate, 7-deazoguanosine, 5-methylcytosine, and inosine.

[0214] In some embodiments, the disclosed mRNA may contain at least one chemical modification, including but not limited to pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0215] In some embodiments, the chemical modification is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0216] In some embodiments, the chemical modification includes N1-methylpseuuridine.

[0217] 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.

[0218] 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.

[0219] Preparation of such analogues is described, for example, in U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642. E. mRNA synthesis

[0220] The mRNA disclosed herein can be synthesized according to any of a variety of methods. For example, the mRNA disclosed herein can be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin. Immunol. [Journal of Immunology Symposium] 25(2): 152-159; Brunelle et al. (2013) MethodsEnzymol. [Enzymological Methods] 530:101-14. In short, IVT is typically performed using the following: a linear or circular DNA template containing a promoter, a library of ribonucleoside triphosphates, a buffer system that may include DTT and magnesium ions, a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions may vary depending on the particular application. The presence of these reagents is generally undesirable in the final mRNA product and can be considered as impurities or contaminants that can be purified or removed to provide uncontaminated and / or homogeneous mRNA suitable for therapeutic use. While mRNA from an in vitro transcription reaction may be required in some embodiments, mRNA from other sources may be used in accordance with this disclosure, including wild-type mRNA produced by bacteria, fungi, plants and / or animals. Immunogenic compositions and vaccines

[0221] This disclosure also relates to immunogenic compositions comprising any one of the modified influenza B HA peptides disclosed herein or an artificial nucleic acid or vector encoding such modified influenza B HA peptides. As used herein, the term "immunogenic composition" refers to a composition that generates an immune response, which may or may not be a protective immune response or protective immunity. The term "immune response" refers to a response of cells of the immune system (such as B cells, T cells, dendritic cells, macrophages, or polymorphonuclear cells) to a stimulus (such as an antigen, immunogen, or vaccine). An immune response may include any cells of the body involved in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate and / or adaptive immune responses. Methods for measuring immune responses are well known in the art and include, for example, measuring the proliferation and / or activity of lymphocytes (such as B or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, etc. An antibody response or humoral response is an immune response that produces antibodies. A "cellular immune response" is an immune response mediated by T cells and / or other leukocytes.

[0222] This document also provides vaccines comprising the immunogenic compositions disclosed herein and pharmaceutically acceptable carriers. As used herein, the term "vaccine" refers to a composition that produces 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 the occurrence of an infection-related illness) or reduces the symptoms of an infection (e.g., influenza virus infection). Vaccines may elicit prophylactic and therapeutic responses. Methods of administration vary depending on the vaccine but may include inoculation, ingestion, inhalation, or other forms of administration. Inoculation can be delivered via any of a variety of routes, including parenteral, intravenous, subcutaneous, intraperitoneal, intradermal, intranasal, inhalation, or intramuscular delivery.

[0223] The term "pharmaceuticalally acceptable" means that a carrier, at the dose and concentration used, will not cause unwanted or harmful effects on the subjects administering it. Such pharmaceutically acceptable carriers and excipients are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 19th edition, Mark Publishers, Easton, PA, 1995; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, eds., Taylor and Francis, 2000; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, ed., Pharmaceutical Press, 2000). The term "carrier" refers to a diluent, adjuvant, excipient, or medium administered with the composition. For example, saline solutions and aqueous solutions of glucose and glycerol can be used as liquid carriers, particularly for injectable solutions. The exact formulation should be suitable for the method of administration. Preferably, the modified influenza B HA peptide and / or nucleic acid (e.g., mRNA) molecule is formulated and administered as a sterile solution. The sterile solution is prepared by sterile filtration or by other methods known in the art. The solution can then be lyophilized or filled into a drug dosing container. The pH of the solution is typically in the range of pH 3.0 to 9.5, for example, pH 5.0 to 7.5.

[0224] Therefore, in some embodiments, this document provides a composition comprising: any modified influenza B HA peptide disclosed herein, a trimeric influenza B HA peptide complex comprising three copies of any modified influenza B HA peptide disclosed herein, an artificial nucleic acid encoding any modified influenza B HA peptide disclosed herein, or a vector comprising such an artificial nucleic acid. In some embodiments, this document provides a composition comprising one or more mRNA molecules encapsulated in lipid nanoparticles (LNPs), wherein the one or more mRNAs encapsulate any modified influenza B HA peptide disclosed herein. In some embodiments, such a composition is an immunogenic composition.

[0225] In some embodiments, this document also provides an immunogenic composition or vaccine comprising any modified influenza B HA peptide disclosed herein. In some embodiments, this document provides an immunogenic composition or vaccine comprising: a trimeric influenza B HA peptide complex comprising three copies of any modified influenza B HA peptide disclosed herein. In some embodiments, this document provides an immunogenic composition or vaccine comprising: an artificial nucleic acid molecule, or a vector comprising such an artificial nucleic acid molecule encoding any modified influenza B HA peptide disclosed herein. In some embodiments, this document provides an immunogenic composition or vaccine comprising one or more messenger RNA (mRNA) molecules encoding any modified influenza B HA peptide disclosed herein. In some embodiments, one or more mRNA molecules in the immunogenic compositions or vaccines disclosed herein are encapsulated in lipid nanoparticles (LNPs).

[0226] In some embodiments, the immunogenic composition or vaccine may contain other peptides in addition to the modified HAB peptides disclosed herein. In some embodiments, the immunogenic composition or vaccine may contain more than one peptide (e.g., two, three, four, five, six, seven, eight, nine, ten, or more peptides) or mRNA encoding the aforementioned peptides. In some embodiments, the immunogenic composition or vaccine may contain three peptides or mRNA encoding the aforementioned peptides. In some embodiments, the immunogenic composition or vaccine may contain six peptides or mRNA encoding the aforementioned peptides.

[0227] In some embodiments, the immunogenic composition or vaccine comprises a polypeptide derived from two or more (e.g., three, four, five, six, seven, eight, nine, or ten) influenza virus proteins selected from hemagglutinins (e.g., hemagglutinin 1 (HA1) and hemagglutinin 3 (HA3)) and neuraminidase (NA) or mRNA encoding the aforementioned polypeptide. In some embodiments, the immunogenic composition or vaccine comprises one or more (e.g., three, four, five, six, seven, eight, or more) polypeptides derived from HA protein, NA protein, and / or HA and NA proteins or mRNA encoding the aforementioned polypeptide. In some embodiments, the polypeptide is derived from different influenza strains.

[0228] In some embodiments, the immunogenic composition or vaccine comprises one or more polypeptides of influenza A virus, influenza B virus, or influenza C virus, or mRNA encoding the aforementioned polypeptides. In some embodiments, the HA polypeptide of influenza A virus is selected from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18. In some embodiments, the NA polypeptide of influenza A virus is selected from subtypes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11.

[0229] In some embodiments, the immunogenic composition or vaccine comprises (i) one or more HA peptides, (ii) one or more NA peptides, or (iii) two, three, four, five, six, seven, eight, nine or more of a combination of one or more HA peptides and NA peptides, or contains mRNA encoding the aforementioned peptides.

[0230] In some embodiments, the immunogenic composition or vaccine comprises (i) one or more HA peptides, (ii) one or more NA peptides, or (iii) two, three, four, five, six, seven, eight, nine or more of the following subtypes of H1N1, H3N2, H2N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2 and H10N7 and / or combinations of B / Yamagata and B / Victoria lineages, or contains mRNA encoding the aforementioned peptides.

[0231] In some embodiments, the immunogenic composition or vaccine comprises mRNA encoding an influenza H3 HA peptide, mRNA encoding an influenza H1 HA peptide, and mRNA encoding a modified influenza B HA peptide derived from the influenza B / Victoria lineage disclosed herein.

[0232] In some embodiments, the immunogenic composition or vaccine comprises mRNA encoding an influenza H3 HA peptide, an influenza N2 NA peptide, an influenza H1 HA peptide, an influenza N1 NA peptide, an mRNA encoding a modified influenza B HA peptide derived from the influenza B / Victoria lineage disclosed herein, and an mRNA encoding an NA peptide derived from the influenza B / Victoria lineage.

[0233] In some embodiments, the immunogenic composition or vaccine comprises mRNA encoding an influenza H3 HA peptide, mRNA encoding an influenza H1 HA peptide, mRNA encoding an influenza B HA peptide from the influenza B / Victoria lineage, and mRNA encoding an influenza B HA peptide from the influenza B / Yamagata lineage, wherein at least one of the influenza B HA peptide from the influenza B / Victoria lineage and the influenza B HA peptide from the influenza B / Yamagata lineage is a modified influenza B HA peptide according to this disclosure.

[0234] In some embodiments, the immunogenic composition or vaccine comprises mRNA encoding influenza H3 HA peptide, mRNA encoding influenza N2 NA peptide, mRNA encoding influenza H1 HA peptide, mRNA encoding influenza N1 NA peptide, mRNA encoding influenza B HA peptide from the influenza B / Victoria lineage, mRNA encoding NA peptide from the influenza B / Victoria lineage, mRNA encoding influenza B HA peptide from the influenza B / Yamagata lineage, and mRNA encoding NA peptide from the influenza B / Yamagata lineage, wherein at least one of the influenza B HA peptide from the influenza B / Victoria lineage and the influenza B HA peptide from the influenza B / Yamagata lineage is a modified influenza B HA peptide according to this disclosure.

[0235] In some embodiments, the immunogenic composition or vaccine comprises an influenza H3 HA peptide, an influenza H1 HA peptide, and a modified influenza B HA peptide derived from the influenza B / Victoria lineage disclosed herein.

[0236] In some embodiments, the immunogenic composition or vaccine comprises an influenza H3 HA peptide, an influenza N2 NA peptide, an influenza H1 HA peptide, an influenza N1 NA peptide, a modified influenza B HA peptide derived from the influenza B / Victoria lineage disclosed herein, and an NA peptide from the influenza B / Victoria lineage.

[0237] In some embodiments, the immunogenic composition or vaccine comprises an influenza H3 HA peptide, an influenza H1 HA peptide, an influenza B HA peptide from the influenza B / Victoria lineage, and an influenza B HA peptide from the influenza B / Yamagata lineage, wherein at least one of the influenza B HA peptide from the influenza B / Victoria lineage and the influenza B HA peptide from the influenza B / Yamagata lineage is a modified influenza B HA peptide according to this disclosure.

[0238] In some embodiments, the immunogenic composition or vaccine comprises an influenza H3 HA peptide, an influenza N2 NA peptide, an influenza H1 HA peptide, an influenza N1 NA peptide, an influenza B HA peptide from the influenza B / Victoria lineage, an NA peptide from the influenza B / Victoria lineage, an influenza B HA peptide from the influenza B / Yamagata lineage, and an NA peptide from the influenza B / Yamagata lineage, wherein at least one of the influenza B HA peptide from the influenza B / Victoria lineage and the influenza B HA peptide from the influenza B / Yamagata lineage is a modified influenza B HA peptide according to this disclosure.

[0239] Each RNA molecule may be present in the compositions disclosed herein in an amount that effectively induces an immune response in a subject administered the composition. In some embodiments, each RNA molecule may be present in the vaccine or immunogenic compositions disclosed herein in an amount ranging from, for example, from about 0.1 μg to about 150 μg, such as from about 5 μg to about 120 μg, from about 10 μg to about 60 μg, or from about 15 μg to about 45 μg (inclusive of all values ​​and subranges therein). In some embodiments, each RNA molecule may be present in the vaccine or immunogenic compositions in an amount sufficient to encode, for example, from about 5 μg to about 120 μg, such as from about 10 μg to about 60 μg, or from about 15 μg to about 45 μg of a modified influenza B HA peptide.

