Hybrid multivalent influenza vaccine containing hemagglutinin and neuraminidase and methods of using same

Abstract

Disclosed herein is a hybrid multivalent vaccine or immunogenic composition comprising: (i) one or more influenza virus proteins selected from one or more influenza virus hemagglutinin (HA) proteins, one or more influenza virus neuraminidase (NA) proteins, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding one or more influenza virus proteins selected from one or more influenza virus HA proteins, one or more influenza virus NA proteins, or a combination thereof. Also disclosed are methods of using the vaccine or immunogenic composition.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and is based on the filing date of U.S. Provisional Application No. 63 / 276,247, filed November 5, 2021, the entire contents of which are incorporated by reference herein.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The name of this copy of XML, created on October 14, 2022, is 0171_0068_PCT_Sequence_Listing.xml and is 2,408 bytes in size.

[0003] Field of the Disclosure Disclosed herein are hybrid multivalent influenza vaccines or immunogenic compositions and methods of using the hybrid multivalent influenza vaccines or immunogenic compositions for inducing immunity to both influenza virus hemagglutinin (HA) and influenza neuraminidase (NA), which contain both influenza virus antigens and ribonucleic acid molecules encoding influenza virus antigens. [Background technology]

[0004] Background of the Disclosure Influenza is caused by a virus that primarily attacks the upper respiratory tract, including the nose, throat and bronchi, and rarely the lungs. Infection usually lasts about a week. It is characterized by the sudden onset of high fever, muscle pain, headache and severe fatigue, dry cough, sore throat and rhinitis. Most people recover within 1-2 weeks without needing any medical treatment. However, in young children, the elderly and those suffering from medical conditions such as lung disease, diabetes, cancer, kidney or heart problems, influenza poses a serious risk. In these individuals, infection can lead to severe complications of underlying diseases, pneumonia and death, but healthy adults and older children can be affected as well. The annual seasonal influenza epidemic is thought to cause 3-5 million severe illnesses and 250,000-500,000 deaths worldwide each year.

[0005] Influenza viruses are members of the Orthomyxoviridae family. There are three main subtypes of influenza viruses, called influenza A, influenza B, and influenza C. Influenza virions contain a segmented negative-sense RNA genome that encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (M1), proton ion channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2). HA, NA, M1, and M2 are membrane-associated, while NP, PB1, PB2, PA, and NS2 are nucleocapsid-associated proteins. The HA and NA proteins are envelope glycoproteins and are primarily involved in virus attachment to cells and entry of viral particles, as well as release from cells, respectively.

[0006] Certain known licensed influenza vaccine compositions are inactivated or live attenuated vaccines containing whole virions or virions treated with agents that dissolve lipids ("split" vaccines), purified glycoproteins expressed in cell culture ("subunit vaccines"). Other types of vaccines, such as those based on RNA / DNA or viral vectors, have been developed. These vaccines provide protection, in part, by inducing the production of antibodies against influenza antigens such as HA. The antigenic evolution of influenza viruses by mutation, also called antigenic drift, results in modifications in HA and, to a lesser extent, NA. Thus, the amino acid sequences of major influenza antigens, including HA and NA, are highly variable across a particular group, subtype and / or strain.

[0007] Thus, available vaccines can only protect against strains with surface glycoproteins that contain identical or cross-reactive epitopes. To provide a broader antigenic spectrum, conventional vaccines contain components from several different virus strains, including strains from both influenza A and B. The selection of strains for use in current seasonal influenza vaccines is reviewed annually to keep up with antigenic drift and combat rapidly evolving virus strains, and is based on World Health Organization (WHO) recommendations. These recommendations reflect international epidemiological observations.

[0008] Due to the reassortment procedures used to generate the high-yielding virus strains used for manufacturing, current influenza virus seeds for vaccine production must be shown to have the appropriate HA antigen. However, there is currently no requirement or restriction on the NA content in influenza vaccines. There is evidence that NA levels in vaccines are highly variable. Kendal et al., Further Studies of Neuraminidase Content of Inactivated Influenza Vaccines and the Neuraminidase Antibody Responses After Vaccination of Immunologically Primed and Unprimed Populations, INFECTION AND IMMUNITY 1980;29(3):966-971 reported that NA-specific activity can vary by approximately 40-fold for different lots. Kendal et al. also noted a rapid decline in NA activity during 6 months of storage. As a result, antibody responses to NA were less frequent (mean seroconversion rate 18%) compared to HA responses (seroconversion rate 64%).

[0009] Moreover, although there is growing evidence that NA-specific antibodies correlate with resistance to disease in humans, current vaccination strategies focus almost exclusively on HA antigens or entirely on HA antigens, as in the case of the FLUBLOK® tetravalent vaccine containing recombinant HA protein. Furthermore, there is limited data available regarding the immunological response to NA during influenza infection, especially when compared with the data for HA (Wong et al., Hemagglutinin and Neuraminidase Antibodies Are Induced in Age- and Subtype-Dependent Manner after Influenza Virus Infection, JOURNAL OF VIROLOGY 2020;94(7):e01385-19). Influenza viruses naturally contain about one-tenth as much NA on the viral surface compared to HA, and established processes for concentrating HA antigens may not be suitable for maintaining NA in its enzymatically active and tetrameric conformation. Thus, while currently available inactivated influenza virus vaccines may contain NA, the quantity and quality vary widely and are not uniform. Furthermore, it has been described that NA is immunosubdominant when presented to the immune system together with HA (Krammer, The human antibody response to influenza A virus infection and vaccination, NATURE REVIEWS IMMUNOLOGY 2019;19:383-397). In other words, HA is known to be immunodominant over NA. Ibid. This phenomenon of immunodominance observed in conventional influenza vaccines remains an obstacle to the development of multivalent vaccines that can successfully achieve a multivalent immune response against multiple antigens or epitopes, especially for multivalent vaccines containing immunodominant proteins such as HA and / or when the valency in the vaccine is increased.Woodruff et al., B Cell Competition for Restricted T Cell Help Suppresses Rare-Epitope Responses, CELL REPORTS 2018;25:321~27.

[0010] Thus, the ability to supplement standard of care influenza strains in a vaccine with additional antigens including at least one mRNA molecule encoding influenza virus HA or influenza virus NA, which may confer enhanced and / or broader breadth of protection against circulating influenza strains by inducing a robust immune response against both HA and NA, is desirable. However, combining influenza virus HA and influenza virus NA into a hybrid multivalent vaccine composition that includes both influenza proteins and the ribonucleic acid encoding same, and enhances the NA and / or HA immune response, conferring enhanced and / or broader breadth of protection against circulating influenza strains, especially when compared to currently available standard of care influenza vaccines, can be a challenging task. Summary of the Invention [Means for solving the problem]

[0011] The present disclosure provides a vaccine or immunogenic composition comprising: (i) one or more influenza virus proteins selected from one or more influenza virus hemagglutinin (HA) proteins, one or more influenza virus neuraminidase (NA) proteins, or a combination thereof; and (ii) one or more ribonucleic acid molecules encoding one or more influenza virus proteins selected from one or more influenza virus HA proteins, one or more influenza virus NA proteins, or a combination thereof.

[0012] In certain embodiments, the one or more influenza virus proteins are recombinant influenza virus proteins, and in certain embodiments, the one or more influenza virus proteins are present in an inactivated influenza virus (IIV). In certain embodiments, the one or more ribonucleic acid molecules are mRNA molecules. Thus, in certain embodiments, disclosed herein is a vaccine or immunogenic composition comprising: (i) one or more recombinant influenza virus proteins selected from a recombinant HA protein, a recombinant NA protein, or a combination thereof; and (ii) one or more mRNA molecules encoding one or more influenza virus proteins selected from a recombinant HA protein, a recombinant NA protein, or a combination thereof.

[0013] In one aspect of the disclosure, the vaccine or immunogenic composition disclosed herein comprises not more than eight, such as eight, or not more than four, such as four, influenza virus proteins in (i) and a ribonucleic acid molecule in (ii) encoding not more than eight, such as eight, or not more than four, such as four, influenza virus proteins. In a particular embodiment, the vaccine or immunogenic composition is an 8-valent vaccine or immunogenic composition, and in a particular embodiment, the vaccine or immunogenic composition is a 16-valent vaccine or immunogenic composition. Other multivalent vaccines or immunogenic compositions are also described herein.

[0014] In various embodiments of the vaccine or immunogenic composition disclosed herein, the one or more influenza virus proteins of (i) comprise 1 to 8 influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, influenza virus HA from B / Yamagata lineage, influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from B / Victoria lineage, or influenza virus NA from B / Yamagata lineage; and (ii) one or more ribonucleic acid molecules encoding 1 to 8 influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, influenza virus HA from B / Yamagata lineage, influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from B / Victoria lineage, or influenza virus NA from B / Yamagata lineage.

[0015] In certain embodiments, the one or more influenza proteins in (i) comprise four recombinant influenza virus HA proteins and the one or more ribonucleic acid molecules encode four influenza virus NA proteins. In certain embodiments, the one or more ribonucleic acid molecules encode four full-length influenza virus NA proteins (e.g., wild-type or machine-learned NA). In certain embodiments, the one or more influenza proteins in (i) comprise four recombinant influenza virus NA proteins and the one or more ribonucleic acid molecules encode four influenza virus HA proteins. In certain embodiments, the one or more ribonucleic acid molecules encode four full-length influenza virus HA proteins (e.g., wild-type or machine-learned NA).

[0016] In certain embodiments, the one or more influenza virus proteins in (i) comprise a first influenza virus HA protein (wherein the first influenza virus HA protein is an H1 HA); a second influenza virus HA protein (wherein the second influenza virus HA protein is an H3 HA); a third influenza virus HA protein from a B / Victoria influenza virus lineage; and a fourth influenza virus HA protein from a B / Yamagata influenza virus lineage, and in certain embodiments, the one or more ribonucleic acid molecules encode a first influenza virus NA protein (wherein the first influenza virus NA protein is an N1 NA); a second influenza virus NA protein (wherein the second influenza virus NA protein is an N2 NA); a third influenza virus NA protein from a B / Victoria influenza virus lineage; and a fourth influenza virus NA protein from a B / Yamagata influenza virus lineage. In certain embodiments, each of the first, second, third, and fourth influenza virus HA proteins is a recombinant influenza virus HA. In certain embodiments, the one or more ribonucleic acid molecules encode four full-length influenza virus NA proteins (e.g., wild-type or machine-learned NA).

[0017] Also disclosed herein are embodiments in which the one or more influenza virus proteins in (i) include a first influenza virus NA protein, where the first influenza virus NA protein is N1 NA; a second influenza virus NA protein, where the second influenza virus NA protein is N2 NA; a third influenza virus NA protein from a B / Victoria influenza virus lineage; and a fourth influenza virus NA protein from a B / Yamagata influenza virus lineage, and in certain embodiments, the one or more ribonucleic acids encode a first influenza virus HA protein, where the first influenza virus HA protein is H1 HA; a second influenza virus HA protein, where the second influenza virus HA protein is H3 HA; a third influenza virus HA protein from a B / Victoria influenza virus lineage; and a fourth influenza virus HA protein from a B / Yamagata influenza virus lineage.

[0018] In certain embodiments, each of the first, second, third and fourth influenza virus NA proteins is a recombinant influenza virus NA. In certain embodiments, each of the first, second, third and fourth influenza virus NA proteins is a modified recombinant influenza virus NA.