[0240] The LNP compositions disclosed herein can be provided as cryo-liquid or lyophilized forms. Various cryoprotectants can be used, including, but not limited to, sucrose, trehalose, glucose, mannitol, mannose, dextrose, etc. The cryoprotectant can constitute 5%-30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains trehalose, for example, 5%-30% (e.g., 10%) (w / v). Once formulated with a cryoprotectant, the LNP composition can be frozen (or lyophilized and cryopreserved) at -20°C to -80°C. The LNP composition can be provided to the patient in an aqueous buffer solution: thawed if previously frozen, or reconstituted at the bedside in an aqueous buffer solution if previously lyophilized. The buffer solution is preferably isotonic and suitable for, for example, intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate-buffered saline (PBS).

[0241] In some embodiments, the compositions disclosed herein are immunogenic compositions capable of evoking an immune response against influenza B virus in a subject.

[0242] In some embodiments, the immunogenic compositions or vaccines disclosed herein may further comprise one or more carriers, targeting ligands, stabilizing agents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients to stabilize the modified influenza B HA peptide contained therein, or the mRNA molecule encoding it, and / or LNPs encapsulating such mRNA molecules, or to facilitate the administration of the immunogenic composition or vaccine. Examples of such excipients include, but are not limited to, parabens, thimerosal, thimerosal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., ethylenediaminetetraacetic acid or EDTA). I. Lipid nanoparticles

[0243] The term "lipid nanoparticle" or "LNP" refers to particles having at least one nanometer-scale size (e.g., 1-1,000 nm) comprising one or more lipids, such as cationic lipids and / or non-cationic lipids, and one or more excipients selected from neutral lipids, anionic lipids, zwitterionic lipids, ionizable lipids, steroids, and polymer-conjugated lipids (e.g., PEGylated lipids). Examples of suitable lipids include, but are not limited to, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). LNP compositions encapsulating RNA are known in the art, for example those described in PCT Publications WO 2021 / 237084 and WO 2022 / 099003, the entire contents of which are incorporated herein by reference.

[0244] Any known LNP formulation may be used in the embodiments disclosed herein. In some embodiments, LNP comprises four classes of lipids: (i) ionizable lipids (e.g., cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids; and (iv) accessory lipids. A. Cationic lipids

[0245] Ionizable lipids promote mRNA encapsulation and can be cationic lipids. Cationic lipids provide a positively charged environment at low pH to promote the efficient encapsulation of negatively charged mRNA drug substances. Exemplary cationic lipids are shown in Table 3 below. Table 3. Cationic lipids.

[0246] Cationic lipids may be selected from the group consisting of: [ckkE10] / [OF-02], [(6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butyrate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanoic acid (L319); 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoic acid 9-heptadecyl Ester (SM-102); [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); [3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl](Z)-octadec-9-enoate (DODAP); 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS); [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptane-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl] N-[2-(dimethylamino)ethyl]carbamate (DC-Chol); tetra(8-methylnonyl)3,3′,3′′,3′′′-(((methylazonyl)bis(propane-3,1-diyl))bis(azonyl))tetrapropionate (306Oi10); (2-(dioctylammonyl)ethyl)decyl phosphate (9A1P9); 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidone-1-yl)propyl)-2,5-dihydro-1H-imidazol-2-carboxylic acid ethyl ester (A2-Iso5-2DC18); bis(2-(dodecane) 3,3′-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazatanediyl)azirandiyl)dipropionate (BAME-O16B); 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azirandiyl)bis(dodecyl-2-ol) (C12-200); 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);9,9′,9″,9′′′,9′′′′,9′′′′′-((((benzene-1,3,5-tricarbonyl)tri(azanediyl))tri(propane-3,1-diyl))tri(azanetriyl))hexa(octyl-3-yl)hexyl nonanoate (FTT5); (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetra(ethane-2,1-diyl)(9Z,9′Z,9″Z,9′′′Z,12Z,12′Z,12″Z,12′′′Z)-tetra(octadec-9,12-di) Acrylates (OF-Deg-Lin); TT3; N1,N3,N5-tris(3-(eicosylamino)propyl)phenyl-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylformamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); 8-((2-hydroxyethyl)(8-(nonoxy)-8-oxooctyl)amino)octanoic acid heptadecanyl-9-yl ester (lipid 5); IM-001; and combinations thereof.

[0247] In some embodiments, the cationic lipid is biodegradable. In various embodiments, the cationic lipid is not biodegradable. In some embodiments, the cationic lipid is cleavable. In some embodiments, the cationic lipid is not cleavable.

[0248] Cationic lipids are described in further detail in Dong et al. (PNAS. Proceedings of the National Academy of Sciences 111(11):3955-60. 2014); Fenton et al. (Adv. Mater. Advanced Materials 28:2939. 2016); U.S. Patent No. 9,512,073; and U.S. Patent No. 10,201,618, each of which is incorporated herein by reference. B. Polyethylene glycol-modified lipids

[0249] Polyethylene glycol-modified lipid components provide control over the particle size and stability of nanoparticles. The addition of such components can prevent complex aggregation and provides a means to increase the cycle life of lipid-nucleic acid drug compositions and their delivery to target tissues (Klibanov et al., FEBS Letters [European Federation of Biochemical Societies Letters] 268(1):235-7. 1990). These components can be selected for rapid in vivo exchange from the drug composition (see, for example, US Patent No. 5,885,613).

[0250] The PEGylated lipids under consideration include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length, covalently linked to lipids having one or more alkyl chains of length C6-C20 (e.g., C8, C10, C12, C14, C16, or C18), such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipids are 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (DMG-PEG); 1,2-distearyl-sn-glycerol-3-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycerol-3-phosphatidylethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearyl-rac-glycerol-polyethylene glycol (DSG-PEG); PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159); and combinations thereof.

[0251] In some embodiments, the PEG has a high molecular weight, for example, 2000-2400 g / mol. In some embodiments, the PEG is PEG2000 (or PEG-2K). In some embodiments, the PEGylated lipids described herein are DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(PEG)-2000]-N,N-bistetradecylacetamide). In some embodiments, the PEGylated lipids described herein are DMG-PEG2000. C. Cholesterol-based lipids

[0252] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticles. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example: DC-Choi (N,N-dimethyl-N-ethylformamidocholesterol), 1,4-bis(3-N-oleenylaminopropyl)piperazine (Gao et al., BiochemBiophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Patent 5,744,335), and imidazole cholesterol ester (“ICE”; WO 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-stigmasterol, sitosterol, fucosterol, stigmasterol (stigmasterane-5,22-dien-3-ol), ergosterol; 3β-hydroxy-5,24-cholestadiene; lanosterol (8,24-lanosterdien-3β-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24,25-dihydrolanosterol); yeast sterol (5α-cholest-8,24-dien-3β-ol); cholesterol Lathosterol (5α-cholesterol-7-en-3β-ol); diosgenin ((3β,25R)-spirost-5-en-3-ol); campesterol (campesterol-5-en-3β-ol); campestanol (5α-campestanol-3β-ol); 24-methylene cholesterol (5,24(28)-cholestadien-24-methylen-3β-ol); cholesterol margarate (cholesterol-5-en-3β-yl margarate); cholesterol oleate; cholesterol stearate; and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipids used in the LNP are cholesterol. D. Helper lipids

[0253] The assisting lipid enhances the structural stability of the LNP and demonstrates its escape from endosomes. It improves the uptake and release of the mRNA drug payload. In some embodiments, the assisting lipid is a zwitterionic lipid with fusion-promoting properties for enhancing the uptake and release of the drug payload. Examples of accessory lipids are 1,2-dioleoyl-SN-glycerol-3-phosphatidylethanolamine (DOPE); 1,2-distearyl-sn-glycerol-3-phosphatidylcholine (DSPC); 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS); 1,2-ditransoleoyl-sn-glycerol-3-phosphatidylethanolamine (DEPE); and 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycerol-3-phosphatidylcholine (DLPC), 1,2-distearylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycerol-3-phosphatidylethanolamine (DLPE).

[0254] Other exemplary cofactor lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramides, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof. In some embodiments, the cofactor lipid is DOPE. In some embodiments, the cofactor lipid is DSPC.

[0255] In various embodiments, the LNP of the present invention comprises (i) a cationic lipid selected from OF-02, cKK-E10, 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 ratio of lipid components

[0256] The molar ratios of the components described are important for the effectiveness of LNPs in delivering mRNA. The molar ratio of cationic lipids, PEGylated lipids, cholesterol-based lipids, and cofactor lipids is A : B : C : D, where A + B + C + D = 100%. In some embodiments, the molar ratio of cationic lipids to total lipids (i.e., A) in the LNP 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 to 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 cholesterol-based lipids to 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 auxiliary lipids 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 (polyglycolic acid lipids + cholesterol) component has the same molar amount as the auxiliary lipids. In some embodiments, the molar ratio of cationic lipids to auxiliary lipids contained in the LNP is greater than 1.

[0257] In some embodiments, the LNP disclosed herein includes: i) Cationic lipids with a molar ratio of 35% to 55% or 40% to 50% (e.g., cationic lipids with molar ratios of 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54% or 55%). ii) Polyethylene glycol (PEG) conjugated (polyethylene glycol-modified) lipids in a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., PEG-modified lipids in molar ratios 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) Cholesterol-based lipids with a molar ratio of 20% to 45%, 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., cholesterol-based lipids with molar ratios 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) Associate lipids in molar ratios of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., associate lipids in molar ratios 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%). All molar ratios are relative to the total lipid content of the LNP.

[0258] In some embodiments, the LNP comprises: 40% cationic lipids; 1.5% PEGylated lipids; 28.5% cholesterol-based lipids; and 30% auxiliary lipids.

[0259] In some embodiments, the PEGylated lipid is dimyristic-PEG2000 (DMG-PEG2000).

[0260] In various embodiments, the cholesterol-based lipid is cholesterol.

[0261] In some embodiments, the auxiliary lipid is 1,2-dioleoyl-SN-glycerol-3-phosphatidylethanolamine (DOPE).

[0262] In some embodiments, the LNP comprises: OF-02 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0263] In some embodiments, the LNP comprises: cKK-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0264] In some embodiments, the LNP comprises: GL-HEPES-E3-E10-DS-3-E18-1 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0265] In some embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0266] In some embodiments, the LNP comprises: GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DOPE in a molar ratio of 5% to 35%.

[0267] In some embodiments, the LNP comprises: SM-102 in a molar ratio of 35% to 55%; DMG-PEG2000 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DSPC in a molar ratio of 5% to 35%.

[0268] In some embodiments, the LNP comprises: ALC-0315 in a molar ratio of 35% to 55%; ALC-0159 in a molar ratio of 0.25% to 2.75%; cholesterol in a molar ratio of 20% to 50%; and DSPC in a molar ratio of 5% to 35%.

[0269] In some embodiments, the LNP comprises: 40% OF-02; 1.5% DMG-PEG2000; 28.5% cholesterol; and 30% DOPE.

[0270] In some embodiments, the LNP comprises: 40% cKK-E10 in molar ratio; 1.5% DMG-PEG2000 in molar ratio; 28.5% cholesterol in molar ratio; and 30% DOPE in molar ratio.

[0271] In some embodiments, the LNP comprises: 40% GL-HEPES-E3-E10-DS-3-E18-1 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio.

[0272] In some embodiments, the LNP comprises: 40% GL-HEPES-E3-E12-DS-4-E10 in a molar ratio; 1.5% DMG-PEG2000 in a molar ratio; 28.5% cholesterol in a molar ratio; and 30% DOPE in a molar ratio.

[0273] In some embodiments, the LNP comprises: 40% GL-HEPES-E3-E12-DS-3-E14; 1.5% DMG-PEG2000; 28.5% cholesterol; and 30% DOPE.

[0274] In some embodiments, the LNP comprises: 50% of 9-heptadecyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); 10% of 1,2-distearyl-sn-glycerol-3-phosphorylcholine (DSPC); 38.5% of cholesterol; and 1.5% of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000).