[0019] In certain embodiments disclosed herein, at least one of the one or more influenza virus proteins comprises an influenza virus HA protein and / or an influenza virus NA protein having a molecular sequence identified or designed from a machine learning model, and in certain embodiments, at least one of the one or more ribonucleic acid molecules encodes one or more influenza virus proteins having a molecular sequence identified or designed from a machine learning model.

[0020] In certain embodiments, the H1 HA is from an H1N1 influenza virus strain, the H3 HA is from an H3N2 influenza virus strain, the N1 NA is from an H1N1 influenza virus strain, and / or the N2 NA is from an H3N2 influenza virus strain. In certain embodiments, the H1 HA and N1 NA are from the same H1N1 influenza virus strain, and / or the H3 HA and N2 NA are from the same H3N2 influenza virus strain.

[0021] According to certain embodiments, the vaccine or immunogenic composition further comprises an adjuvant, such as a squalene in water adjuvant or a liposome-based adjuvant. In certain embodiments, the one or more ribonucleic acid molecules are encapsulated in lipid nanoparticles (LNPs) and the vaccine or immunogenic composition does not further comprise an adjuvant.

[0022] In certain embodiments, the one or more ribonucleic acid molecules comprise at least one chemically modified nucleotide, which in certain embodiments comprises pseudouridine, in particular N1-methylpseudouridine, 2'-fluororibonucleotides, 2'-methoxyribonucleotides and / or phosphorothioate linkages.

[0023] In certain embodiments where the one or more influenza virus HA proteins are recombinant influenza virus HA proteins, the recombinant influenza virus HA proteins are produced in cultured insect cells by a baculovirus expression system, and in certain embodiments where the one or more influenza virus NA proteins are recombinant influenza virus NA proteins, the recombinant influenza virus NA proteins are produced in Chinese hamster ovary (CHO) cells.

[0024] In a particular embodiment, the influenza virus protein in (i) and / or the ribonucleic acid molecule in (ii) are derived from a standard of care influenza strain.

[0025] In various embodiments disclosed herein, one or more ribonucleic acid molecules are encapsulated in the LNPs, and in certain embodiments, the LNPs comprise a cationic lipid, a polyethylene glycol-conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid. In certain aspects, the vaccine or immunogenic composition comprises at least two, e.g., at least four, ribonucleic acid molecules encapsulated in the same LNPs. In certain embodiments, the LNPs comprise a cationic lipid in a molar ratio of 35% to 45%, e.g., 40%, a PEGylated lipid in a molar ratio of 0.25% to 2.75%, e.g., 1.5%, a cholesterol-based lipid in a molar ratio of 25% to 35%, e.g., 28.5%, and a helper lipid in a molar ratio of 25% to 35%, e.g., 30%, all of which are relative to the total lipid content of the LNPs.

[0026] In certain embodiments, the cationic lipid is selected from the group including OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10 and GL-HEPES-E3-E12-DS-3-E14, such as cKK-E10, and in certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000. In certain embodiments, the cholesterol-based lipid is cholesterol and the helper lipid is dioleoyl-SN-glycero-3-phosphoethanolamine. In certain embodiments disclosed herein, the LNP comprises cKK-E10, e.g., at a molar ratio of 40%, dimyristoyl-PEG2000, e.g., at a molar ratio of 1.5%, cholesterol, e.g., at a molar ratio of 28.5%, and dioleoyl-SN-glycero-3-phosphoethanolamine, e.g., at a molar ratio of 30%.

[0027] In certain embodiments, the LNPs comprise (i) ALC-0315 as a cationic lipid, (ii) N,N ditetradecylacetamido-polyethylene glycol (e.g., ALC-0159) as a PEGylated lipid, (iii) DSPC as a helper lipid, and (iv) cholesterol. In certain embodiments, the LNPs comprise (i) ALC-0315 as a cationic lipid in a molar ratio of about 25% to about 65%, e.g., about 46.3%; (ii) N,N ditetradecylacetamido-polyethylene glycol (e.g., ALC-0159) as a PEGylated lipid in a molar ratio of about 0.5% to about 2.6%, e.g., 1.6%; (iii) DSPC as a helper lipid in a molar ratio of about 5% to about 15%, e.g., 9.4%; and (iv) cholesterol in a molar ratio of about 20% to about 60%, e.g., 42.7%.

[0028] In certain embodiments of the vaccine or immunogenic compositions disclosed herein, each of the influenza virus proteins in (i) is present in the composition in an amount ranging from about 0.1 μg to about 90 μg, such as from about 1 μg to about 60 μg or from about 5 μg to about 45 μg, and in certain embodiments, each of the ribonucleic acid molecules is present in the composition in an amount ranging from about 0.1 μg to about 150 μg, such as from about 1 μg to about 60 μg or from about 5 μg to about 45 μg. In certain embodiments, the compositions are formulated for intramuscular injection.

[0029] In another aspect, disclosed herein is a vaccine comprising the immunogenic composition disclosed herein and a pharmaceutical carrier.

[0030] Another aspect of the disclosure is directed to a method of immunizing a subject against influenza virus, the method comprising administering to the subject an immunologically effective amount of a vaccine as disclosed herein. Also disclosed herein is a vaccine as disclosed herein for use in a method of immunizing a subject against influenza virus. Also disclosed herein is an immunogenic composition as disclosed herein for the manufacture of a vaccine for use in a method of immunizing a subject against influenza virus. In certain embodiments, the method or use prevents influenza virus infection in a subject, and in certain embodiments, the method or use generates a protective immune response in the subject, such as an HA antibody response and / or an NA antibody response. In certain embodiments, the subject is a human, and in certain embodiments, the vaccine is administered or prepared to be administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0031] Another aspect of the present disclosure is directed to a method for reducing one or more symptoms of influenza virus infection, comprising administering to a subject a prophylactically effective amount of the vaccine disclosed herein.Also disclosed is a vaccine as disclosed herein for use in the method for reducing one or more symptoms of influenza virus infection.Also disclosed is an immunogenic composition as disclosed herein for the manufacture of a vaccine for use in the method for reducing one or more symptoms of influenza virus infection.

[0032] Another aspect of the present disclosure is directed to a method of enhancing or broadening a protective immune response in a subject, the method comprising administering to the subject an immunologically effective amount of a vaccine disclosed herein, which enhances the vaccine efficacy of a standard of care influenza virus vaccine composition by an amount ranging from about 5% to about 100%, such as at least about 20%, or from about 40% to about 80%, such as from about 40% to about 60%. Also disclosed is a vaccine as disclosed herein for use in a method of enhancing or broadening a protective immune response in a subject, the method comprising administering to the subject an immunologically effective amount of a vaccine disclosed herein, which enhances the vaccine efficacy of a standard of care influenza virus vaccine composition by an amount ranging from about 5% to about 100%, such as at least about 20%, or from about 40% to about 80%, such as from about 40% to about 60%. Also disclosed is an immunogenic composition as disclosed herein for the manufacture of a vaccine for use in a method of enhancing or broadening a protective immune response in a subject, the method comprising administering to a subject an immunologically effective amount of a vaccine as disclosed herein, which improves the vaccine efficacy of a standard of care influenza virus vaccine composition by an amount ranging from about 5% to about 100%, such as at least about 20%, or from about 40% to about 80%, such as from about 40% to about 60%. In certain embodiments, the standard of care influenza virus vaccine composition is an inactivated influenza virus composition comprising an inactivated influenza virus from an H1N1 strain, an H3N2 strain, a B / Victoria lineage, and a B / Yamagata lineage. In certain embodiments, the standard of care influenza virus vaccine composition comprises a recombinant influenza virus HA from an H1N1 strain, an H3N2 strain, a B / Victoria lineage, and a B / Yamagata lineage.

[0033] In various embodiments, the methods or uses and compositions disclosed herein treat or prevent disease caused by either or both seasonal and pandemic influenza strains. In certain embodiments of the methods or uses disclosed herein, the subject is a human, and the human is 6 months or older, under 18 years of age, at least 6 months and under 18 years of age, at least 18 years and under 65 years of age, at least 6 months and under 5 years of age, at least 5 years and under 65 years of age, at least 60 years of age, or at least 65 years of age. In certain embodiments, the methods or uses disclosed herein comprise administering to the subject two doses of the vaccine, 2-6 weeks apart, such as 4 weeks apart. [Brief description of the drawings]

[0034] [Figure 1A] FIG. 1A is a graph showing N1 heterologous panel binding levels in ferret sera at day 42 against N1 influenza virus strains and Tet standards following administration of a hybrid octavalent vaccine composition as described in Example 3. [Figure 1B] FIG. 1B is a graph showing NB homology panel binding levels in ferret sera at day 42 against NB influenza virus strains and Tet standards following administration of a hybrid octavalent vaccine composition as described in Example 3. [Figure 1C] FIG. 1C is a graph showing N2 heterologous panel binding levels in ferret sera at day 42 against N2 influenza virus strains and Tet standards following administration of a hybrid octavalent vaccine composition as described in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Some viruses can substantially change the structure of their envelope glycoprotein components. For example, influenza viruses constantly change the amino acid sequence of their envelope glycoproteins. Either major amino acid mutations (antigenic shift) or minor mutations (antigenic drift) can give rise to new epitopes, allowing the virus to evade the immune system. Antigenic mutations are the main cause of repeated influenza epidemics. Antigenic variants within a subtype (e.g., H1 or H3) emerge and are gradually selected as the dominant virus, while the preceding virus is suppressed by specific antibodies that arise in the population. In general, neutralizing antibodies against one variant become less and less effective as successive variants arise. The immune response to variants within a subtype may depend on the host's previous experience.

[0036] HA and NA evolve quite differently. For example, it has been shown that for all genes of influenza viruses, including the gene for HA, the rate of silent nucleotide substitution is faster than the rate of coding nucleotide substitution (Webster, RG, et al., Evolution and ecology of influenza A viruses, MICROBIOL REVS. 1992; 56(1): 152-179). However, HA has a much higher rate of coding change than the internal proteins. The higher rate of coding nucleotide change in the HA gene compared to other genes has been taken as evidence that immune selection is an important factor in its evolution (Palese, P., et al., Variation of Influenza A, B, and C Viruses, SCIENCE 1982; 215(4539): 1468-74). Using reassortment antigens to eliminate any nonspecific steric hindrance, Kilbourne et al. studied the evolutionary rates of epidemiologically important HA and NA antigens isolated from humans over a 10-year period and found that HA evolves more rapidly than NA (Kilbourne, ED, et al., Independent and disparate evolution in nature of influenza virus A hemagglutinin and neuraminidase glycoproteins, PNAS 1990;87(2):786-790). This was shown for both type A H1N1 and H3N2 viruses and confirmed by subsequent experiments with more recent strains. The reason for the apparent difference in evolutionary rates is unclear but may be due to the fact that antibodies against HA neutralize the virus and prevent infection. This may put greater selective pressure on HA to maintain itself in partially immune populations. Thus, because NA undergoes more gradual antigenic drift when compared to HA, a vaccine or immunogenic composition containing both HA and NA may confer broader protection (in the form of NA antibodies) against influenza strains containing antigenically drifted HA antigens.