[0275] In some embodiments, the LNP comprises: 46.3% (4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 9.4% 1,2-distearate-sn-glycerol-3-phosphorylcholine (DSPC); 42.7% cholesterol; and 1.6% 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

[0276] In some embodiments, the LNP comprises: 47.4% molar of [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 10% molar of 1,2-distearate-sn-glycerol-3-phosphorylcholine (DSPC); 40.9% molar of cholesterol; and 1.7% molar of 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

[0277] In some embodiments, the LNP comprises: 40% IM-001; 1.5% DMG-PEG2000; 28.5% cholesterol; and 30% DOPE.

[0278] To calculate the actual amount of each lipid to be incorporated into the LNP formulation, the molar amount of the cationic lipid is first determined based on the 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 LNP. Next, the molar amount of each other lipid is calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid.

[0279] Therefore, in some embodiments, this document provides a composition comprising artificial messenger RNA (mRNA) encoding any influenza B HA polypeptide disclosed herein encapsulated in an LNP, wherein the LNP comprises a cationic lipid. In some embodiments, the cationic lipid comprises OF-02. In some embodiments, the cationic lipid comprises cKK-E10. In some embodiments, the cationic lipid comprises GL-HEPES-E3-E10-DS-3-E18-1. In some embodiments, the cationic lipid comprises GL-HEPES-E3-E12-DS-4-E10. In some embodiments, the cationic lipid comprises GL-HEPES-E3-E12-DS-3-E14. In some embodiments, the cationic lipid comprises (4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315). In some embodiments, the cationic lipid comprises IM-001.

[0280] In some embodiments, the LNP encapsulating the artificial mRNA disclosed herein further comprises PEGylated lipids, cholesterol-based lipids, and cofactor lipids. In some embodiments, the PEGylated lipids comprise DMG-PEG2000. In some embodiments, the cholesterol-based lipids comprise cholesterol. In some embodiments, the cofactor lipids comprise DOPE. In some embodiments, the LNP comprises a molar ratio of about 35% to about 55% cationic lipids, a molar ratio of about 0.25% to about 2.75% PEGylated lipids, a molar ratio of about 20% to about 45% cholesterol-based lipids, and a molar ratio of about 5% to about 35% cofactor lipids, wherein all molar ratios are relative to the total lipid content of the LNP. In some embodiments, the LNP comprises a molar ratio of about 40% cationic lipids, a molar ratio of about 1.5% PEGylated lipids, a molar ratio of about 28.5% cholesterol-based lipids, and a molar ratio of about 30% cofactor lipids, wherein all molar ratios are relative to the total lipid content of the LNP.

[0281] In some embodiments, this document provides a composition comprising artificial messenger RNA (mRNA) encoding an influenza B HA peptide SEQ ID NO:3 encapsulated in an LNP, wherein the LNP comprises about 40% GL-HEPES-E3-E12-DS-4-E10, about 1.5% DMG-PEG2000, about 28.5% cholesterol, and about 30% DOPE in a molar ratio. In some embodiments, this document provides a composition comprising artificial messenger RNA (mRNA) encoding an influenza B HA peptide SEQ ID NO: 5 encapsulated in an LNP, wherein the LNP comprises about 40% GL-HEPES-E3-E12-DS-4-E10, about 1.5% DMG-PEG2000, about 28.5% cholesterol, and about 30% DOPE in a molar ratio. In some embodiments, this document provides a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 4 encapsulated in an LNP, wherein the LNP comprises approximately 40% GL-HEPES-E3-E12-DS-4-E10, approximately 1.5% DMG-PEG2000, approximately 28.5% cholesterol, and approximately 30% DOPE in a molar ratio. In some embodiments, this document provides a composition comprising an artificial messenger RNA (mRNA) comprising the nucleic acid sequence of SEQ ID NO: 6 encapsulated in an LNP, wherein the LNP comprises approximately 40% GL-HEPES-E3-E12-DS-4-E10, approximately 1.5% DMG-PEG2000, approximately 28.5% cholesterol, and approximately 30% DOPE in a molar ratio. II. Methods for manufacturing LNP vaccines

[0282] LNPs can be prepared using a variety of techniques known in the art. For example, multilayer vesicles (MLVs) can be prepared using conventional techniques, such as by depositing selected lipids onto the inner wall of a suitable container or vessel (by dissolving the lipids in a suitable solvent and then evaporating the solvent to leave a thin film inside the vessel) or by spray drying. An aqueous phase can then be added to the vessel with vortexing, which allows the MLV to form. Unilayer vesicles (ULVs) can then be formed by homogenizing, sonicating, or extruding the multilayer vesicles. Alternatively, ULVs can be formed using detergent removal techniques.

[0283] Various methods are described in patent applications US 2011 / 0244026, US 2016 / 0038432, US 2018 / 0153822, US 2018 / 0125989, and US 2021 / 0046192, and can be used to manufacture LNP vaccines. One exemplary method requires encapsulating mRNA by mixing it with a mixture of lipids without first pre-forming the lipids into lipid nanoparticles, as described in patent application US 2016 / 0038432. Another exemplary method requires encapsulating mRNA by mixing pre-formed LNPs with mRNA, as described in patent application US 2018 / 0153822.

[0284] In some embodiments, a method for preparing LNPs loaded with mRNA includes heating one or more solutions to a temperature above ambient temperature, wherein the one or more solutions are solutions containing pre-formed lipid nanoparticles, solutions containing mRNA, and mixed solutions containing mRNA encapsulated with LNPs. In some embodiments, the method includes heating one or both of the mRNA solution and the pre-formed LNP solution prior to a mixing step. In some embodiments, the method includes heating one or more of the solution containing the pre-formed LNPs, the solution containing mRNA, and the solution containing mRNA encapsulated with LNPs during a mixing step. In some embodiments, the method includes heating the LNP-encapsulated mRNA after a mixing step. In some embodiments, the one or more solutions are heated to temperatures of or above the following: 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 therein. In some embodiments, one or more solutions are heated to the following temperature ranges: about 25°C-70°C, about 30°C-70°C, about 35°C-70°C, about 40°C-70°C, about 45°C-70°C, about 50°C-70°C, or about 60°C-70°C, including all values ​​and sub-ranges therebetween. In some embodiments, the temperature is about 65°C.

[0285] Various methods can be used to prepare mRNA solutions suitable for the present invention. In some embodiments, mRNA can be directly dissolved in the buffer solution described herein. In some embodiments, the mRNA solution can be generated by mixing the mRNA stock solution with the buffer solution prior to mixing with the lipid solution for encapsulation. In some embodiments, the mRNA solution can be generated by mixing the mRNA stock solution with the buffer solution immediately prior to mixing with the lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution may contain mRNA in water or a buffer solution at a concentration of 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.

[0286] In some embodiments, a pump is used to mix the mRNA stock solution with the buffer solution. 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 of at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x that of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100–6000 ml / min (e.g., about 100–300 ml / min, 300–600 ml / min, 600–1200 ml / min, 1200–2400 ml / min, 2400–3600 ml / min, 3600–4800 ml / min, 4800–6000 ml / min, or 60–420 ml / min, including all values ​​and subranges therein). In some embodiments, the buffer solution is mixed at or greater than the following flow rates: approximately 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min, including all values ​​and subranges therein.

[0287] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10 to 600 ml / min (e.g., about 5 to 50 ml / min, about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min, including all values ​​and subranges therein). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min, including all values ​​and subranges therebetween.

[0288] The process of incorporating desired mRNA into lipid nanoparticles is referred to as “addition”. An exemplary method is described in Lasic et al., FEBS Lett. [FEBS Communications] (1992) 312:255-8. The nucleic acid incorporated into the LNP can be entirely or partially located within the internal space of the lipid nanoparticle, within the bilayer of the lipid nanoparticle membrane, or associated with the outer surface of the lipid nanoparticle membrane. Incorporating mRNA into lipid nanoparticles is also referred to herein as “encapsulation,” in which the nucleic acid is completely or substantially contained within the internal space of the lipid nanoparticle.

[0289] Suitable LNPs can be prepared in various sizes. In some embodiments, reduced lipid nanoparticle size is associated with more efficient mRNA delivery. The selection of an appropriate LNP size can take into account the target cell or tissue site and, to some extent, the application for which the lipid nanoparticles will be used.

[0290] Several methods known in the art can be used to regulate the size of lipid nanoparticle clusters. The preferred method described herein utilizes the Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of an LNP sample is mixed with 990 μl of 10% trehalose. This solution is placed in a cuvette and then placed in the Zetasizer machine. The z-mean diameter (nm), or cumulative mean, is considered to be the average size of the LNPs in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) using dynamic light scattering (DLS) and cumulative analysis of autocorrelation functions. The average LNP diameter can be reduced by sonicating the formed LNPs. Intermittent sonication cycles can be alternated with quasi-elastic light scattering (QELS) assessments to guide efficient lipid nanoparticle synthesis.

[0291] In some embodiments, the majority of purified LNPs (i.e., LNPs greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, including all values ​​and subranges therebetween) are about 70-150 nm in size (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm, including all values ​​and subranges therebetween). In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles are about 70-150 nm in size (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 therein).

[0292] In some embodiments, the average diameter of the LNP is 30-200 nm. In various embodiments, the average diameter of the LNP is 80-150 nm.

[0293] In some embodiments, the average size of the LNP in the composition of the present invention is 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.

[0294] In some embodiments, the size of the LNP in the compositions of the present invention, which is greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (inclusive of all values ​​and subranges therein), ranges from about 40 nm to 90 nm (e.g., about 45 nm to 85 nm, about 50 nm to 80 nm, about 55 nm to 75 nm, about 60 nm to 70 nm, inclusive of all values ​​and subranges therein) or about 50 nm to 70 nm (e.g., 55 nm to 65 nm), is particularly suitable for pulmonary delivery via nebulization.

[0295] In some embodiments, the measure of dispersibility or molecular size heterogeneity (PDI) of LNPs in the pharmaceutical compositions provided by the present invention is less than about 0.5. In some embodiments, the PDI of LNPs is 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 therein. The PDI can be measured using a Zetasizer machine as described above.

[0296] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (inclusive of all values ​​and subranges therein) of purified LNPs in the pharmaceutical compositions provided herein encapsulate the mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical compositions encapsulate the mRNA within each individual particle. In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 50% to 99%; or greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or 99%, inclusive of all values ​​and subranges therein. Typically, the encapsulation efficiency of the lipid nanoparticles used herein is at least 90% (e.g., at least 91%, 92%, 93%, 94%, or 95%, inclusive of all values ​​and subranges therein).

[0297] In some embodiments, the N / P ratio of the LNP is between 1 and 10. In some embodiments, the N / P ratio of the lipid nanoparticles is greater than 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 another embodiment, the typical N / P ratio of the LNP described herein is 4.

[0298] In some embodiments, the pharmaceutical composition according to the invention contains at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg (inclusive of all values ​​and subranges therein) of encapsulated mRNA. In some embodiments, the pharmaceutical composition contains about 0.1 μg to 1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, or at least about 1000 μg (inclusive of all values ​​and subranges therein) of encapsulated mRNA.

[0299] In some embodiments, mRNA can be prepared by chemical synthesis or by in vitro transcription (IVT) using a DNA template. For example, in IVT, a cDNA template is used to generate mRNA transcripts, and the DNA template is degraded by a DNase. The transcripts are purified by deep filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding caps and tails, and the modified RNA is purified again by deep filtration and TFF.

[0300] The mRNA is then prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of the LNP. The amphiphilic solution for dissolving the four lipid components of the LNP can be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, a citrate, phosphate, acetate, or succinate buffer and can 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 salts (e.g., sodium, potassium, and / or calcium salts). In a particular embodiment, the aqueous buffer has 1 mM citrate, 150 mM NaCl, and a pH of 4.5.