[0037] Since influenza viruses naturally contain about 10 times less NA on the viral surface compared to HA, and since established processes for concentrating HA antigens may not be suitable for maintaining NA in its enzymatically active and tetrameric conformation, the amount of NA detectable in a vaccine composition, such as an inactivated virus vaccine, may vary considerably. Thus, the addition of recombinant NA or mRNA encoding NA to a vaccine or immunogenic composition as disclosed herein may allow better control over the amount of NA contained in the vaccine or immunogenic composition. The creation of stable NA by recombinant or through mRNA encoding NA and its addition to an HA antigen, such as a recombinantly produced HA antigen or mRNA encoding HA antigen, may allow a better balancing of both HA and NA immune responses in subjects receiving the vaccine or immunogenic composition when compared to currently available vaccines, which may in turn provide enhanced and / or broader protection against circulating influenza strains.

[0038] Thus, disclosed herein are hybrid multivalent influenza vaccines or immunogenic compositions comprising influenza virus HA or NA in addition to one or more ribonucleic acid encoding one or more influenza virus HA or NA, including, for example, hybrid multivalent influenza vaccine compositions comprising one or more ribonucleic acid (e.g., mRNA) molecules encoding influenza virus HA (e.g., recombinant HA) and one or more influenza virus NA proteins.

[0039] definition In order to make this disclosure more readily understandable, certain terms are first defined below. Throughout this specification, additional definitions of the following terms and other terms may be set forth. In the event that a definition of a term set forth below conflicts with a definition in an application or patent incorporated by reference, the meaning of the term shall be understood using the definition set forth in this application.

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

[0041] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in and of itself, imply any importance, priority or order of one claim element relative to another claim element, or the temporal order in which operations of a method are performed, but is merely used as a label (but for the purposes of the use of ordinal terms) to distinguish one claim element having a certain name from another claim element having the same name, in order to distinguish the claim elements.

[0042] Adjuvant: As used herein, the term "adjuvant" refers to a substance or combination of substances that can be used to enhance the immune response to an antigenic component of a vaccine or immunogenic composition.

[0043] Antigen: As used herein, the term "antigen" refers to an agent that elicits an immune response when exposed to or administered to an organism; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell). In some embodiments, the antigen elicits a humoral response in the organism (e.g., including production of antigen-specific antibodies); alternatively or additionally, in some embodiments, the antigen elicits a cellular response in the organism (e.g., involving T cells whose receptors specifically interact with the antigen). One of skill in the art will appreciate that a particular antigen may elicit an immune response in one or more members of a target organism (e.g., mice, ferrets, rabbits, primates, humans), but not in all members of the target organism's 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%, 99% of members of the target species. In some embodiments, the antigen binds to an antibody and / or a T cell receptor and may or may not induce a specific physiological response in the organism. In some embodiments, for example, the antigen may bind to an antibody and / or a T cell receptor in vitro, regardless of whether such interactions occur in vivo. In some embodiments, the antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. Antigens include forms of NA and HA as described herein.

[0044] Approximately: As used herein, when applied to one or more subject values, the term "approximately" or "about" refers to a value similar to a stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise specified or clear from the context (except where such number exceeds 100% of possible values).

[0045] Carrier: As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. In some exemplary embodiments, the carrier may include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. In some embodiments, the carrier is or includes one or more solid ingredients.

[0046] Epitope: As used herein, the term "epitope" includes any moiety that is specifically recognized, in whole or in part, by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts a relevant three-dimensional structure. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a structure. In some embodiments, at least some of such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative structure (e.g., linearized).

[0047] Excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to impart or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, sorbitol, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0048] H1: As used herein, "H1" refers to influenza virus subtype 1 hemagglutinin (HA). Influenza A viruses are further divided into groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refer to classification of viruses based on the sequences of two proteins on the surface of the virus, HA and neuraminidase (NA). Currently, 18 HA subtypes (H1-H18) are recognized. Thus, H1 is distinct from other HA subtypes, including H2-H18.

[0049] H3: As used herein, "H3" refers to influenza virus subtype 3 HA. H3 is therefore distinct from other HA subtypes, including H1, H2, and H4-H18.

[0050] Immune response: As used herein, the term "immune response" refers to the reaction 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 cell of the body involved in a 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 proliferation and / or activity of lymphocytes (such as B cells or T cells), measuring secretion of cytokines or chemokines, measuring inflammation, measuring antibody production, and the like. An antibody response or humoral response is an immune response in which antibodies are produced. A "cellular immune response" is one that is mediated by T cells and / or other white blood cells.

[0051] Immunogen: As used herein, the term "immunogen" or "immunogenic" refers to a compound, composition, or substance that, under appropriate conditions, is capable of stimulating an immune response, such as the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. As used herein, the term "immunogenic composition" refers to a composition that produces an immune response that may or may not be a protective immune response. As used herein, "immunizing" means inducing a protective immune response in a subject against an infectious disease (e.g., influenza).

[0052] Immunologically effective amount: As used herein, the term "immunologically effective amount" means an amount sufficient to immunize a subject.

[0053] In some embodiments: As used herein, the term "in some embodiments" refers to embodiments of all aspects of the present disclosure, unless the context clearly dictates otherwise.

[0054] Machine learning: As used herein, the term "machine learning" refers to the use of algorithms that improve automatically through experience and / or the use of data. Machine learning can include building predictive models, such as models of influenza antigenicity, to enable prediction of data, including the use of algorithms designed to select candidate antigens through predictive models. Target strains can be identified and then selection algorithms can be built. Examples of machine learning algorithms and methods can be found, for example, in PCT applications WO 2021 / 080990A1 (entitled Systems and Methods for Designing Vaccines) and WO 2021 / 080999A1 (entitled Systems and Methods for Predicting Biological Responses), both of which are incorporated herein by reference in their entireties. Machine learning, as used herein, can also include the application of computational tools to analyze and interpret data, for example, bioinformatics analysis, such as phylogenetic analysis. Similarly, "machine learning influenza virus HA" refers to an influenza virus HA identified or designed by machine learning, and "machine learning influenza virus NA" refers to an influenza virus NA identified or designed by machine learning. "Machine learning model" refers to a model that uses an algorithm that improves automatically through experience and / or by use of data to predict data, such as candidate antigens.

[0055] Modified: As used herein, the term "modified," such as modified HA or NA, refers to any HA or NA protein or nucleic acid that has a different amino acid or nucleic acid sequence when compared to the wild-type form of the protein or nucleic acid. For example, modified influenza NA refers to influenza NA that has an amino acid or nucleic acid sequence that is different from the wild-type NA protein or nucleic acid sequence. Modified influenza NA can include one or more amino acid deletions and / or substitutions compared to wild-type influenza NA.

[0056] Monomeric influenza virus neuraminidase: Wild-type influenza virus neuraminidase (NA) is a tetramer of four identical monomers. Each NA monomer in wild-type influenza NA consists of four distinct structural domains: an enzymatic head region, a stalk region, a transmembrane region, and a cytoplasmic tail. As used herein, the term "monomeric influenza virus neuraminidase" refers to an NA monomer that can combine with three other NA monomers to form a tetrameric NA. As described herein, a modified monomeric influenza virus neuraminidase can include the head region of influenza virus NA, but includes a heterologous tetramerization domain or a fraction thereof, and / or lacks at least a portion of one or more of the cytoplasmic tail, the transmembrane region, and the stalk region.

[0057] N1: As used herein, "N1" refers to influenza virus subtype 1 neuraminidase (NA). Influenza A viruses are classified into groups 1 and 2. Groups 1 and 2 are further classified into subtypes, which refers to classification of viruses based on the sequence of two proteins on the surface of the virus, HA and neuraminidase (NA). Currently, eleven NA subtypes (N1-N11) are recognized. Thus, N1 is distinct from other NA subtypes, including N2-N11. N2: As used herein, "N2" refers to influenza virus subtype 2 neuraminidase (NA). Thus, N2 is distinct from other NA subtypes, including N1 and N3-N11.

[0058] Influenza B strains are divided into two lineages: B / Yamagata and B / Victoria.

[0059] Pandemic strain: A "pandemic" influenza strain is one that has caused or is capable of causing a pandemic infection in a population of subjects, such as a human population. In some embodiments, a pandemic strain is causing a pandemic infection. In some embodiments, such a pandemic infection includes epidemic infection across multiple regions, and in some embodiments, a pandemic infection includes infection across regions that are separated from each other (e.g., by mountains, by bodies of water, as parts of separate continents, etc.) such that infection would not normally be transmitted between them.

[0060] Prevention: The term "prevention," as used herein, refers to preventing, avoiding disease onset, delaying the onset, and / or reducing the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., infection with an influenza virus). In some embodiments, prevention is assessed on a population basis, and an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the occurrence, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition.

[0061] Recombinant: As used herein, the term "recombinant" is intended to refer to a polypeptide that is designed, engineered, prepared, expressed, produced or isolated by recombinant means (e.g., an HA and / or NA polypeptide described herein), e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide isolated from a recombination, combinatorial polypeptide library, or a polypeptide prepared, expressed, produced or isolated by any other means, including splicing selected sequence elements together. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources. In some embodiments, one or more of such selected sequence elements result from a combination of multiple (e.g., two or more) known sequence elements that do not naturally occur in the same polypeptide (e.g., two epitopes from two separate HA or NA polypeptides). The recombinant HA is rHA and the recombinant NA is rNA.

[0062] Seasonal strain: A "seasonal" influenza strain is one that has caused or is capable of causing seasonal infections (e.g., annual epidemics) in a population of interest, such as a human population. In some embodiments, a seasonal strain is causing seasonal infections.

[0063] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is often measured in terms of the percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. Homologs or variants of a given gene or protein have a relatively high degree of sequence identity when aligned using standard methods.

[0064] The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may, but do not necessarily, be randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually performed by comparing said sequences over segments or "windows of comparison" after optimal alignment in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs which employ said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0065] The percentage of identity is obtained by determining the number of corresponding identical positions in the sequences being compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence) and multiplying the result by 100.

[0066] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, in consecutive nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0067] A nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional and / or structural characteristic of the given sequence, e.g., in some instances, is functionally and / or structurally equivalent to the given sequence. In some embodiments, a nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally and / or structurally equivalent to the given sequence.

[0068] Standard care strain: The World Health Organization (WHO) selects influenza strains to be included in seasonal vaccine formulations each year based on intensive surveillance efforts. As used herein, the term "standard care strain" or "SOC strain" refers to influenza strains selected by the World Health Organization (WHO) for inclusion in seasonal vaccine formulations. Standard care strains can include past standard care strains, current standard care strains, or future standard care strains.

[0069] Subject: As used herein, the term "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human. In some embodiments, "subject" refers to a non-human animal. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the non-human subject is a mammal (e.g., a rodent, mouse, rat, rabbit, ferret, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, the subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In some embodiments, the subject is an adult, an adolescent, or an infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject."

[0070] Tetrameric NA molecule: As used herein, the term "tetrameric NA molecule" refers to a compound containing four NA monomer polypeptide units. In some embodiments, each monomeric NA molecule in a certain tetrameric NA compound contains a globular head domain, a stalk region, a hydrophobic transmembrane domain, and a short N-terminal cytoplasmic domain. In some embodiments, one or more of these domains or regions of a given monomeric NA molecule are shortened, absent, or modified compared to a reference wild-type monomeric NA molecule.

[0071] Tetramerization domain: As used herein, the term "tetramerization domain" refers to an amino acid sequence that encodes a domain that results in tetrameric assembly of a polypeptide or protein. A tetramerization domain that is not native to a particular protein may be referred to as an artificial or heterologous tetramerization domain. Exemplary tetramerization domains include, but are not limited to, sequences from Tetrabrachion, GCN4 leucine zipper, or vasodilator-stimulated phosphoprotein (VASP).

[0072] Vaccine composition: As used herein, the term "vaccine composition" or "vaccine" refers to a composition that generates a protective immune response in a subject. As used herein, a "protective immune response" refers to an immune response that protects a subject from infection (prevents infection or prevents the development of a disease associated with infection) or reduces the symptoms of infection (e.g., infection with influenza virus). Vaccines can induce both prophylactic (preventive) and therapeutic responses. Methods of administration vary depending on the vaccine, but can include inoculation, ingestion, inhalation, or other forms of administration. Inoculation can be delivered by any of a number of routes, including intravenously, subcutaneously, intraperitoneally, intradermally, intranasally, by inhalation, or parenterally, such as intramuscularly. Vaccines can be administered with adjuvants to enhance the immune response.

[0073] Vaccination: As used herein, terms such as "vaccinate" refer to the administration of a vaccine composition to generate a protective immune response in a subject, e.g., a response against a disease-causing pathogen, such as an influenza virus. Vaccination can occur before, during, and / or after exposure to the disease-causing pathogen, and / or before, during, and / or after the onset of one or more symptoms, in some embodiments before, during, and / or immediately after exposure to the pathogen. In some embodiments, vaccination involves multiple administrations of the vaccine composition at appropriate time intervals.

[0074] Vaccine efficacy: As used herein, the term "vaccine efficacy" or "vaccine effectiveness" refers to a measure of the percentage reduction in evidence of disease among subjects administered a vaccine composition. For example, 50% vaccine efficacy indicates a 50% reduction in the number of disease cases among a group of vaccinated subjects when compared to a group of non-vaccinated subjects or a group of subjects administered a different vaccine.

[0075] Wild-type (WT): As understood in the art, the term "wild-type" generally refers to the normal form of a protein or nucleic acid as found in nature. For example, wild-type HA and NA polypeptides are found in natural isolates of influenza viruses. A variety of different wild-type HA and NA sequences can be found in the NCBI influenza virus sequence database.

[0076] Nomenclature for influenza viruses All nomenclatures used to classify influenza viruses are those commonly used by those skilled in the art. Thus, influenza virus types or groups refer to three major types of influenza: influenza A, influenza B, or influenza C, which infect humans. Influenza A and B cause significant morbidity and mortality annually. It is understood by those skilled in the art that the designation of a virus as a particular type is related to sequence differences in the respective M1 (matrix) protein or P (nucleoprotein). Influenza A viruses are further divided into groups 1 and 2. These groups are further divided into subtypes, which refers to classification of the virus based on the sequences of two proteins on the surface of the virus, HA and NA. Currently, there are 18 recognized HA subtypes (H1-H18) and 11 recognized NA subtypes (N1-N11). Group 1 contains N1, N4, N5, and N8, and H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18. Group 2 contains N2, N3, N6, N7, and N9, and H3, H4, H7, H10, H14, and H15. N10 and N11 have been identified in influenza-like genomes isolated from bats (Wu et al., Trends in Microbiology, 2014, 22(4):183-91). There are potentially 198 different influenza A subtype combinations, but only about 131 subtypes have been detected in nature. Current subtypes of influenza A viruses commonly circulating in human populations that cause seasonal pandemics include A(H1N1) and A(H3N2).

[0077] Influenza A subtypes can be further divided into different genetic "clades" and "subclades". For example, A subtype A(H1N1) contains clade 6B.1 and subclade 6B.1A. A subtype A(H3N2) contains clades 3C.2A and 3C.3A, and subclades 3C.2A1, 3C.2A2, 3C2A3, and 3C.2A4. Similarly, B subtype Victoria contains clade V1A and subclades V1A.1, V1A.2, and V1A.3, while B subtype Yamagata contains clades Y1, Y2, and Y3. Finally, the term strain refers to viruses within a subtype that differ from each other in having minor genetic variations in their genomes.

[0078] For convenience, certain abbreviations may be used to refer to the protein constructs and parts thereof described herein. For example, HA may refer to influenza hemagglutinin protein. H1 refers to HA from influenza subtype 1 strain. H3 refers to HA from influenza subtype 3 strain. Similarly, NA may refer to influenza neuraminidase protein or parts thereof. N2 refers to neuraminidase from influenza subtype 2 strain. The term tet-NA or rTET-NA refers to recombinant NA containing a heterologous tetramerization domain that forms tetrameric NA when expressed in cells. HA refers to hemagglutinin or parts thereof.

[0079] Hemagglutinin (HA) Hemagglutinin (HA), along with NA, is one of the two major influenza surface proteins. The function of both NA and HA involves interaction with sialic acid, a terminal molecule that binds to sugar moieties on glycoproteins or glycolipids expressed on the surface of cells. Binding of HA to sialic acid on the cell surface induces endocytosis of the virus by the cell, allowing the virus to enter and infect the cell. Sialic acid is also added to HA and NA as part of the glycosylation process that occurs within the infected cell.

[0080] HA is thought to mediate influenza virus attachment to host cells and virus-cell membrane fusion during viral penetration into cells. Antigenic variation in the HA molecule is responsible for frequent influenza epidemics and limited control of infection by immunization.

[0081] HA exists as a trimer in mature influenza viruses. Each HA monomer consists of two polypeptides (HA1 and HA2) linked by disulfide bonds. These polypeptides are derived by cleavage of a single precursor protein, HA0, during influenza virus maturation. In part because these molecules are tightly folded, HA0 and mature HA1 and HA2 differ slightly in their conformational and antigenic characteristics. Furthermore, HA0 is more stable and resistant to denaturation and proteolysis.

[0082] Isolation, propagation and purification of influenza virus strains for cloning the desired HA gene can be carried out by any method known in the art, such as the method disclosed in U.S. Pat. No. 5,762,939, incorporated herein by reference.

[0083] The methods and compositions of the disclosure may involve the use of any form of HA, including wild-type HA, modified non-wild-type HA, HA derived from a seasonal or pandemic influenza virus strain, recombinant HA, HA present in inactivated influenza virus (IIV) and / or reassortant viruses, HA having a molecular sequence identified or designed from a machine learning model, HA encoded by a ribonucleic acid molecule, and / or any other form of HA known in the art.

[0084] The primary HA gene product is a full-length unprocessed HA (rHA0), which is not secreted but remains associated with the peripheral membrane of infected cells. In insect cells, this rHA0 is glycosylated with N-linked high-mannose glycans, and there is evidence that rHA0 forms trimers post-translationally and then accumulates in the cytoplasmic plasma membrane.

[0085] The rHA0 can be selectively extracted from the peripheral membrane using a non-denaturing non-ionic detergent or other methods known in the art for purifying recombinant proteins from cells, e.g., insect cells, such as affinity or gel chromatography, antigen binding, DEAE ion exchange, or lentil lectin affinity chromatography. The purified rHA0 can then be resuspended in an isotonic buffer solution. In certain embodiments, the rHA0 is purified to at least about 80%, e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0086] Influenza virus HA proteins disclosed herein include influenza virus HA present in inactivated virions. In certain embodiments, the influenza virus HA protein is present in a reassortant virus. In certain embodiments, the inactivated and / or reassortant virus is a split inactivated virus. In certain embodiments, influenza virus HA present in an inactivated and / or reassortant virus is disclosed herein, wherein the HA is selected from an H1 HA from a standard care influenza virus, an H3 HA from a standard care influenza virus, an HA from a standard care influenza virus strain from the B / Victoria lineage, or an HA from a standard care influenza virus from the B / Yamagata lineage.

[0087] In certain embodiments disclosed herein, the influenza virus HA is one or more machine learning influenza virus HAs, such as recombinant machine learning influenza virus HAs having molecular sequences identified or designed from a machine learning model. In certain embodiments, the machine learning recombinant influenza virus HAs can be selected from one or more of H1 HA, H3 HA, HA from the B / Victoria lineage, HA from the B / Yamagata lineage, or combinations thereof. In selecting the one or more machine learning influenza virus HAs, any machine learning algorithm or model can be used, including, for example, those described herein.

[0088] Influenza virus HA disclosed herein may be formulated and packaged alone or in combination with other influenza virus HA antigens and / or influenza virus NA as discussed below. In certain embodiments, the vaccine or immunogenic composition comprises one, two, three, four, five, six, seven, eight or more influenza virus HA antigens. In certain embodiments, the vaccine or immunogenic composition comprises four influenza virus HAs to create a quadrivalent vaccine or immunogenic composition. In certain embodiments, four influenza virus HAs are formulated with four influenza virus NA antigens to create an octavalent vaccine or immunogenic composition. In certain embodiments, four influenza virus HA antigens, such as four recombinant influenza virus HA antigens, are formulated with ribonucleic acid molecules encoding four influenza virus NA antigens to create an octavalent vaccine or immunogenic composition.

[0089] The influenza virus HA present in the vaccine or immunogenic composition disclosed herein may include influenza virus HA from a standard of care influenza virus strain and / or any combination of machine learning influenza virus HA as disclosed herein. For example, in certain embodiments, the influenza virus HA may be a wild type influenza HA, a non-wild type influenza HA, an HA from a seasonal or pandemic influenza virus strain, and / or any other form of influenza HA known in the art. In certain embodiments, a recombinant influenza virus HA is disclosed herein, where the HA is selected from an H1 HA from a standard of care influenza virus, an H3 HA from a standard of care influenza virus, an HA from a standard of care influenza virus strain from the B / Victoria lineage, or an HA from a standard of care influenza virus from the B / Yamagata lineage.

[0090] In certain embodiments disclosed herein, the influenza virus HA is from a pandemic strain or a strain with pandemic potential, including, for example, H1, H2, H3, H5, H7, H9 and / or H10.

[0091] Hemagglutinin activity can be measured using techniques known in the art, including, for example, hemagglutinin inhibition assay (HAI). HAI applies the process of hemagglutination, called hemagglutination, in which sialic acid receptors on the surface of red blood cells (RBCs) bind to hemagglutinin glycoproteins found on the surface of influenza viruses (and some other viruses), creating a network or lattice structure of interconnected RBCs and virus particles that occurs in a concentration-dependent manner on the virus particles. This is a physical measurement taken as a proxy for the ability of the virus to bind to similar sialic acid receptors on cells that target pathogens in the body. The introduction of anti-viral antibodies generated in a human or animal immune response to another virus (which may be genetically similar or different from the virus used to bind to RBCs in the assay) alters the concentration of the virus enough to disrupt the virus-RBC interaction and change the concentration at which hemagglutination is observed in the assay. One goal of HAI can be to characterize the concentration of antibodies in an antiserum or other sample that contains the antibodies, relative to their ability to inhibit hemagglutination in the assay. The highest dilution of antibody that prevents hemagglutination is called the HAI titer (ie, the measured response).