[0301] An exemplary non-limiting method for preparing mRNA-LNP compositions involves mixing a buffered mRNA solution with an ethanol solution of lipids in a controlled, homogeneous manner, wherein the lipid:mRNA ratio is maintained throughout the mixing process. In this illustrative example, the mRNA is presented in an aqueous buffer containing citrate monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to a solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., cationic lipids, PEGylated lipids, cholesterol-based lipids, and accessory lipids) is dissolved in ethanol. The aqueous mRNA solution and the ethanol lipid solution are mixed at a 4:1 volume ratio in a T-shaped mixer with a near-pulseless pump system. The resulting mixture is then subjected to downstream purification and buffer exchange. Buffer exchange can be performed using a dialysis cassette or a TFF system. A TFF can be used to concentrate and buffer exchange the resulting nascent LNPs immediately after formation via the T-mixing process. The percolation process is a continuous operation, and the volume is kept constant by adding an appropriate buffer at the same rate as the percolation stream. adjuvant

[0302] In some embodiments, the immunogenic compositions or vaccines disclosed herein contain an adjuvant. In other embodiments, the immunogenic compositions or vaccines disclosed herein do not contain an adjuvant. Similarly, in some embodiments, the immunogenic compositions or vaccines disclosed herein may be administered together with an adjuvant to enhance the immune response. In other embodiments, the immunogenic compositions or vaccines may be administered without an adjuvant. As used herein, the term "adjuvant" refers to a substance or combination of substances that can be used to enhance an immune response to an antigenic component of a vaccine or immunogenic composition. Adjuvants may include suspensions of minerals (alum, aluminum salts, including, for example, aluminum hydroxide / aluminum hydroxide (AlOOH), aluminum phosphate (AlPO4), aluminum hydroxyphosphate sulfate (AAHS), and / or potassium aluminum sulfate) on which antigens are adsorbed; or water-in-oil emulsions in which the antigen solution is emulsified in mineral oil (e.g., Freund's incomplete adjuvant), sometimes including killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., those including CpG motifs) can also be used as adjuvants (e.g., 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 biomolecules such as lipids and co-stimulatory 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-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, and 41 BBL.

[0303] In some embodiments, the adjuvant is a squalene-based adjuvant comprising an oil-in-water adjuvant emulsion containing at least the following: squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant, and a hydrophobic nonionic surfactant. In some embodiments, the emulsion is thermally reversible, optionally wherein approximately 90% of the population, by volume of oil droplets, is smaller than approximately 200 nm.

[0304] In some embodiments, the polyoxyethylene alkyl ether has the formula CH3-(CH2). x -(O-CH2-CH2) n -OH, where n is an integer from 10 to 60, and x is an integer from 11 to 17. In some embodiments, the polyoxyethylene alkyl ether surfactant is polyoxyethylene (12) hexadecyl ether.

[0305] In some embodiments, approximately 90% of the population size, based on oil droplet volume, is less than approximately 160 nm. In some embodiments, approximately 90% of the population size, based on oil droplet volume, is less than approximately 150 nm. In some embodiments, approximately 50% of the population size, based on oil droplet volume, is less than approximately 100 nm. In some embodiments, approximately 50% of the population size, based on oil droplet volume, is less than approximately 90 nm.

[0306] In some embodiments, the adjuvant further comprises at least one sugar alcohol, including but not limited to glycerol, erythritol, xylitol, sorbitol and mannitol.

[0307] In some embodiments, the hydrophilic nonionic surfactant has a hydrophilic / lipophilic balance (HLB) value greater than or equal to about 10. In some embodiments, the hydrophobic nonionic surfactant has an HLB value less than about 9. In some embodiments, the hydrophilic nonionic surfactant has an HLB value greater than or equal to about 10, and the hydrophobic nonionic surfactant has an HLB value less than about 9.

[0308] In some embodiments, the hydrophobic nonionic surfactant is a sorbitol ester (such as sorbitol monooleate) or a mannitol diester surfactant. In some embodiments, the amount of squalene is between about 5% and about 45%. In some embodiments, the amount of polyoxyethylene alkyl ether surfactant is between about 0.9% and about 9%. In some embodiments, the amount of hydrophobic nonionic surfactant is between about 0.7% and about 7%. In some embodiments, the adjuvant comprises: i) about 32.5% squalene, ii) about 6.18% polyoxyethylene (12) cetearyl ether, iii) about 4.82% sorbitol monooleate, and iv) about 6% mannitol.

[0309] In some embodiments, the adjuvant further comprises alkyl polysaccharide glycosides and / or cryoprotectants, such as sugars, particularly dodecyl maltodextrin and / or sucrose.

[0310] In some embodiments, the adjuvant comprises AF03, as described in Klucker et al., J. Pharm. Sci. [Journal of Pharmaceutical Sciences], 2012, 101(12):4490-4500 (incorporated herein by reference in its entirety). In some embodiments, the adjuvant comprises a liposome-based adjuvant, such as SPA14. SPA14 is a liposome-based adjuvant (AS01-like) comprising a Toll-like receptor 4 (TLR4) agonist (E6020) and a saponin (QS21).

[0311] In some embodiments, the vaccine or immunogenic composition does not contain an adjuvant. In some embodiments, one or more mRNA molecules encapsulated in an LNP may be used to adjuvant one or more modified influenza B HA peptides in the vaccine or immunogenic composition. See, for example, Shirai et al., Vaccines, 2020, 8(433):1-18. In other embodiments, the vaccine or immunogenic composition further contains an adjuvant. application

[0312] The immunogenic compositions or vaccines disclosed herein can be formulated for administration in any manner known in the field of drug delivery, such as oral, parenteral, intravenous, intramuscular, subcutaneous, intradermal, transdermal, intrathecal, submucosal, sublingual, rectal, vaginal, etc. In some embodiments, the immunogenic compositions or vaccines disclosed herein are formulated for sublingual, intramuscular, intradermal, subcutaneous, intravenous, intranasal, inhalation, or intraperitoneal administration.

[0313] In some embodiments, the immunogenic compositions or vaccines disclosed herein are formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular administration. In some embodiments, the immunogenic compositions or vaccines disclosed herein are formulated for sublingual administration. In some embodiments, the immunogenic compositions or vaccines are formulated for intramuscular administration. The immunogenic compositions or vaccines disclosed herein may also be formulated for intranasal or inhalation administration. The immunogenic compositions or vaccines disclosed herein may also be formulated for any other intended route of administration.

[0314] In some embodiments, the immunogenic compositions or vaccines disclosed herein are formulated for intradermal injection, intranasal administration, or intramuscular injection. General considerations for the formulation and manufacture of agents administered via these routes can be found, for example, Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference. Currently, oral or nasal spray or aerosol routes (e.g., by inhalation) are most commonly used for the direct delivery of therapeutic agents to the lungs and respiratory system. In some embodiments, the immunogenic compositions or vaccines disclosed herein are administered using a device that delivers a dose of the vaccine or immunogenic composition. Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include short needle devices, such as those described in the following patents: U.S. Patent Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, and 5,417,662, all of which are incorporated herein by reference. Intradermal compositions can also be administered by devices that limit the effective penetration length of the needle into the skin, such as those described in WO 1999 / 34850 (incorporated herein by reference) and their functional equivalents.

[0315] In addition, jet injection devices are also suitable, which deliver liquid vaccines or immunogenic compositions to the dermis via a liquid jet syringe or a needle that pierces the stratum corneum and generates a jet that reaches the dermis. Jet injection devices are described in, for example, U.S. Patent Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335, 5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941,880, and 4,940,460. 1997 / 37705 and WO 1997 / 13537, all of which are incorporated herein by reference. Additionally, a standard syringe can be used in the classic Mantoux method for intradermal administration.

[0316] Preparations intended for parenteral administration typically include sterile aqueous or non-aqueous 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 buffer media. Parenteral media include sodium chloride solutions, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextran), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0317] The immunogenic compositions or vaccines disclosed herein may be packaged in containers, such as pre-filled syringes, vials, or autoinjectors. In some embodiments, the immunogenic compositions or vaccines disclosed herein are packaged in pre-filled syringes. In some embodiments, the immunogenic compositions or vaccines disclosed herein are packaged in vials. In some embodiments, the immunogenic compositions or vaccines disclosed herein are packaged in autoinjectors. In other embodiments, the immunogenic compositions or vaccines disclosed herein are packaging boxes for patient-friendly autoinjector and infusion pump devices.

[0318] Prefilled syringes offer several advantages over other types of packaging, such as convenience, affordability, accuracy, sterility, and safety. Therefore, in some embodiments, this document provides a prefilled syringe containing about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 mL of any immunogenic composition or vaccine disclosed herein. How to use

[0319] This document also provides methods for administering the immunogenic compositions or vaccines described herein to subjects. These methods can be used to vaccinate subjects to prevent them from contracting influenza B virus, reduce the likelihood of them contracting influenza B virus, or reduce the likelihood of them developing severe illness due to influenza B virus infection. Similarly, this disclosure provides any vaccine or immunogenic composition described herein for administering an anti-influenza B virus vaccine to subjects. The use of any immunogenic composition described herein in the manufacture of a vaccine for administering an anti-influenza B virus vaccine to subjects is also disclosed. In some embodiments, the method or use of vaccination includes administering an immunologically effective amount of any immunogenic composition or vaccine described herein to a subject in need.

[0320] As used herein, the terms "immunely effective amount" or "therapeuticly effective amount" refer to an amount sufficient to immunize a subject. In some embodiments, the immunely effective amount or therapeutically effective amount is capable of inducing protective immunity against an infectious disease, including but not limited to an increase in antibody titers and / or T-cell immunity against the infectious disease. In some embodiments, the immunely effective amount or therapeutically effective amount of the vaccine or immunogenic composition disclosed herein increases the protective immunity of 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.

[0321] Therefore, in some embodiments, this disclosure provides a method for immunizing a subject, the method comprising administering to a subject in need any of the vaccines or immunogenic compositions described herein. In some embodiments, this disclosure provides a method for immunizing a subject, the method comprising administering to a subject in need an immunogenically effective amount of any of the vaccines or immunogenic compositions described herein. As used herein, “immunize” means inducing a protective immune response in a subject against influenza B virus infection. Similarly, this disclosure provides any of the vaccines or immunogenic compositions described herein for immunizing a subject against influenza B virus infection. Use of any of the immunogenic compositions described herein is also disclosed for manufacturing a vaccine for immunizing a subject against influenza B virus infection.

[0322] In some embodiments, the method or use prevents a subject from contracting influenza B virus infection or illness caused by influenza B virus infection. In some embodiments, the method or use reduces the likelihood of a subject contracting influenza B 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. In some embodiments, the method or use reduces the likelihood of a subject developing severe illness due to influenza B virus infection by approximately 0.1%, approximately 0.5%, approximately 1%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99%, approximately 100%, including all values ​​and subranges therebetween, compared to a subject who has not received the vaccine or immunogenic composition disclosed herein. In some embodiments, the method or use induces a protective immune response in the subject. In some embodiments, the protective immune response is an antibody response.

[0323] In some embodiments, a method for alleviating one or more symptoms of influenza B virus infection is also provided, the method comprising administering to a subject in need any of the vaccines or immunogenic compositions described herein. In some embodiments, a method for alleviating one or more symptoms of influenza B virus infection is provided herein, the method comprising administering to a subject in need a preventatively effective amount of any of the vaccines or immunogenic compositions described herein.

[0324] This disclosure provides any vaccine or immunogenic composition described herein for the purpose of alleviating one or more symptoms of influenza B virus infection. It also discloses any immunogenic composition described herein for the manufacture of a vaccine for the purpose of alleviating one or more symptoms of influenza B virus infection in a subject.

[0325] In some embodiments, the methods or uses disclosed herein reduce one or more symptoms of influenza B 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 therein, compared to subjects who have not received the vaccine or immunogenic composition disclosed herein.