[0092] Another approach to measure HA antibody responses is to measure a potentially larger set of antibodies elicited by human or animal immune responses, which may not necessarily affect hemagglutination in HAI assays. A common approach for this is to utilize ELISA technology, where a viral antigen (e.g., hemagglutinin) is immobilized on a solid surface, and then antibodies from antisera are allowed to bind to the antigen. The readout measures the catalysis of an exogenous enzyme substrate conjugated to either the antibodies from the antisera, or to other antibodies that themselves bind to the antibodies of the antisera. Catalysis of the substrate produces a readily detectable product. There are many variations of this type of in vitro assay. One such variation is called antibody forensics (AF), a multiplex bead array technique that allows a single serum sample to be measured simultaneously against many antigens. These measurements characterize concentration and total antibody recognition when compared to HAI titers, which are believed to be more specifically related to interference with sialic acid binding by the hemagglutinin molecule. Thus, antisera antibodies may in some cases have proportionally higher or lower measurements than the corresponding HAI titers for the hemagglutinin molecules of one virus compared to the hemagglutinin molecules of another virus; in other words, these two measurements, AF and HAI, may not be linearly correlated.

[0093] Another method of measuring HA antibody responses involves virus neutralization assays (e.g., microneutralization assays), where antibody titers are measured in permissive cell cultures after incubating virus with serial dilutions of antibody / serum samples, by reduction in plaques, foci and / or fluorescent signal, depending on the specific neutralization assay technique.

[0094] Each influenza virus HA may be included in the compositions disclosed herein in an amount effective to induce an immune response in a subject to which the composition is administered. In certain embodiments, each influenza virus HA is present in the vaccine or immunogenic composition disclosed herein in an amount ranging from about 0.1 μg to about 500 μg, such as about 5 μg to about 120 μg, about 1 μg to about 60 μg, about 10 μg to about 60 μg, about 15 μg to about 60 μg, about 40 μg to about 50 μg, about 42 μg to about 47 μg, about 5 μg to about 45 μg, about 15 μg to about 45 μg, about 0.1 μg to about 90 μg, about 5 μg to about 90 μg, about 10 μg to about 90 μg, or about 15 μg to about 90 μg. In certain embodiments, each recombinant HA may be present in a vaccine or immunogenic composition disclosed herein in an amount of about 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg or about 90 μg.

[0095] Neuraminidase (NA) Along with HA, neuraminidase (NA) is the second major influenza surface protein. NA removes sialic acid from cellular glycoproteins and glycolipids and from newly synthesized HA and NA on nascent virions. Removal of sialic acid by NA promotes efficient release of virus particles from the surface of infected cells by preventing their aggregation. It also prevents the virus from binding via HA to already infected dead cells, which promotes further spread of the viral infection. When NA is present in an immunogenic form, either in conventional vaccines or on intact virions, it is a minor component and is therefore subordinate to continuing antigenic competition with the immunodominant HA. Due to a competitive mechanism, immunogenic responses to NA appear to be partially suppressed in favor of the more frequently occurring HA antigens (Johanssen et al., Immunologic response to influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin, J. IMMUNOL. 1987;139(6):2010-2014; and Kilbourne, Comparative Efficacy of Neuraminidase-Specific and Conventional Influenza Virus Vaccines in Induction of Antibody to Neuraminidase in Humans, J. INFECT. DIS. 1976;134(4):384-94). As a result, the effects of NA immunity can generally be masked by neutralizing HA antibodies.

[0096] The methods and compositions of the present disclosure may include the use of any form of NA, including wild-type NA, modified, non-wild-type NA, NA from seasonal or pandemic influenza virus strains, recombinant NA, NA present in IIV and / or reassortant viruses, NA having a molecular sequence identified or designed from a machine learning model, NA encoded by a ribonucleic acid molecule, and / or any other form of NA known in the art.

[0097] A wild-type influenza virus neuraminidase The compositions and methods disclosed herein may, in certain embodiments, include the use of a tetrameric NA polypeptide comprising four wild-type monomeric NA molecules. NA is a type II transmembrane glycoprotein that assembles on the viral surface as a tetramer of four identical monomers. The molecular weight of the wild-type monomer is generally about 55-72 kDa, depending on the influenza subtype; the molecular weight of the tetramer is generally about 240-260 kDa, depending on the influenza subtype. Each monomer consists of four distinct structural domains: an enzymatic head region, a stalk region, a transmembrane region, and a cytoplasmic tail. The largest domain is the head region, which is anchored to the viral membrane by the stalk region, which is connected to the transmembrane region and finally to the N-terminal cytoplasmic domain.

[0098] The stalk region among different influenza A virus subtypes, including N1 and N2, can vary significantly in size and amino acid structure (Blok et al., Variation in the membrane-insertion and 'stalk' sequences in eight subtypes of influenza type A virus neuraminidase, BIOCHEMISTRY 1982, 21(17):4001-4007). Differences in stalk length are thought to modulate the distance of the enzymatic head domain and affect the ability of NA to access sialic acids on cell surface receptors, with shorter stalk regions correlating with lower sialidase activity (Da Silva et al., Assembly of Subtype 1 Influenza Neuraminidase is Driven by Both the Transmembrane and Head Domains, J BIOL CHEM 2013, 288(1):644-53; and McAuley et al., Influenza Virus Neuraminidase Structure and Functions, FRONTIERS IN MICROBIOLOGY 2019, 10(39)). Despite variability between the stalk regions of different subtypes, the NA stalk regions also share several structural features, including at least one cysteine ​​residue and a potential glycosylation site. Cysteine ​​residues may participate in the formation of disulfide bonds between NA monomers, helping to form stabilized NA tetramers, while glycosylation sites may contribute to tetramer stabilization (McAuley et al., 2019). For example, the conserved cysteine ​​residue at amino acid position 78 of N2 NA is thought to be involved in the tetramer assembly mechanism (Shtyrya et al., Influenza virus neuraminidase: structure and function, ACTA NATURAE 2009;1(2):26-32).

[0099] The enzymatic head region is composed of four monomers. Each monomer in the head forms a conserved six-bladed propeller structure. Each blade has four antiparallel β-sheets stabilized by disulfide bonds and connected by loops of various lengths. McAuley et al.,2019. Tetramerization of monomers is important for the formation of the active site and the synthesis of enzymatically active NA. Dai et al.,Identification of Residues That Affect Oligomerization and / or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins,J.VIROLOGY 2016,90(20):9457-70.

[0100] Although the amino acid sequence and length of NA can vary significantly between different influenza A virus NA subtypes, such as N1 and N2, and especially between the NA stalk regions of different influenza A virus NA subtypes, the length of the amino acid sequence of N2 from different influenza strains is generally about 469 amino acids, with some strains having about one or two (or more) amino acid insertions or deletions, generally in the head region. When referring to specific amino acid residues in wild-type N2, the specific amino acid residue numbers are based on N2 numbering as understood in the art. The N-terminal cytoplasmic tail generally corresponds to amino acids 1-6 of the wild-type N2 sequence, while the transmembrane domain generally corresponds to amino acids 7-35 of the wild-type N2 sequence. For example, in the wild-type NA sequence of strain A / PERTH / 16 / 2009 (SEQ ID NO:1), the cytoplasmic region corresponds to amino acids 1-6 of SEQ ID NO:1, while the transmembrane region corresponds to amino acids 7-35 of SEQ ID NO:1. The N2 stalk region is generally about 46 amino acids in length, beginning at approximately amino acid 36 and ending at approximately amino acid 82 of the wild-type N2 sequence. For example, in the wild-type N2 sequence of strain A / PERTH / 16 / 2009 (SEQ ID NO: 1), the stalk region corresponds to approximately amino acid 36 to amino acid 82 of SEQ ID NO: 1. However, the exact boundary between the end of the N2 stalk region and the beginning of the N2 head region has not been resolved by x-ray crystallography.

[0101] b. Recombinant and / or modified influenza virus neuraminidase The methods and compositions of the present disclosure may include the use of modified forms of influenza virus NA, including modified recombinant NA, modified NA having a molecular sequence identified or designed from a machine learning model, and / or modified NA encoded by a ribonucleic acid molecule.

[0102] In certain embodiments, the influenza virus NA comprises four modified monomeric NA molecules that form a soluble tetrameric NA when expressed in a host cell. In one aspect, the modified monomeric NA molecule comprises the head region and heterologous oligomerization domain of the influenza virus NA, but lacks at least a portion of one or more of the cytoplasmic tail, transmembrane region, and stalk region of the influenza virus NA.

[0103] For example, the modified monomeric NA may include a heterologous tetramerization domain that replaces one or more of the cytoplasmic tail, transmembrane region, and stalk region of the influenza virus NA, or replaces all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region of the influenza virus NA. In certain embodiments, the heterologous tetramerization domain is a tetramerization domain, for example, as disclosed in US Patent Application Publication No. 2013 / 0034578, which is hereby incorporated by reference in its entirety. Schmidt et al.,PLos ONE,2011,6(2):e16284;Da Silva et al.,J Biol Chem,2013,288(1):644-53;Dai et al.,2016,J.Virology,90(20):9457-70;Bosch et al. al., 2010, J. Virology, 84(19):10366-74; see also Prevato et al., 2015, PLos ONE, 10(8): e0135474. In other embodiments, the heterologous tetramerization domain is a peptide found at the extreme C-terminus of a lamprey VLR-B antibody (i.e., the domain referred to as "C-TERM" in Figure 11C of WO 2008 / 016854, which is hereby incorporated by reference in its entirety) as described in WO 2016 / 097769, which is hereby incorporated by reference in its entirety, such as SEQ ID NO: 1 or SEQ ID NO: 2 of WO 2016 / 097769.

[0104] In certain embodiments, the modified monomeric influenza virus NA comprises a signal peptide, a heterotetramerization domain and a head region of an influenza virus NA, and expression of the modified monomeric influenza virus NA in a host cell results in secretion of a tetrameric NA.

[0105] The wild-type NA protein is a membrane-bound protein that contains a transmembrane domain. To create a soluble NA protein, the transmembrane domain can be deleted and a signal peptide can be added. The signal peptide targets the recombinant NA protein to the secretory pathway so that the recombinant NA protein is secreted from the host cell in which the recombinant NA is expressed. When the modified monomeric NA nucleic acid is translated into a polypeptide inside the host cell, the polypeptide contains a signal peptide. However, during post-translational processing, the signal peptide is cleaved, and the secreted polypeptide no longer contains the signal peptide. Although the modified monomeric NA itself may contain a signal peptide after translation to target the modified monomeric NA to the secretory pathway, the signal peptide is removed through post-translational processing, and the soluble tetrameric NA obtained from the host cell expressing the modified monomeric NA is composed of four modified NA monomers that no longer contain the signal peptide.

[0106] In certain embodiments, for example, the tetrameric NA comprises four modified monomeric influenza virus NAs, which comprise an influenza virus NA head region and a heterologous tetramerization domain.

[0107] In certain embodiments, all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region of an influenza virus NA may be replaced by a signal peptide and a heterologous tetramerization domain. A modified NA comprising a heterologous tetramerization domain may lack the entire NA stalk region, or it may lack substantially all of the NA stalk region, i.e., the modified NA construct may include the C-terminal portion of the NA stalk region. For example, a modified NA comprising a heterologous tetramerization domain may include about 1-13 of the most C-terminal amino acids of the NA stalk region. As understood in the art, the most C-terminal amino acid of the stalk region is the residue immediately adjacent to the NA head region. As a further example, a modified NA comprising a heterologous tetramerization domain construct may include 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 of the most C-terminal amino acids of the NA stalk region. As a further example, a modified NA containing a heterologous tetramerization domain construct may contain about eight of the most C-terminal amino acids of the NA stalk region.

[0108] In certain embodiments, the heterologous tetramerization domain is a Staphylothermus marinus tetrabrachion tetramerization domain, a GCN4 leucine zipper tetramerization domain, a paramyxovirus phosphoprotein tetramerization domain, or a human vasodilator-stimulated phosphoprotein (VASP) tetramerization domain.

[0109] As a further example, as disclosed in PCT International Application No. PCT / US2022 / 039980, which is hereby incorporated by reference in its entirety, it has been discovered that modified monomeric influenza virus subtype 2 neuraminidase (N2) lacking all or substantially all of the stalk domain can form soluble tetrameric NA when expressed in cells, even without the addition of a heterologous tetramerization domain. Although not all N2 strains lacking all or substantially all of the stalk domain produce detectable amounts of soluble tetrameric NA, most of the N2 strains tested produced detectable amounts of soluble tetrameric NA, indicating that the shortened stalk design strategy can be broadly applied to NA proteins from various N2 influenza strains. Depending on the N2 strain used, this modified monomeric NA design strategy can result in the production of predominantly tetrameric NA or a mixture of monomeric NA and tetramers when expressed in a host cell. Thus, certain N2 strains and certain stalk deletion mutants of specific N2 strains, when expressed in cells, produce higher yields of soluble tetrameric NA. In any instance, when such modified NA constructs are expressed in host cells, it may be desirable to purify the tetrameric NA produced.

[0110] As used herein, "substantially all of the stalk region" of influenza virus subtype 2 neuraminidase (N2) refers to amino acid 36 through at least amino acid 69 of the stalk region of influenza virus N2. Thus, a modified N2 lacking substantially all of the cytoplasmic tail, transmembrane region, and stalk region may lack amino acids 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, or 1-81 of influenza virus subtype 2 NA. In other words, a modified N2 as described herein may include up to the 13 most C-terminal amino acids of the stalk region of influenza virus subtype 2 NA, which generally refers to amino acids 70-82 of N2. In certain embodiments, the entire cytoplasmic tail, transmembrane region, and stalk region (e.g., amino acids 1-82) are removed from the modified N2.

[0111] In some embodiments, for example, a tetrameric NA comprises four modified influenza virus subtype 2 neuraminidase molecules, where the modified influenza virus neuraminidase comprises the head region of influenza virus neuraminidase and lacks all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region of influenza virus neuraminidase, where the tetrameric NA does not contain a heterologous tetramerization domain. In some of these embodiments, all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region of influenza virus neuraminidase are replaced by a signal peptide. The signal peptide is normally cleaved during post-translational processing such that the secreted NA polypeptide generally does not contain the signal peptide. In some of these embodiments, for example, amino acid 1 through at least amino acids 70-82 of wild-type N2 influenza virus NA may be replaced by a signal peptide. These modified N2 constructs, in which all or substantially all of the cytoplasmic domain, transmembrane domain and stalk region are replaced by a signal peptide and form tetrameric NAs when expressed in cells, are also described in further detail in PCT International Application No. PCT / US2022 / 039980, which is hereby incorporated by reference in its entirety.

[0112] The tetrameric NA molecules formed by these modified monomeric NAs are generally substantially soluble in a fluid sample and generally catalytically active (e.g., capable of enzymatically cleaving the glycosidic bond of neuraminic acid). However, the tetrameric NA molecules may also be catalytically inactive, for example due to mutations.

[0113] Neuraminidase activity can be measured using techniques known in the art, including, for example, the MUNANA assay, the ELLA assay, or the NA-Star® assay (ThermoFisher Scientific, Waltham, Mass.). In the MUNANA assay, 2'-(4-methylumbelliferyl)-alpha-DN-acetylneuraminic acid (MUNANA) is used as a substrate. Any enzymatically active neuraminidase contained in the sample cleaves the MUNANA substrate and releases 4-methylumbelliferone (4-MU), a fluorescent compound. Thus, the amount of neuraminidase activity in the test sample correlates with the amount of 4-MU released, which can be measured using fluorescence intensity (RFU, relative fluorescence units).

[0114] For the purpose of determining the neuraminidase activity of the soluble tetrameric NA of the present disclosure, the MUNANA assay should be performed using the following conditions: Mix the soluble tetrameric NA with buffer [33.3 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5), 4 mM CaCl2, 50 mM BSA] and substrate (100 μM MUNANA) and incubate at 37 ° C for 1 h with shaking; Stop the reaction by adding alkaline pH solution (0.2 M Na2CO3); Measure the fluorescence intensity using excitation and emission wavelengths of 355 and 460 nm, respectively; Calculate the enzyme activity relative to the 4MU reference. If necessary, an equivalent assay can be used to measure neuraminidase enzyme activity.

[0115] Also disclosed herein are one or more influenza virus NAs identified or designed using a machine learning model as described herein, including recombinant machine learning influenza virus NAs ("machine learning influenza virus NAs"). In certain embodiments, the machine learning influenza virus NAs may be selected from one or more of N1 NA, N2 NA, NA from the B / Victoria lineage, NA from the B / Yamagata lineage, or combinations thereof. Any machine learning algorithm or model may be used when selecting one or more machine learning influenza virus NAs, including, for example, those described herein.

[0116] As discussed herein, the influenza virus NA disclosed herein can be formulated and packaged, alone or in combination with other influenza virus NA antigens and / or influenza virus HA. In certain embodiments, the vaccine or immunogenic composition comprises one, two, three, four, five, six, seven, eight or more influenza virus NA antigens. In certain embodiments, the influenza virus NA is formulated with three additional influenza virus NA antigens to create a quadrivalent vaccine or immunogenic composition. In certain embodiments, four influenza virus NA are formulated with four influenza virus HA antigens to create an octavalent vaccine or immunogenic composition. In certain embodiments, ribonucleic acid molecules encoding four influenza virus NA antigens are formulated with four influenza virus HA antigens, such as four recombinant influenza virus HA antigens, to create an octavalent vaccine or immunogenic composition.

[0117] The influenza virus NA present in the vaccine or immunogenic composition disclosed herein may include any combination of influenza virus NA from a standard of care influenza virus strain and / or machine learning influenza virus NA as disclosed herein. For example, in certain embodiments, the influenza virus NA may be wild type influenza NA, non-wild type influenza NA, NA from a seasonal or pandemic influenza virus strain and / or any other form of influenza NA known in the art. In certain embodiments, recombinant influenza virus NA is disclosed herein, where the NA is selected from N1 NA from a standard of care influenza virus, N2 NA from a standard of care influenza virus, NA from a standard of care influenza virus strain from the B / Victoria lineage, or NA from a standard of care influenza virus from the B / Yamagata lineage.

[0118] In certain embodiments disclosed herein, the influenza virus NA is from a pandemic strain or a strain with pandemic potential, including, for example, N1, N2, N7 and / or N9.

[0119] Each influenza virus NA may be present in the compositions disclosed herein in an amount effective to induce an immune response in a subject to which the composition is administered. In certain embodiments, each influenza virus NA may be present in the vaccine or immunogenic composition disclosed herein in an amount ranging from, for example, 1 μg to about 500 μg, for example, from about 5 μg to about 120 μg, from about 1 μg to about 60 μg, from about 10 μg to about 60 μg, from about 15 μg to about 60 μg, from about 5 μg to about 45 μg, from about 15 μg to about 45 μg, from about 0.1 μg to about 90 μg, from about 5 μg to about 90 μg, from about 10 μg to about 90 μg, from about 15 μg to about 90 μg, from about 5 μg to about 25 μg, or from about 10 μg to about 20 μg, or from about 12 μg to 18 μg. In certain embodiments, each recombinant NA may be present in a vaccine or immunogenic composition disclosed herein in an amount of about 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg or about 90 μg.

[0120] Ribonucleic acid encoding HA or NA The vaccine or immunogenic composition disclosed herein comprises one or more ribonucleic acid molecules, e.g., mRNA molecules, encoding one or more influenza virus HAs or one or more influenza virus NAs, as disclosed herein. In certain embodiments, the ribonucleic acid molecules, e.g., mRNA, may encode influenza virus NAs, e.g., any one of a combination of N1 NA, N2 NA, NA from B / Victoria lineage, or NA from B / Yamagata lineage. In certain embodiments, the one or more ribonucleic acid molecules encode N1 NA, N2 NA, NA from B / Victoria lineage, and NA from B / Yamagata lineage. Generally, the ribonucleic acid molecules, e.g., mRNA, encode full-length NAs (e.g., wild-type or machine-learned NAs), although they may also encode modified NAs.

[0121] In certain embodiments, the ribonucleic acid molecule, such as an mRNA, may encode an influenza virus HA, such as any one of a combination of an H1 HA, an H3 HA, an HA from the B / Victoria lineage, or an HA from the B / Yamagata lineage. In certain embodiments, the one or more ribonucleic acid molecules encode an H1 HA, an H3 HA, an HA from the B / Victoria lineage, and an HA from the B / Yamagata lineage. Typically, the ribonucleic acid molecule, such as an mRNA, encodes a full-length HA (e.g., a wild-type or machine-generated HA), although they may also encode modified HAs.

[0122] In certain embodiments, the ribonucleic acid molecule is encapsulated in a lipid-nanoparticle (LNP).

[0123] Representative mRNAs and LNPs are disclosed, for example, in PCT application WO 2022 / 099003, entitled "Lipid Nanoparticles for Delivering mRNA Vaccines," the entire contents of which are incorporated by reference herein.

[0124] Any known LNP formulation may be used in the embodiments disclosed herein. In certain embodiments, LNPs include a mixture of four types of lipids: ionized (e.g., cationic) lipids, polyethylene glycol (PEG)-conjugated lipids, cholesterol-based lipids, and helper lipids such as phospholipids. LNPs are used to encapsulate ribonucleic acid molecules (e.g., mRNA). The encapsulated mRNA molecules may be composed of natural ribonucleotides, chemically modified nucleotides, or a combination thereof, and may individually or collectively code for one or more proteins.

[0125] Ionizable lipids facilitate mRNA encapsulation and may be cationic lipids, which provide a positively charged environment at low pH and facilitate efficient encapsulation of negatively charged mRNA drug substances.

[0126] Contemplated PEGylated lipids include C6-C PEG-Ceramides, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). 20 (For example, 8, C 10 , C 12 , C 14 , C 16 Or C 18 Examples of PEGylated lipids include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length covalently attached to lipids having alkyl chains up to 5 kDa in length. In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG); N,N-ditetradecylacetamide-polyethylene glycol (e.g., ALC-0159); or 1-monomethoxypolyethylene glycol-2,3-dimyristylglycerol (e.g., PEG2000-DMG).

[0127] PEG preferably has a high molecular weight, for example, 2000-2400 g / mol. In some embodiments, PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000 or C8 PEG2000. The PEGylated lipid component provides control over the particle size and stability of the nanoparticles. The addition of such components can prevent complex aggregation, increase circulation lifetime, and provide a means for increasing delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al., FEBS Letters (1990) 268(1):235-7). These components can be selected to be rapidly exchanged from the pharmaceutical composition in vivo (see, for example, U.S. Pat. No. 5,885,613).