[0326] In some embodiments, the vaccine or immunogenic composition, along with optional adjuvants, may be administered before or after the onset of one or more symptoms of influenza B virus infection. That is, in some embodiments, the vaccine or immunogenic composition described herein may be administered prophylactically to prevent influenza B virus infection or to improve symptoms of potential influenza B virus infection.

[0327] In some embodiments, a subject is at risk of infection if they will come into contact with other individuals or animals known or suspected of being infected with influenza B virus and / or if they will be in a place where influenza B virus infection is known or believed to be prevalent or endemic. In some embodiments, a vaccine or immunogenic composition is administered to a subject who has influenza B virus infection or who exhibits one or more symptoms commonly associated with influenza B virus infection. In some embodiments, a subject is known or believed to have been exposed to influenza B virus infection.

[0328] The vaccine or immunogenic composition according to this disclosure may be administered in any amount or dose suitable for achieving the desired outcome. In some embodiments, the desired outcome is to induce a durable adaptive immune response against influenza B virus. In some embodiments, the desired outcome is to reduce the intensity, severity, and / or frequency of one or more symptoms associated with influenza B virus infection, and / or to delay their onset. In some embodiments, the desired outcome is to provide a vaccine or immunogenic composition with consistent RNA quality. The required dose may vary depending on the subject, including the subject's species, age, weight, and general condition, the severity of the infection being treated, the specific composition used, and its administration method.

[0329] In some embodiments, the vaccine or immunogenic composition described herein is administered to subjects, wherein these subjects can be any member of the animal kingdom. In some embodiments, the subjects are non-human animals. In some embodiments, non-human subjects are poultry (e.g., chickens or birds), reptiles, amphibians, fish, insects, and / or worms. In some embodiments, non-human subjects are mammals (e.g., ferrets, rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs).

[0330] In some embodiments, the vaccine or immunogenic composition described herein is administered to a human subject. In some embodiments, the human subject is 6 months or older, 6 months to 35 months, at least 2 years old, at least 3 years old, 36 months to 8 years old, 9 years or older, at least 6 months and less than 5 years old, at least 6 months and less than 18 years old, or at least 3 years and less than 18 years old. In some embodiments, the human subject is an infant (less than 36 months old). In some embodiments, the human subject is a child or adolescent (less than 18 years old). In some embodiments, the human subject is a child at least 6 months old and less than 5 years old. In some embodiments, the human subject is at least 5 years old and less than 60 years old. In some embodiments, the human subject is at least 5 years old and less than 65 years old. In some embodiments, the human subject is an older adult (at least 60 years old or at least 65 years old). In some embodiments, the human subject is a non-older adult (at least 18 years old and less than 65 years old or at least 18 years old and less than 60 years old).

[0331] The methods and uses of the vaccine or immunogenic composition described herein include administering a single dose (i.e., without a booster dose) to a subject. In some embodiments, the methods and uses of the vaccine or immunogenic composition described herein include a primitivism-booster vaccination strategy. Primitivism-booster vaccination includes administering a primitivism vaccine or immunogenic composition, followed by administering a booster vaccine or immunogenic composition to the subject after a period of time. An immune response is “initiated” after administration of the primitivism vaccine or immunogenic composition and “boosted” after administration of the booster vaccine or immunogenic composition. The primitivism vaccine or immunogenic composition may include the vaccine or immunogenic composition described herein and optional adjuvants. Similarly, the booster vaccine or immunogenic composition may include the vaccine or immunogenic composition described herein and optional adjuvants. The primitivism vaccine or immunogenic composition may, but does not necessarily have to, be the same as the booster vaccine or immunogenic composition. Administration of the booster vaccine or immunogenic composition typically occurs several weeks or months after administration of the primitivism 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 some embodiments, the recipients of the primary-boost vaccination are unvaccinated subjects, typically unvaccinated infants or children.

[0332] 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 may be administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally. Other applications

[0333] The modified influenza B HA peptides disclosed herein can have other applications, such as in in vitro methods for preparing trimeric influenza B HA peptide complexes. Therefore, this document provides an in vitro method for preparing trimeric influenza B HA peptide complexes, comprising expressing an artificial nucleic acid molecule encoding any of the modified influenza B HA peptides disclosed herein in host cells to prepare the trimeric influenza B HA peptide complex. In some embodiments, the artificial nucleic acid molecule encoding the modified influenza B HA peptide is part of a vector. In other embodiments, the expression of the modified influenza B HA peptide is achieved by culturing host cells in a cell culture medium. Therefore, in some embodiments, this document provides an in vitro method for preparing trimeric influenza B HA peptide complexes, comprising culturing host cells in a cell culture medium and expressing the trimeric influenza B HA peptide complex. In some embodiments, the in vitro method disclosed herein further includes the step of purifying the trimeric influenza B HA peptide complex from the cell culture medium.

[0334] In some embodiments, the trimeric influenza B HA peptide complex prepared according to the in vitro methods disclosed herein exhibits higher pre-fusion conformational stability compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications. In some embodiments, the pre-fusion conformational stability is measured by the increased binding of the trimeric influenza B HA peptide complex to a stem region-specific antibody (e.g., CR9114) compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications. In some embodiments, the pre-fusion conformational stability is measured by the increased binding ratio of the stem region-specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications. In some embodiments, the stability of the pre-fusion conformation is measured by the increased binding of the trimeric influenza B HA peptide complex to a stem region-specific antibody (e.g., CR9114) and the increased binding ratio of the stem region-specific antibody (e.g., CR9114) to an RBS-specific antibody (e.g., R95-1D05) compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications.

[0335] In some embodiments, the trimeric influenza B HA peptide complex prepared according to the in vitro methods disclosed herein exhibits stronger immunogenicity compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications. Immunogenicity can be measured using any method known in the art. For example, in some embodiments, a hemagglutination inhibition assay (HAI) is used to measure immunogenicity. The first set of representative embodiments disclosed herein

[0336] Example 1. A modified influenza B hemagglutinin (HA) polypeptide comprising a head region and a stem region, wherein the modified influenza B HA polypeptide comprises one or more modifications selected from the following: a) At least one proline mutation in the stem region, wherein the at least one proline mutation disrupts at least one helical structure in the stem region of the modified influenza B HA polypeptide in the fusion conformation. b) At least two cysteine ​​mutations, wherein the at least two cysteine ​​mutations form disulfide bridges in the modified influenza B HA peptide; c) One or more amino acid mutations in the head region and / or stem region, wherein the one or more amino acid mutations stabilize the modified influenza B HA peptide in the pre-fusion conformation through interface stabilization; d) One or more amino acid mutations in the head region and / or stem region, wherein the one or more amino acid mutations inactivate one or more pH sensors in the head region and / or stem region; e) At least one amino acid mutation in the head region, wherein the at least one amino acid mutation introduces at least one N-linked glycosylation motif in the head region; f) At least one amino acid mutation in the stem region, wherein the at least one amino acid mutation introduces or disrupts an N-linked glycosylation motif in the stem region; and g) At least one amino acid mutation in the head region, wherein the at least one amino acid mutation results in reduced sialic acid binding of the modified influenza B HA peptide compared to a control influenza B HA peptide which lacks at least one amino acid mutation that reduces sialic acid binding of the modified influenza B HA peptide.

[0337] Example 2. A modified influenza B HA polypeptide as described in Example 1, comprising at least one of two modifications listed in a) to g).

[0338] Example 3. A modified influenza B HA polypeptide as described in Example 1 or 2, comprising at least one of three modifications listed in a) to g).

[0339] Example 4. A modified influenza B HA polypeptide as described in any one of Examples 1-3, comprising at least one of four modifications listed in a) to g).

[0340] Example 5. A modified influenza B HA polypeptide as described in any one of Examples 1-4, comprising at least one of the five modifications listed in a) to g).

[0341] Example 6. A modified influenza B HA polypeptide as described in any one of Examples 1-5, comprising at least one of the six modifications listed in a) to g).

[0342] Example 7. A modified influenza B HA polypeptide as described in any one of Examples 1-6, comprising at least one of each of the modifications listed in a) to g).

[0343] Example 8. A modified influenza B HA polypeptide as described in any one of Examples 1-7, wherein the one or more modifications stabilize the modified influenza B HA polypeptide in its pre-fusion conformation.

[0344] Example 9. The modified influenza B HA peptide as described in Example 8, wherein the stabilization of the pre-fusion conformation is measured by the increased binding of the modified influenza B HA peptide to stem region-specific antibodies compared to the wild-type influenza B HA peptide and / or by the increased binding ratio of the stem region-specific antibodies to RBS-specific antibodies compared to the wild-type influenza B HA peptide.

[0345] Example 10. A modified influenza B HA polypeptide as described in any one of Examples 1-9, wherein the head region is the segment of the modified influenza B HA polypeptide at approximately amino acid positions 57-307, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0346] Example 11. A modified influenza B HA polypeptide as described in any one of Examples 1-10, wherein the stem region is the segment of the modified influenza B HA polypeptide at approximately amino acid positions 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0347] Example 12. A modified influenza B HA polypeptide as described in any one of Examples 1-11, wherein the at least one proline mutation is located at amino acid positions 363, 366, 371, 372, 376, 380, 383, 390, 391, 393, 395, 397, 399, 421, 430, 431, 434, 436 and / or 490, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0348] Example 13. The modified influenza B HA polypeptide as described in Example 12, wherein the at least one proline mutation is located at amino acid positions 372, 397, 399, 421, 430, 431, 434 and / or 436.

[0349] Example 14. A modified influenza B HA polypeptide as described in any one of Examples 1-13, wherein the at least two cysteine ​​mutations are located at amino acid positions 20 and 387, 35 and 408, 36 and 415, 37 and 411, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 232 and 433, 2 33 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 380 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 396 and 514, 401 and 475, 430 and 437, 430 and 438, and / or 430 and 439, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0350] Example 15. A modified influenza B HA polypeptide as described in Example 14, wherein the at least two cysteine ​​mutations are located at amino acid positions 20 and 387, 36 and 415, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 233 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 401 and 475, 430 and 437, 430 and 438, and / or 430 and 439.

[0351] Example 16. A modified influenza B HA polypeptide as described in any one of Examples 1-15, wherein the modified influenza B HA polypeptide is stabilized by interface stabilization in a pre-fusion conformation. The one or more amino acid mutations include at least one cavity-filling mutation in the stem region and / or one or more amino acid mutations in the head region and / or stem region to form a polar interaction with adjacent amino acid residues.

[0352] Example 17. A modified influenza B HA polypeptide as described in Example 16, wherein the at least one cavity-filling mutation is located at amino acid positions 460, 467 and / or 474, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0353] Example 18. A modified influenza B HA polypeptide as described in Example 17, wherein the at least one cavity-filling mutation is a substitution of lysine (K) at amino acid position 460, a substitution of phenylalanine (F) or tyrosine (Y) at amino acid position 467, and / or a substitution of glutamine (Q) at amino acid position 474.

[0354] Example 19. A modified influenza B HA polypeptide as described in Example 16, wherein the polar interaction includes a salt bridge or a hydrogen bond.

[0355] Example 20. A modified influenza B HA polypeptide as described in any one of Examples 1-19, wherein the modified influenza B HA polypeptide is stabilized by interface stabilization in the pre-fusion conformation where the one or more amino acid mutations are located at amino acid positions 18, 121, 188, 226, 228, 408, 435 and / or 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0356] Example 21. A modified influenza B HA polypeptide as described in any one of Examples 1-20, wherein the one or more amino acid mutations that inactivate one or more pH sensors in the head region and / or stem region are located at amino acid positions 226, 228, 237, 239, 383, 388, 391, 401, 405, 408, 435, 460, 474 and / or 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0357] Example 22. The modified influenza B HA peptide as described in Example 21, wherein the one or more amino acid mutations that inactivate one or more pH sensors in the head region and / or stem region are located at amino acid positions 226, 228, 237, 239, 383, 388, 401, 405, 408, 435, 460 and / or 475.

[0358] Example 23. A modified influenza B HA polypeptide as described in any one of Examples 1-22, wherein the head region contains a receptor binding site (RBS), and wherein the at least one N-linked glycosylation motif introduced in the head region is located in or adjacent to the RBS of the head region.

[0359] Example 24. The modified influenza B HA polypeptide as described in Example 23, wherein the RBS is a region of the modified influenza B HA polypeptide consisting of residues at amino acid positions 110, 151-157, 165, 173-175, 208-218, 248, and 254-259, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0360] Example 25. A modified influenza B HA polypeptide as described in any one of Examples 23 or 24, wherein the at least one N-linked glycosylation motif introduced in the head region is generated by introducing at least one substitution at amino acid positions 60, 62, 141, 143, 186, 187, 214, 216, 223 and / or 224, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0361] Example 26. A modified influenza B HA polypeptide as described in any one of Examples 1-25, wherein the at least one N-linked glycosylation motif introduced or disrupted in the stem region is generated by introducing at least one substitution at amino acid positions 28, 336 and / or 349, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0362] Example 27. A modified influenza B HA polypeptide as described in any one of Examples 1-26, wherein the N-linked glycosylation motif contains a concordant sequence of NxS / Ty, wherein x and y are not proline (P).

[0363] Example 28. A modified influenza B HA polypeptide as described in any one of Examples 1-27, wherein the at least one amino acid mutation that reduces sialic acid binding of the modified influenza B HA polypeptide is located at amino acid positions 157, 177, 218 and / or 257, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0364] Example 29. A modified influenza B HA polypeptide comprising amino acid substitutions at the following positions: a) Amino acid positions 383, 475, and 460; b) Amino acid positions 226, 237, 383, 460, and 475; or c) Amino acid positions 383, 401, 405, 408, and 475 For example, indexing can be done by referring to the amino acid sequence of SEQ ID NO: 1.

[0365] Example 30. The modified influenza B HA polypeptide as described in Example 29, comprising the following amino acid substitutions: a) H383L, H475L and A460K; b) K226M, H237L, H383L, A460K, and H475L; or c) H383M, S401V, A405V, K408M and H475M.

[0366] Example 31. A modified influenza B HA polypeptide as described in any one of Examples 1-30, wherein the modified influenza B HA polypeptide is derived from influenza B / Yamagata virus.

[0367] Example 32. The modified influenza B HA peptide as described in Example 31, wherein the influenza B / Yamagata virus is B / Phuket / 3073 / 2013.

[0368] Example 33. A modified influenza B HA polypeptide as described in any one of Examples 1-30, wherein the modified influenza B HA polypeptide is derived from the B / Victoria influenza virus.

[0369] Example 34. The modified influenza B HA peptide as described in Example 33, wherein the influenza B / Victoria virus is B / Austria / 1359417 / 2021.

[0370] Example 35. A modified influenza B HA polypeptide as described in any one of Examples 1-34, further comprising a signal peptide.

[0371] Example 36. A modified influenza B HA polypeptide as described in Example 35, wherein the signal peptide is an influenza HA signal peptide.

[0372] Example 37. A trimeric influenza B HA polypeptide complex comprising three copies of the modified influenza B HA polypeptide as described in any one of Examples 1-36.

[0373] Example 38. The trimeric influenza B HA polypeptide complex as described in Example 37, wherein the trimeric influenza B HA polypeptide complex has higher pre-fusion conformational stability compared to the trimeric influenza B HA polypeptide complex prepared from a control influenza B HA polypeptide without one or more modifications.

[0374] Example 39. The trimeric influenza B HA polypeptide complex as described in Example 38, wherein the stability of the pre-fusion conformation is measured by the increased binding of the trimeric influenza B HA polypeptide complex to stem region-specific antibodies compared to a control influenza B HA polypeptide complex prepared without one or more modifications, and / or by the increased binding ratio of the stem region-specific antibodies to RBS-specific antibodies compared to a control influenza B HA polypeptide complex prepared without one or more modifications.

[0375] Example 40. A trimeric influenza B HA polypeptide complex as described in any one of Examples 37-40, wherein the trimeric influenza B HA polypeptide complex exhibits stronger immunogenicity compared to a trimeric influenza B HA polypeptide complex prepared from a control influenza B HA polypeptide without one or more modifications.

[0376] Example 41. The trimeric influenza beta-HA polypeptide complex as described in Example 40, wherein immunogenicity was measured using a hemagglutination inhibition assay.

[0377] Example 42. An artificial nucleic acid encoding a modified influenza B HA polypeptide as described in any one of Examples 1-36.

[0378] Example 43. A vector comprising the artificial nucleic acid as described in Example 42.

[0379] Example 44. A host cell comprising the vector as described in Example 43.

[0380] Example 45. A composition comprising a modified influenza B HA polypeptide as described in any one of Examples 1-36, a trimer influenza B HA polypeptide complex as described in any one of Examples 37-41, an artificial nucleic acid as described in Example 42, or a carrier as described in Example 43.

[0381] Example 46. A composition comprising one or more messenger RNA (mRNA) molecules encapsulated in lipid nanoparticles (LNPs), wherein the one or more mRNAs encapsulate a modified influenza B HA polypeptide as described in any one of Examples 1-36.

[0382] Example 47. A composition as described in Example 45 or 46, wherein the composition is an immunogenic composition.

[0383] Example 48. A vaccine comprising the composition as described in Example 47, and a pharmaceutically acceptable carrier.

[0384] Example 49. The vaccine as described in Example 48 further comprises an adjuvant.

[0385] Example 50. A method for immunizing a subject, the method comprising administering a vaccine as described in Example 48 or 49 to a subject in need.

[0386] Example 51. The method as described in Example 50, wherein the method prevents the subject from being infected with influenza B virus, reduces the likelihood of the subject being infected with influenza B virus, or reduces the likelihood of the subject developing a serious illness due to influenza B virus infection.

[0387] Example 52. The method as described in Example 50 or 51, wherein the subject is a human.

[0388] Example 53. The method as described in Example 52, wherein the person is 6 months or older, less than 18 years old, at least 6 months and less than 18 years old, at least 18 years and less than 65 years old, at least 6 months and less than 5 years old, at least 5 years and less than 65 years old, at least 60 years old, or at least 65 years old.

[0389] Example 54. The method as described in any one of Examples 50-53, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0390] Example 55. A method for alleviating one or more symptoms of influenza B virus infection, the method comprising administering a vaccine as described in Example 48 or 49 to a subject in need.

[0391] Example 56. An in vitro method for preparing a trimeric influenza beta-HA polypeptide complex, the method comprising culturing host cells as described in Example 44 in a cell culture medium and expressing the trimeric influenza beta-HA polypeptide complex.

[0392] Example 57. The in vitro method as described in Example 56, wherein the trimeric influenza B HA peptide complex has higher pre-fusion conformational stability compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications.

[0393] Example 58. The in vitro method as described in Example 57, wherein the stability of the pre-fusion conformation is measured by the increased binding of the trimeric influenza B HA peptide complex to the stem region-specific antibody compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications, and / or by the increased binding ratio of the stem region-specific antibody to the RBS-specific antibody compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications.

[0394] Example 59. The in vitro method as described in any one of Examples 56-58, wherein the trimeric influenza B HA peptide complex has stronger immunogenicity compared to a trimeric influenza B HA peptide complex prepared from a control influenza B HA peptide without one or more modifications.

[0395] Example 60. An in vitro method as described in Example 59, wherein a hemagglutination inhibition assay is used to measure immunogenicity.

[0396] Example 61. The in vitro method as described in any one of Examples 56-60, further comprising the step of purifying the trimeric influenza beta-HA polypeptide complex from the cell culture medium. The second set of representative embodiments disclosed herein

[0397] Example 1. A modified influenza B hemagglutinin (HA) polypeptide comprising a head region and a stem region, wherein the modified influenza B HA polypeptide comprises one or more modifications selected from the following: a) At least one proline mutation in the stem region, wherein the at least one proline mutation disrupts at least one helical structure in the stem region of the modified influenza B HA polypeptide in the fusion conformation. b) At least two cysteine ​​mutations, wherein the at least two cysteine ​​mutations form disulfide bridges in the modified influenza B HA peptide; c) One or more amino acid mutations in the head region and / or stem region, wherein the one or more amino acid mutations stabilize the modified influenza B HA peptide in the pre-fusion conformation through interface stabilization; d) One or more amino acid mutations in the head region and / or stem region, wherein the one or more amino acid mutations inactivate one or more pH sensors in the head region and / or stem region; e) At least one amino acid mutation in the head region, wherein the at least one amino acid mutation introduces at least one N-linked glycosylation motif in the head region; f) At least one amino acid mutation in the stem region, wherein the at least one amino acid mutation introduces or disrupts an N-linked glycosylation motif in the stem region; and g) At least one amino acid mutation in the head region, wherein the at least one amino acid mutation results in reduced sialic acid binding of the modified influenza B HA peptide compared to a control influenza B HA peptide which lacks at least one amino acid mutation that reduces sialic acid binding of the modified influenza B HA peptide.

[0398] Example 2. A modified influenza B HA polypeptide as described in Example 1, comprising at least one of two, three, four, five, six, or each of the modifications listed in a) to g).

[0399] Example 3. A modified influenza B HA peptide as described in Example 1 or 2, wherein the one or more modifications stabilize the modified influenza B HA peptide in a pre-fusion conformation, optionally wherein the stabilization of the pre-fusion conformation is measured by the increased binding of the modified influenza B HA peptide to stem region-specific antibodies compared to the wild-type influenza B HA peptide and / or by the increased binding ratio of the stem region-specific antibodies to RBS-specific antibodies compared to the wild-type influenza B HA peptide.

[0400] Example 4. The modified influenza B HA polypeptide as described in any one of Examples 1-3, wherein: (i) The head region is the segment of the modified influenza B HA polypeptide at approximately amino acid positions 57-307, as indexed by referring to the amino acid sequence of SEQ ID NO: 1; and / or (ii) The stem region is the segment of the modified influenza B HA polypeptide at approximately amino acid positions 16-56 and 308-547, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0401] Example 5. A modified influenza B HA polypeptide as described in any one of Examples 1-4, wherein: (i) The at least one proline mutation is located at amino acid positions 363, 366, 371, 372, 376, 380, 383, 390, 391, 393, 395, 397, 399, 421, 430, 431, 434, 436 and / or 490, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; and / or (ii) The at least two cysteine ​​mutations are located at amino acid positions 20 and 387, 35 and 408, 36 and 415, 37 and 411, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 232 and 433, 233 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 380 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 396 and 514, 401 and 475, 430 and 437, 430 and 438, and / or 430 and 439, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0402] Example 6. A modified influenza B HA peptide as described in any one of Examples 1-5, wherein the modified influenza B HA peptide is stabilized by interface stabilization in the pre-fusion conformation. The one or more amino acid mutations include at least one cavity-filling mutation in the stem region and / or one or more amino acid mutations in the head region and / or stem region to form polar interactions with adjacent amino acid residues; optionally wherein: (i) The at least one cavity-filling mutation is located at amino acid positions 460, 467, and / or 474, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; for example, lysine (K) is substituted at amino acid position 460, phenylalanine (F) or tyrosine (Y) is substituted at amino acid position 467, and / or glutamine (Q) is substituted at amino acid position 474; and / or (ii) The polar interaction includes salt bridges or hydrogen bonds.