[0128] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNPs include one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Pat. No. 5,744,335), imidazole cholesterol ester ("ICE"; WO 2011 / 068810), β-sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNPs is cholesterol.

[0129] The helper lipid enhances the structural stability of the LNP and aids the LNP in endosomal escape. It improves the uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is a zwitterionic lipid with fusogenic properties to enhance the uptake and release of the drug payload. In certain embodiments, the helper lipid is a phospholipid. Examples of helper lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE) and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0130] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or combinations thereof.

[0131] In certain embodiments disclosed herein, the LNPs comprise (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, GL-HEPES-E3-E12-DS-3-E14, ALC-0315, or SM-102; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0132] In certain embodiments disclosed herein, the LNPs comprise (i) ALC-0315 as a cationic lipid, (ii) N,N ditetradecylacetamido-polyethylene glycol (e.g., ALC-0159) as a PEGylated lipid, (iii) DSPC as a helper lipid, and (iv) cholesterol. In certain embodiments, the LNPs comprise (i) ALC-0315 as a cationic lipid in a molar ratio of about 25% to about 65%, e.g., about 46.3%; (ii) N,N ditetradecylacetamido-polyethylene glycol (e.g., ALC-0159) as a PEGylated lipid in a molar ratio of about 0.5% to about 2.6%, e.g., 1.6%; (iii) DSPC as a helper lipid in a molar ratio of about 5% to about 15%, e.g., 9.4%; and (iv) cholesterol in a molar ratio of about 20% to about 60%, e.g., 42.7%.

[0133] The molar ratios of the LNP components above may enhance the effectiveness of the LNP in delivering mRNA. The molar ratios of cationic lipid, PEGylated lipid, cholesterol-based lipid and helper lipid are A:B:C:D (where A+B+C+D=100%). In some embodiments, the molar ratio of cationic lipid in the LNP to total lipid (i.e., A) is 35-50%. In some embodiments, the molar ratio of PEGylated lipid component to total lipid (i.e., B) is 0.25-2.75%. In some embodiments, the molar ratio of cholesterol-based lipid to total lipid (i.e., C) is 20-50%. In some embodiments, the molar ratio of helper lipid to total lipid (i.e., D) is 5-35%. In some embodiments, the (PEGylated lipid+cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNP has a molar ratio of cationic lipid to helper lipid greater than 1.

[0134] To calculate the actual amount of each lipid contained in the LNP formulation, first, the molar amount of the cationic lipid is 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 the LNP). Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid.

[0135] In certain embodiments, the LNPs contain cationic lipids, PEGylated lipids, cholesterol-based lipids, and helper lipids in a molar ratio of 40:1.5:28.5:30. In further particular embodiments, the LNPs contain (i) 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 in a molar ratio of 40:1.5:28.5:30.

[0136] If desired, the LNP or LNP formulation may be multivalent. In some embodiments, the LNP may carry ribonucleic acid molecules (e.g., mRNAs) encoding two or more antigens, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens, from the same or different pathogens. For example, the LNP may carry multiple ribonucleic acid molecules (e.g., mRNAs), each encoding a different antigen; or may carry a polycistronic mRNA that can be translated into two or more antigens (e.g., each antigen coding sequence is separated by a nucleotide linker that codes for a self-cleaving peptide, such as a 2A peptide). LNPs carrying different ribonucleic acid molecules (e.g., mRNAs) typically contain (encapsulate) multiple copies of each mRNA molecule. For example, LNPs carrying or encapsulating two different ribonucleic acid molecules (e.g., mRNAs) typically carry multiple copies of each of the two different ribonucleic acid molecules (e.g., mRNAs).

[0137] In some embodiments, a single LNP formulation can include multiple species (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of LNPs, each of which carries a different ribonucleic acid molecule (eg, mRNA).

[0138] In some embodiments, a vaccine or immunogenic composition disclosed herein comprises a ribonucleic acid molecule encoding a polypeptide derived from one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) influenza virus proteins selected from H1 HA, H3 HA, HA from B / Victoria lineage, and / or HA from B / Yamagata lineage. In further embodiments, a vaccine or immunogenic composition disclosed herein contains four ribonucleic acid molecules (e.g., mRNA), where a first ribonucleic acid molecule encodes an H1 HA derived from a first standard of care influenza virus strain, a second ribonucleic acid molecule encodes an H3 HA derived from a second standard of care influenza virus strain, a third ribonucleic acid molecule encodes an HA derived from a third standard of care influenza virus strain from B / Victoria lineage, and a fourth ribonucleic acid molecule encodes an HA derived from a fourth standard of care influenza virus strain from B / Yamagata lineage.

[0139] In some embodiments, a vaccine or immunogenic composition disclosed herein comprises a ribonucleic acid molecule encoding a polypeptide derived from one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) influenza virus proteins selected from N1 HA, N2 HA, HA from B / Victoria lineage, and / or HA from B / Yamagata lineage. In further embodiments, a vaccine or immunogenic composition disclosed herein contains four ribonucleic acid molecules (e.g., mRNAs), a first ribonucleic acid molecule encoding an N1 HA derived from a first standard of care influenza virus strain, a second ribonucleic acid molecule encoding an N2 HA derived from a second standard of care influenza virus strain, a third ribonucleic acid molecule encoding an HA derived from a third standard of care influenza virus strain derived from B / Victoria lineage, and a fourth ribonucleic acid molecule encoding an HA derived from a fourth standard of care influenza virus strain derived from B / Yamagata lineage.

[0140] In certain embodiments, the vaccine or immunogenic composition disclosed herein may comprise one or more self-amplifying ribonucleic acids, such as one or more self-amplifying mRNAs encoding influenza virus HA or one or more self-amplifying mRNAs encoding influenza virus NA. Antigen expression from conventional mRNA is proportional to the number of mRNA molecules successfully delivered from the vaccine or immunogenic composition to a subject. However, self-amplifying mRNAs comprise genetically engineered replicons derived from self-replicating viruses, and therefore can be added to the vaccine or immunogenic composition in lower doses than conventional mRNAs while achieving comparable results.

[0141] The self-amplifying mRNA may encode any of the influenza virus HAs disclosed herein, including, for example, H3 HA, H1 HA, HA from B / Victoria lineage, and / or HA from B / Yamagata lineage. In certain embodiments, the self-amplifying mRNA may encode any of the influenza virus NAs disclosed herein, including, for example, N1 NA, N2 NA, NA from B / Victoria lineage, and / or NA from B / Yamagata lineage.

[0142] The ribonucleic acid molecule (e.g., mRNA) may be unmodified (i.e., containing only the natural ribonucleotides A, U, C, and / or G linked by phosphodiester bonds) or may be chemically modified (e.g., containing nucleotide analogs such as pseudouridine (e.g., N-1-methylpseudouridine), 2'-fluororibonucleotides, and 2'-methoxyribonucleotides, and / or phosphorothioate bonds). The ribonucleic acid molecule (e.g., mRNA) may include a 5' cap and a poly-A tail. In certain embodiments, the one or more ribonucleic acid molecules comprise one or more modified nucleotides, and in certain embodiments the one or more modified nucleotides are selected from pseudouridine, methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. In certain embodiments, all uridines in the ribonucleic acid molecule are replaced by pseudouridines, such as methylpseudouridine, such as 1N-methylpseudouridine.

[0143] Each ribonucleic acid molecule may be present in the compositions disclosed herein in an amount effective to induce an immune response in a subject to which the composition is administered. In certain embodiments, each ribonucleic acid molecule may be present in the vaccine or immunogenic composition disclosed herein in an amount ranging from, for example, about 0.1 μg to about 150 μg, e.g., about 5 μg to about 120 μg, about 10 μg to about 60 μg, or about 15 μg to about 45 μg. In certain embodiments, each ribonucleic acid molecule is present in the vaccine or immunogenic composition in an amount sufficient to encode influenza virus HA or NA, e.g., about 5 μg to about 120 μg, e.g., about 10 μg to about 60 μg, or about 15 μg to about 45 μg.

[0144] To stabilize the nucleic acid and / or LNP (e.g., to extend the shelf life of a vaccine or immunogenic composition), to facilitate administration of the LNP pharmaceutical composition, and / or to enhance in vivo expression of the nucleic acid, the nucleic acid and / or LNP may be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharma- ceutical acceptable excipients. Examples of such excipients are parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0145] The LNP compositions of the present disclosure may be provided in a frozen liquid form or in a lyophilized form. A variety of cryoprotectants may be used, including but not limited to sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant may comprise 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition includes trehalose, for example, 5-30% (e.g., 10%) (w / v). Once formulated with the cryoprotectant, the LNP composition may be frozen (or lyophilized and cryopreserved) at −20° C. to −80° C. The LNP composition may be provided to the patient in a buffered aqueous solution (thawed if previously frozen, or reconstituted in a buffered aqueous solution at the bedside if previously lyophilized). The buffer is preferably isotonic, e.g., suitable for intramuscular or intradermal injection. In some embodiments, the buffer is phosphate buffered saline (PBS).

[0146] Vaccine or immunogenic composition In certain embodiments, disclosed herein is a vaccine or immunogenic composition comprising: (i) one or more influenza virus proteins selected from one or more influenza virus HA proteins, one or more influenza virus NA proteins, or a combination thereof; and (ii) one or more ribonucleic acid molecules encoding one or more influenza virus proteins selected from one or more influenza virus HA proteins, one or more influenza virus NA proteins, or a combination thereof.

[0147] In a particular embodiment, the vaccine or immunogenic composition comprises one to eight (such as one, two, three, four, five, six, seven or eight) influenza virus proteins selected from influenza virus HA proteins, influenza virus NA proteins or a combination thereof, and one to eight (such as one, two, three, four, five, six, seven or eight) ribonucleic acid molecules encoding one to eight (such as one, two, three, four, five, six, seven or eight) influenza virus HA proteins, one to eight (such as one, two, three, four, five, six, seven or eight) influenza virus NA proteins, or in a particular embodiment, the vaccine or immunogenic composition is a pentavalent vaccine, such as a pentavalent vaccine or immunogenic composition comprising four influenza virus HA proteins and one ribonucleic acid molecule encoding an influenza virus NA protein, or comprising four influenza virus NA proteins and one ribonucleic acid molecule encoding an influenza virus HA protein. In a particular embodiment, the vaccine or immunogenic composition is a hexavalent vaccine or immunogenic composition. In certain embodiments, the vaccine or immunogenic composition is a 7-valent vaccine or immunogenic composition. In certain embodiments, the vaccine or immunogenic composition is an 8-valent vaccine or immunogenic composition. In certain embodiments, the vaccine or immunogenic composition is a 9-valent vaccine or immunogenic composition, a 10-valent vaccine or immunogenic composition, an 11-valent vaccine or immunogenic composition, a 12-valent vaccine or immunogenic composition, a 13-valent vaccine or immunogenic composition, a 14-valent vaccine or immunogenic composition, a 15-valent vaccine or immunogenic composition, or a 16-valent vaccine or immunogenic composition. In certain embodiments, the vaccine or immunogenic composition is a multivalent vaccine or immunogenic composition comprising more than 16 different influenza virus HA proteins, influenza virus NA proteins, and / or ribonucleic acid molecules encoding influenza virus HA and / or influenza virus NA proteins.