[0403] Example 7. A modified influenza B HA polypeptide as described in any one of Examples 1-6, wherein the modified influenza B HA polypeptide is stabilized by interface stabilization in the pre-fusion conformation with one or more amino acid mutations located at amino acid positions 18, 121, 188, 226, 228, 408, 435 and / or 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0404] Example 8. A modified influenza B HA polypeptide as described in any one of Examples 1-7, wherein the one or more amino acid mutations that inactivate one or more pH sensors in the head region and / or stem region are located at amino acid positions 226, 228, 237, 383, 388, 391, 401, 405, 408, 435, 460, 474 and / or 475, as indexed by reference to the amino acid sequence of SEQ ID NO:1.

[0405] Example 9. A modified influenza B HA polypeptide as described in any one of Examples 1-8, wherein the head region contains a receptor binding site (RBS), and wherein the at least one N-linked glycosylation motif introduced in the head region is located in or adjacent to the RBS of the head region, optionally wherein the at least one N-linked glycosylation motif introduced in the head region is generated by introducing at least one substitution at amino acid positions 60, 62, 141, 143, 186, 187, 214, 216, 223 and / or 224, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0406] Example 10. A modified influenza B HA polypeptide as described in any one of Examples 1-9, wherein the at least one N-linked glycosylation motif introduced or disrupted in the stem region is generated by introducing at least one substitution at amino acid positions 28, 336 and / or 349, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0407] Example 11. A modified influenza B HA polypeptide as described in any one of Examples 1-10, wherein the at least one amino acid mutation that reduces the sialic acid binding of the modified influenza B HA polypeptide is located at amino acid positions 157, 177, 218 and / or 257, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0408] Example 12. A trimeric influenza B HA polypeptide complex comprising three copies of the modified influenza B HA polypeptide as described in any one of Examples 1-11.

[0409] Example 13. An artificial nucleic acid, such as mRNA, encoding a modified influenza B HA polypeptide as described in any one of Examples 1-11.

[0410] Example 14. A vector comprising the artificial nucleic acid as described in Example 13.

[0411] Example 15. A host cell comprising the vector as described in Example 14.

[0412] Example 16. A composition, such as an immunogenic composition, comprising a modified influenza B HA polypeptide as described in any one of Examples 1-11, a trimer influenza B HA polypeptide complex as described in Example 12, mRNA as described in Example 13, or a vector as described in Example 14.

[0413] Example 17. A method for immunizing a subject with the composition as described in Example 16.

[0414] Example 18. An in vitro method for preparing a trimeric influenza beta-HA polypeptide complex, the method comprising culturing host cells as described in Example 15 in a cell culture medium and expressing the trimeric influenza beta-HA polypeptide complex. The third set of representative embodiments disclosed herein

[0415] Example 1. An artificial messenger ribonucleic acid (mRNA) encoding an influenza B HA polypeptide, wherein the influenza B HA polypeptide comprises: a) at least one proline substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one proline substitution is located at amino acid positions 363, 366, 371, 372, 376, 380, 383, 390, 391, 393, 395, 397, 399, 421, 430, 431, 434, 436 and / or 490, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) At least two cysteine ​​substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least two cysteine ​​substitutions are located at amino acid positions 20 and 387, 35 and 408, 36 and 415, 37 and 411, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 232 and 433, 2 33 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 380 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 396 and 514, 401 and 475, 430 and 437, 430 and 438 and / or 430 and 439, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; c) At least one cavity-filling amino acid substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one cavity-filling amino acid substitution is located at amino acid positions 460, 467 and / or 474, as indexed by reference to the amino acid sequence of SEQ ID NO:1; d) One or more interface-stabilizing amino acid substitutions relative to the corresponding wild-type influenza B HA peptide, wherein the one or more interface-stabilizing amino acid substitutions are located at amino acid positions 18, 121, 188, 226, 228, 408, 435 and / or 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; e) One or more pH sensor knockout amino acid substitutions relative to the corresponding wild-type influenza B HA peptide, wherein the one or more pH sensor knockout amino acid substitutions are located at amino acid positions 226, 228, 237, 239, 383, 388, 391, 401, 405, 408, 435, 460, 474 and / or 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; f) At least one amino acid substitution relative to the corresponding wild-type influenza B HA peptide, wherein the at least one amino acid substitution produces or disrupts the N-linked glycosylation motif in the influenza B HA peptide and is located at amino acid positions 28, 60, 62, 141, 143, 186, 187, 214, 216, 223, 224, 336 and / or 349, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; and / or g) At least one amino acid substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one amino acid substitution is located at amino acid positions 157, 177, 218 and / or 257, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0416] Example 2. Artificial mRNA as described in Example 1, wherein the influenza B HA polypeptide comprises: a) Two proline substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the two proline substitutions are located at amino acid positions 430 and 436, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; or b) Five amino acid substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the five amino acid substitutions are located at amino acid positions 383, 401, 405, 408 and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0417] Example 3. Artificial mRNA as described in Example 2, wherein the influenza B HA polypeptide comprises the following amino acid substitutions: a) A430P and N436P; or b) H383M, S401V, A405V, K408M, and H475M, For example, indexing can be done by referring to the amino acid sequence of SEQ ID NO: 1.

[0418] Example 4. Artificial mRNA as described in any one of Examples 1-3, wherein the influenza B HA polypeptide is derived from the B / Victoria influenza virus.

[0419] Example 5. Artificial mRNA as described in Example 5, wherein the B / Victoria influenza virus is B / Austria / 1359417 / 2021.

[0420] Example 6. An artificial mRNA as described in any one of Examples 1-5, wherein the influenza B HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

[0421] Example 7. Artificial mRNA as described in Example 6, wherein the influenza B HA polypeptide contains the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

[0422] Example 8. An artificial mRNA as described in any one of Examples 1-7, comprising a nucleic acid sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

[0423] Example 9. An artificial mRNA as described in Example 8, comprising the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

[0424] Example 10. An artificial mRNA as described in any one of Examples 1-9, comprising a 5'-cap structure and / or a 3'-multiple (A) sequence.

[0425] Example 11. An artificial mRNA as described in any one of Examples 1-10, comprising at least one chemically modified nucleotide and / or phosphate thioester bond.

[0426] Example 12. The artificial mRNA as described in Example 11, wherein the at least one chemically modified nucleotide comprises pseudouridine, 2'-fluororibonucleotide or 2'-methoxyribonucleotide, optionally wherein the pseudouridine is N1-methylpseudouridine.

[0427] Example 13. A composition comprising artificial mRNA as described in any one of Examples 1-12, encapsulated in lipid nanoparticles (LNPs).

[0428] Example 14. The composition as described in Example 13, wherein the LNP comprises cationic lipids.

[0429] Example 15. The composition as described in Example 14, wherein the cationic lipid comprises OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) or IM-001.

[0430] Example 16. A composition as described in Example 14 or 15, wherein the LNP further comprises polyethylene glycol-conjugated (polyethylene glycolated) lipids, cholesterol-based lipids, and auxiliary lipids.

[0431] Example 17. The composition as described in Example 16, wherein: a) The polyethylene glycol-modified lipid comprises or contains 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000); and / or b) The cholesterol-based lipid contains cholesterol; and / or c) The auxiliary lipid may contain dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE).

[0432] Example 18. The composition as described in Example 16 or 17, wherein: a) The cationic lipid exists in a molar ratio of about 35% to about 55%; b) The polyethylene glycol-modified lipid is present in a molar ratio of about 0.25% to about 2.75%; c) The cholesterol-based lipid is present in a molar ratio of approximately 20% to approximately 45%; and d) The auxiliary lipid is present in a molar ratio of about 5% to about 35%. All molar ratios are relative to the total lipid content of the LNP.

[0433] Example 19. The composition as described in Example 18, wherein: a) The cationic lipid exists at a molar ratio of approximately 40%; b) The polyethylene glycol-modified lipid is present at a molar ratio of approximately 1.5%; c) The cholesterol-based lipid is present at a molar ratio of approximately 28.5%; and d) This auxiliary lipid exists at a molar ratio of approximately 30%. All molar ratios are relative to the total lipid content of the LNP.

[0434] Example 20. The composition as described in Example 19, wherein the artificial mRNA encodes the influenza B HA polypeptide of SEQ ID NO: 3, and wherein the LNP comprises about 40% GL-HEPES-E3-E12-DS-4-E10, about 1.5% DMG-PEG2000, about 28.5% cholesterol, and about 30% DOPE in a molar ratio.

[0435] Example 21. The composition as described in Example 19, wherein the artificial mRNA encodes the influenza B HA polypeptide of SEQ ID NO: 5, and wherein the LNP comprises about 40% GL-HEPES-E3-E12-DS-4-E10, about 1.5% DMG-PEG2000, about 28.5% cholesterol, and about 30% DOPE in a molar ratio.

[0436] Example 22. The composition as described in any one of Examples 13-21, wherein the composition is an immunogenic composition.

[0437] Example 23. An influenza B HA polypeptide comprising one or more amino acid substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the one or more amino acid substitutions comprise: a) Substitution of the two proline residues at amino acid positions 430 and 436, as indexed by referring to the amino acid sequence of SEQ ID NO: 1; or b) Amino acid substitutions at positions 383, 401, 405, 408 and 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1.

[0438] Example 24. The influenza B HA polypeptide as described in Example 23, comprising the following amino acid substitutions: a) A430P and N436P; or b) H383M, S401V, A405V, K408M, and H475M, For example, indexing can be done by referring to the amino acid sequence of SEQ ID NO: 1.

[0439] Example 25. The influenza B HA peptide as described in Example 23 or 24, wherein the influenza B HA peptide is derived from the B / Victoria influenza virus.

[0440] Example 26. The modified influenza B HA peptide as described in Example 25, wherein the influenza B / Victoria virus is B / Austria / 1359417 / 2021.

[0441] Example 27. The influenza B HA polypeptide as described in any one of Examples 23-26, comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5.

[0442] Example 28. The influenza B HA polypeptide as described in Example 27, wherein the influenza B HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

[0443] Example 29. A trimeric influenza B HA polypeptide complex comprising three copies of the influenza B HA polypeptide as described in any one of Examples 23-28.

[0444] Example 30. An artificial nucleic acid encoding an influenza B HA polypeptide as described in any one of Examples 23-28.

[0445] Example 31. An artificial nucleic acid as described in Example 30, wherein the artificial nucleic acid comprises at least one chemically modified nucleotide and / or thiophosphate bond.

[0446] Example 32. A vector comprising artificial nucleic acid as described in Example 30 or 31.

[0447] Example 33. A vector as described in Example 32, wherein the vector is a messenger RNA (mRNA) production vector.

[0448] Example 34. A host cell comprising a vector as described in Example 32 or 33.

[0449] Example 35. A composition comprising the influenza B HA polypeptide as described in any one of Examples 23-28, the trimer influenza B HA polypeptide complex as described in Example 29, the artificial nucleic acid as described in Example 30 or 31, or the carrier as described in Example 32 or 33.

[0450] Example 36. The composition as described in Example 35, wherein the composition is an immunogenic composition.

[0451] Example 37. A vaccine comprising the composition as described in Example 22 or 36, and a pharmaceutically acceptable carrier.

[0452] Example 38. The vaccine as described in Example 37 further comprises an adjuvant.

[0453] Example 39. A vaccine as described in Example 37 or 38, wherein the vaccine is an mRNA vaccine, and wherein the vaccine further comprises mRNA encoding an influenza H3 HA polypeptide and mRNA encoding an influenza H1 HA polypeptide.

[0454] Example 40. A vaccine as described in Example 37 or 38, wherein the vaccine is an mRNA vaccine, and wherein the vaccine further comprises mRNA encoding influenza H3 HA peptide, mRNA encoding influenza H1 HA peptide, mRNA encoding influenza N2 neuraminidase (NA) peptide, mRNA encoding influenza N1 NA peptide and mRNA encoding influenza NA peptide from the influenza B / Victoria lineage.

[0455] Example 41. A vaccine as described in Example 37 or 38, wherein the vaccine is a recombinant vaccine, and wherein the vaccine further comprises influenza H3 HA peptide and influenza H1 HA peptide.

[0456] Example 42. A vaccine as described in Example 37 or 38, wherein the vaccine is a recombinant vaccine, and wherein the vaccine further comprises influenza H3 HA peptide, influenza H1 HA peptide, influenza N2 NA peptide, influenza N1 NA peptide and influenza NA peptide from the influenza B / Victoria lineage.

[0457] Example 43. A method for immunizing a subject, the method comprising administering to a subject in need a vaccine as described in any one of Examples 37-42.

[0458] Example 44. The method as described in Example 43, wherein the method prevents the subject from being infected with influenza B virus, reduces the likelihood of the subject being infected with influenza B virus, or reduces the likelihood of the subject developing a serious illness due to influenza B virus infection.

[0459] Example 45. The method as described in Example 43 or 44, wherein the subject is a human.

[0460] Example 46. The method as described in Example 45, wherein the person is 6 months or older, less than 18 years old, at least 6 months and less than 18 years old, at least 18 years and less than 65 years old, at least 6 months and less than 5 years old, at least 5 years and less than 65 years old, at least 60 years old, or at least 65 years old.

[0461] Example 47. The method as described in any one of Examples 43-46, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0462] Example 48. A method for alleviating ...

Claims

1. An artificial messenger ribonucleic acid (mRNA) encoding an influenza B HA polypeptide, wherein the influenza B HA polypeptide comprises: a) At least one proline substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one proline substitution is located at amino acid positions 363, 366, 371, 372, 376, 380, 383, 390, 391, 393, 395, 397, 399, 421, 430, 431, 434, 436 and / or 490, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; b) At least two cysteine ​​substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least two cysteine ​​substitutions are located at amino acid positions 20 and 387, 35 and 408, 36 and 415, 37 and 411, 125 and 431, 127 and 431, 185 and 223, 186 and 224, 186 and 239, 188 and 241, 232 and 433, 23 3 and 434, 239 and 276, 346 and 465, 367 and 478, 378 and 397, 380 and 397, 383 and 401, 387 and 510, 394 and 507, 394 and 510, 396 and 510, 396 and 514, 401 and 475, 430 and 437, 430 and 438 and / or 430 and 439, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; c) At least one cavity-filling amino acid substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one cavity-filling amino acid substitution is located at amino acid positions 460, 467 and / or 474, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; d) One or more interface-stabilizing amino acid substitutions relative to the corresponding wild-type influenza B HA peptide, wherein the one or more interface-stabilizing amino acid substitutions are located at amino acid positions 18, 121, 188, 226, 228, 408, 435 and / or 460, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; e) One or more pH sensor knockout amino acid substitutions relative to the corresponding wild-type influenza B HA peptide, wherein the one or more pH sensor knockout amino acid substitutions are located at amino acid positions 226, 228, 237, 239, 383, 388, 391, 401, 405, 408, 435, 460, 474 and / or 475, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; f) At least one amino acid substitution relative to the corresponding wild-type influenza B HA peptide, wherein the at least one amino acid substitution generates or disrupts the N-linked glycosylation motif in the influenza B HA peptide and is located at amino acid positions 28, 60, 62, 141, 143, 186, 187, 214, 216, 223, 224, 336 and / or 349, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; and / or g) At least one amino acid substitution relative to the corresponding wild-type influenza B HA polypeptide, wherein the at least one amino acid substitution is located at amino acid positions 157, 177, 218 and / or 257, as indexed by reference to the amino acid sequence of SEQ ID NO:

1.

2. The artificial mRNA of claim 1, wherein the influenza B HA polypeptide comprises: a) Two proline substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the two proline substitutions are located at amino acid positions 430 and 436, as indexed by reference to the amino acid sequence of SEQ ID NO: 1; or b) Five amino acid substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the five amino acid substitutions are located at amino acid positions 383, 401, 405, 408 and 475, as indexed by reference to the amino acid sequence of SEQ ID NO:

1.

3. The artificial mRNA of claim 2, wherein the influenza B HA polypeptide comprises the following amino acid substitutions: a) A430P and N436P; or b) H383M, S401V, A405V, K408M, and H475M, For example, indexing can be done by referring to the amino acid sequence of SEQ ID NO:

1.

4. The artificial mRNA according to any one of claims 1-3, wherein the influenza B HA polypeptide is derived from the B / Victoria influenza virus.

5. The artificial mRNA as described in claim 5, wherein the B / Victoria influenza virus is B / Austria / 1359417 / 2021.

6. The artificial mRNA according to any one of claims 1-5, wherein the influenza B HA polypeptide comprises an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

5.

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

5.

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

6.

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

6.

10. The artificial mRNA according to any one of claims 1-9, comprising a 5'-cap structure and / or a 3'-multiple (A) sequence.

11. The artificial mRNA according to any one of claims 1-10, comprising at least one chemically modified nucleotide and / or phosphate thioester bond.

12. The artificial mRNA of claim 11, wherein the at least one chemically modified nucleotide comprises pseudouridine, 2'-fluororibonucleotide or 2'-methoxyribonucleotide, optionally wherein the pseudouridine is N1-methylpseudouridine.

13. A composition comprising an artificial mRNA as described in any one of claims 1-12 encapsulated in lipid nanoparticles (LNPs).

14. The composition of claim 13, wherein the LNP comprises a cationic lipid.

15. The composition of claim 14, wherein the cationic lipid comprises OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) or IM-001.

16. The composition of claim 14 or 15, wherein the LNP further comprises polyethylene glycol-conjugated (polyethylene glycolated) lipids, cholesterol-based lipids, and auxiliary lipids.

17. The composition of claim 16, wherein: a) The PEGylated lipid comprises or contains 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2000); and / or b) The cholesterol-based lipid contains cholesterol; and / or c) The auxiliary lipid may contain dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE).

18. The composition of claim 16 or 17, wherein: a) The cationic lipid exists in a molar ratio of about 35% to about 55%; b) The PEGylated lipid is present in a molar ratio of about 0.25% to about 2.75%; c) The cholesterol-based lipid is present in a molar ratio of approximately 20% to approximately 45%; and d) The auxiliary lipid is present in a molar ratio of about 5% to about 35%. All molar ratios are relative to the total lipid content of the LNP.

19. The composition of claim 18, wherein: a) The cationic lipid exists at a molar ratio of approximately 40%; b) The polyethylene glycol-modified lipid is present at a molar ratio of approximately 1.5%; c) The cholesterol-based lipid is present at a molar ratio of approximately 28.5%; and d) This auxiliary lipid exists at a molar ratio of approximately 30%. All molar ratios are relative to the total lipid content of the LNP.

20. The composition of claim 19, wherein the artificial mRNA encodes the influenza B HA polypeptide of SEQ ID NO: 3, and wherein the LNP comprises about 40% GL-HEPES-E3-E12-DS-4-E10, about 1.5% DMG-PEG2000, about 28.5% cholesterol, and about 30% DOPE in a molar ratio.

21. The composition of claim 19, wherein the artificial mRNA encodes the influenza B HA polypeptide of SEQ ID NO: 5, and wherein the LNP comprises about 40% GL-HEPES-E3-E12-DS-4-E10, about 1.5% DMG-PEG2000, about 28.5% cholesterol, and about 30% DOPE in a molar ratio.

22. The composition of any one of claims 13-21, wherein the composition is an immunogenic composition.

23. An influenza B HA polypeptide comprising one or more amino acid substitutions relative to the corresponding wild-type influenza B HA polypeptide, wherein the one or more amino acid substitutions comprise: a) Substitution of the two proline residues at amino acid positions 430 and 436, as indexed by referring to the amino acid sequence of SEQ ID NO: 1; or b) Amino acid substitutions at positions 383, 401, 405, 408 and 475, as indexed by reference to the amino acid sequence of SEQ ID NO:

1.

24. The influenza B HA polypeptide of claim 23, wherein the following amino acid substitutions are made: a) A430P and N436P; or b) H383M, S401V, A405V, K408M, and H475M, For example, indexing can be done by referring to the amino acid sequence of SEQ ID NO:

1.

25. The influenza B HA polypeptide as described in claim 23 or 24, wherein the influenza B HA polypeptide is derived from the B / Victoria influenza virus.

26. The influenza B HA polypeptide of claim 25, wherein the influenza B / Victoria virus is B / Austria / 1359417 / 2021.

27. The influenza B HA polypeptide according to any one of claims 23-26, comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

5.

28. The influenza B HA polypeptide of claim 27, wherein the influenza B HA polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

5.

29. A trimeric influenza B HA polypeptide complex comprising three copies of the influenza B HA polypeptide as described in any one of claims 23-28.

30. An artificial nucleic acid encoding an influenza B HA polypeptide as described in any one of claims 23-28.

31. The artificial nucleic acid of claim 30, wherein the artificial nucleic acid comprises at least one chemically modified nucleotide and / or thiophosphate bond.

32. A vector comprising the artificial nucleic acid as described in claim 30 or 31.

33. The vector of claim 32, wherein the vector is a messenger RNA (mRNA) production vector.

34. A host cell comprising the vector as described in claim 32 or 33.

35. A composition comprising the influenza B HA polypeptide as described in any one of claims 23-28, the trimer influenza B HA polypeptide complex as described in claim 29, the artificial nucleic acid as described in claim 30 or 31, or the carrier as described in claim 32 or 33.

36. The composition of claim 35, wherein the composition is an immunogenic composition.

37. A vaccine comprising the composition as described in claim 22 or 36, and a pharmaceutically acceptable carrier.

38. The vaccine of claim 37, further comprising an adjuvant.

39. The vaccine of claim 37 or 38, wherein the vaccine is an mRNA vaccine, and wherein the vaccine further comprises mRNA encoding an influenza H3 HA polypeptide and mRNA encoding an influenza H1 HA polypeptide.

40. The vaccine of claim 37 or 38, wherein the vaccine is an mRNA vaccine, and wherein the vaccine further comprises mRNA encoding influenza H3 HA peptide, mRNA encoding influenza H1 HA peptide, mRNA encoding influenza N2 neuraminidase (NA) peptide, mRNA encoding influenza N1 NA peptide and mRNA encoding influenza NA peptide from the influenza B / Victoria lineage.

41. The vaccine of claim 37 or 38, wherein the vaccine is a recombinant vaccine, and wherein the vaccine further comprises influenza H3 HA peptide and influenza H1 HA peptide.

42. The vaccine of claim 37 or 38, wherein the vaccine is a recombinant vaccine, and wherein the vaccine further comprises influenza H3 HA peptide, influenza H1 HA peptide, influenza N2 NA peptide, influenza N1 NA peptide and influenza NA peptide from the influenza B / Victoria lineage.

43. A method for immunizing a subject, the method comprising administering to a subject in need the vaccine as described in any one of claims 37-42.

44. The method of claim 43, wherein the method prevents the subject from contracting influenza B virus, reduces the likelihood of the subject contracting influenza B virus, or reduces the likelihood of the subject developing a serious illness due to influenza B virus infection.

45. The method of claim 43 or 44, wherein the subject is a human being.

46. ​​The method of claim 45, wherein the person is 6 months or more, less than 18 years old, at least 6 months and less than 18 years old, at least 18 years and less than 65 years old, at least 6 months and less than 5 years old, at least 5 years and less than 65 years old, at least 60 years old, or at least 65 years old.

47. The method of any one of claims 43-46, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

48. A method for alleviating one or more symptoms of influenza B virus infection, the method comprising administering a vaccine as described in any one of claims 37-42 to a subject in need.

49. An in vitro method for preparing a trimeric influenza beta-HA polypeptide complex, the method comprising culturing a host cell as described in claim 34 in a cell culture medium and expressing the trimeric influenza beta-HA polypeptide complex.

50. The in vitro method of claim 49, further comprising the step of purifying the trimeric influenza beta-HA polypeptide complex from the cell culture medium.

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