[0148] In certain embodiments, the vaccine or immunogenic composition comprises 1 to 8 (such as 1, 2, 3, 4, 5, 6, 7 or 8) types of influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from the B / Victoria lineage, influenza virus HA from the B / Yamagata lineage, influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from the B / Victoria lineage or influenza virus NA from the B / Yamagata lineage. In certain embodiments, the vaccine or immunogenic composition comprises one or more ribonucleic acid molecules encoding one to eight (such as one, two, three, four, five, six, seven or eight) influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from the B / Victoria lineage, influenza virus HA from the B / Yamagata lineage, influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from the B / Victoria lineage or influenza virus NA from the B / Yamagata lineage.

[0149] In a particular embodiment, the vaccine or immunogenic composition comprises (i) at least four, e.g., four, influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, influenza virus HA from B / Yamagata lineage, influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from B / Victoria lineage, and influenza virus NA from B / Yamagata lineage; and (ii) one or more ribonucleic acid molecules encoding not more than four, e.g., four, influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, influenza virus HA from B / Yamagata lineage, influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from B / Victoria lineage, and influenza virus NA from B / Yamagata lineage. As described elsewhere, influenza virus HA and NA include, but are not limited to, influenza virus HA and NA from standard of care influenza strains.

[0150] In a particular embodiment, the vaccine or immunogenic composition comprises (i) at least four, e.g., four, influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, and influenza virus HA from B / Yamagata lineage, and (ii) one or more ribonucleic acid molecules encoding no more than four, e.g., four, influenza virus proteins selected from influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from B / Victoria lineage, and influenza virus NA from B / Yamagata lineage. As described elsewhere, the influenza virus HA and NA include, but are not limited to, influenza virus HA and NA from standard of care influenza strains.

[0151] In a particular embodiment, the vaccine or immunogenic composition comprises (i) four influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, and influenza virus HA from B / Yamagata lineage, and (ii) one or more ribonucleic acid molecules encoding four influenza virus proteins selected from influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from B / Victoria lineage, and influenza virus NA from B / Yamagata lineage. As described elsewhere, influenza virus HA and NA include, but are not limited to, influenza virus HA and NA from standard of care influenza strains.

[0152] In further embodiments, the vaccine or immunogenic composition comprises (i) four recombinant influenza virus proteins selected from a recombinant influenza virus H1 HA, a recombinant influenza virus H3 HA, a recombinant influenza virus HA from B / Victoria lineage, and a recombinant influenza virus HA from B / Yamagata lineage, and (ii) one or more ribonucleic acid molecules encoding four influenza virus proteins selected from an influenza virus N1 NA, an influenza virus N2 NA, an influenza virus NA from B / Victoria lineage, and an influenza virus NA from B / Yamagata lineage. As described elsewhere herein, the influenza virus HA and NA include, but are not limited to, influenza virus HA and NA from standard of care influenza strains.

[0153] In a particular embodiment, the vaccine or immunogenic composition comprises (i) four influenza virus proteins selected from influenza virus H1 HA present in IIV, influenza virus H3 HA present in IIV, influenza virus HA from B / Victoria lineage present in IIV, and influenza virus from B / Yamagata lineage present in IIV, and (ii) one or more ribonucleic acid molecules encoding four influenza virus proteins selected from influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from B / Victoria lineage, and influenza virus NA from B / Yamagata lineage. As described elsewhere, the influenza virus HA and NA include, but are not limited to, influenza virus HA and NA from standard of care influenza strains.

[0154] In a particular embodiment, the vaccine or immunogenic composition comprises (i) four influenza virus proteins selected from influenza virus N1 NA, influenza virus N2 NA, influenza virus NA from B / Victoria lineage, and influenza virus NA from B / Yamagata lineage, and (ii) one or more ribonucleic acid molecules encoding four influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, and influenza virus HA from B / Yamagata lineage. As described elsewhere, the influenza virus HA and NA include, but are not limited to, influenza virus HA and NA from standard of care influenza strains.

[0155] In further embodiments, the vaccine or immunogenic composition comprises (i) four recombinant influenza virus proteins selected from recombinant influenza virus N1 NA, recombinant influenza virus N2 NA, recombinant influenza virus NA from B / Victoria lineage, and recombinant influenza virus NA from B / Yamagata lineage, and (ii) one or more ribonucleic acid molecules encoding four influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, and influenza virus HA from B / Yamagata lineage. As described elsewhere, influenza virus HA and NA include, but are not limited to, influenza virus HA and NA from standard of care influenza strains.

[0156] In a particular embodiment, the vaccine or immunogenic composition comprises: (i) four influenza virus proteins selected from influenza virus N1 NA present in IIV, influenza virus N2 NA present in IIV, influenza virus NA from B / Victoria lineage present in IIV, and influenza virus NA from B / Yamagata lineage present in IIV, and (ii) one or more ribonucleic acid molecules encoding four influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, and influenza virus HA from B / Yamagata lineage. As described elsewhere, influenza virus HA and NA include, but are not limited to, influenza virus HA and NA from standard of care influenza strains.

[0157] In certain aspects, the vaccine or immunogenic composition described herein further comprises one or more influenza virus HA and / or NA proteins and / or one or more ribonucleic acid molecules encoding one or more influenza virus HA and / or NA proteins, hi certain embodiments, the one or more influenza virus HA and / or NA proteins are identified or designed using a machine learning model.

[0158] The vaccine or immunogenic composition may also further comprise an adjuvant. As used herein, the term "adjuvant" refers to a substance or vehicle that non-specifically enhances the immune response to an antigen. Adjuvants may include mineral suspensions to which antigens are adsorbed (including alum, aluminum salts (e.g., aluminum hydroxide / oxyhydroxide (AlOOH), aluminum phosphate (AlPO4), aluminum hydroxyphosphate sulfate (AAHS), and / or potassium aluminum sulfate)); or water-in-oil emulsions in which antigen solutions are emulsified in mineral oil (Freund's incomplete adjuvant), which may also contain killed mycobacteria to further enhance antigenicity (Freund's complete adjuvant). Immunostimulatory oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants (see, for example, U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199).Adjuvants also include biological molecules such as lipids and costimulatory molecules. Representative biological adjuvants include AS04 (Didierlaurent, AM et al, AS04, an Aluminum Salt-and TLR4 Agonist-Based Adjuvant System, Induces a Transient Localized Innate Immune Response Leading to Enhanced Adaptive Immunity, 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.

[0159] In certain embodiments, the adjuvant is a squalene-based adjuvant comprising an oil-in-water adjuvant emulsion comprising at least: squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic non-ionic surfactant, and a hydrophobic non-ionic surfactant. In certain embodiments, the emulsion is thermoreversible, and optionally has 90% of the population by volume of oil droplets having a size of less than 200 nm.

[0160] In certain 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 a particular embodiment, the polyoxyethylene alkyl ether surfactant is polyoxyethylene (12) cetostearyl ether.

[0161] In certain embodiments, 90% of the population by volume of the oil droplets have a size of less than 160 nm. In certain embodiments, 90% of the population by volume of the oil droplets have a size of less than 150 nm. In certain embodiments, 50% of the population by volume of the oil droplets have a size of less than 100 nm. In certain embodiments, 50% of the population by volume of the oil droplets have a size of less than 90 nm.

[0162] In certain embodiments, the adjuvant further comprises at least one alditol, including but not limited to glycerol, erythritol, xylitol, sorbitol, and mannitol.

[0163] In certain embodiments, the hydrophilic nonionic surfactant has a hydrophilic / lipophilic balance (HLB) of 10 or more. In certain embodiments, the hydrophobic nonionic surfactant has an HLB of less than 9. In certain embodiments, the hydrophilic nonionic surfactant has an HLB of 10 or more and the hydrophobic nonionic surfactant has an HLB of less than 9.

[0164] In certain embodiments, the hydrophobic non-ionic surfactant is a sorbitan ester, such as sorbitan monooleate, or a mannide ester surfactant. In certain embodiments, the amount of squalene is 5-45%. In certain embodiments, the amount of polyoxyethylene alkyl ether surfactant is 0.9-9%. In certain embodiments, the amount of hydrophobic non-ionic surfactant is 0.7-7%. In certain embodiments, the adjuvant comprises i) 32.5% squalene, ii) 6.18% polyoxyethylene (12) cetostearyl ether, iii) 4.82% sorbitan monooleate, and iv) 6% mannitol.

[0165] In certain embodiments, the adjuvant further comprises an alkyl polyglycoside and / or a cryoprotectant, such as a sugar, in particular dodecyl maltoside and / or sucrose.

[0166] In certain embodiments, the adjuvant comprises AF03 as described in Klucker et al., AF03, an alternative squalene emulsion-based vaccine adjuvant prepared by a phase inversion temperature method, J. PHARM. Sci. 2012, 101(12):4490-4500, which is hereby incorporated by reference in its entirety. In certain embodiments, the adjuvant comprises a liposome-based adjuvant such as SPA14 as described in, for example, WO 2022 / 090359, which is hereby incorporated by reference in its entirety. SPA14 is a liposome-based adjuvant containing a Toll-like receptor 4 (TLR4) agonist (E6020) and a saponin (QS21).

[0167] In certain embodiments, including certain embodiments in which one or more nucleic acids are encapsulated in LNPs, the vaccine or immunogenic composition does not include an adjuvant. In certain embodiments, one or more ribonucleic acid molecules, such as one or more mRNA molecules, are encapsulated in LNPs that can serve to adjuvant one or more influenza virus proteins in the composition. See, e.g., Shirai et al, Lipid Nanoparticle Acts as a Potential Adjuvant for Influenza Split Vaccine without Inducing Inflammatory Responses, VACCINES 2020,8(433):1-18.

[0168] In addition to the NA, HA or mRNA encoding NA and / or HA and optional adjuvants, the vaccine or immunogenic composition may further comprise one or more pharma- ceutically acceptable excipients. In general, the nature of the excipient will depend on the particular mode of administration used. For example, parenteral formulations usually contain an injectable fluid, including pharma- ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like, as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the vaccine or immunogenic composition to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pharma- ceutically acceptable salts for adjusting osmotic pressure, preservatives, stabilizers, buffers, sugars, amino acids, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0169] Typically, the vaccine or immunogenic composition is a sterile liquid solution formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal or intramuscular. The vaccine or immunogenic composition may also be formulated for intranasal or inhalation administration. The vaccine or immunogenic composition may also be formulated for any other intended route of administration.

[0170] In some embodiments, the vaccine or immunogenic composition is formulated for intradermal, intranasal or intramuscular injection. In some embodiments, the injectables are prepared in conventional forms, either as liquid solutions or suspensions, as solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. In some embodiments, the injectable solutions and suspensions are prepared from sterile powders or granules. General considerations in the formulation and manufacture of pharmaceuticals for administration by these routes are discussed, for example, in Remington's Pharmaceutical Sciences, 1999, ed. ... thed., 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 to deliver therapeutics directly to the lungs and respiratory system. In some embodiments, the vaccine or immunogenic composition is administered using a device that delivers a metered amount of the vaccine composition. Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include short needle devices, such as those described in U.S. Pat. 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 may also be administered by devices that limit the effective penetration length of a needle into the skin, such as those described in WO 1999 / 34850, incorporated herein by reference, and functional equivalents thereof. Also suitable are jet injection devices that deliver liquid vaccines to the dermis via a liquid jet injector or via a needle that pierces the stratum corneum and produces a jet that reaches the dermis.Jet injection devices are described, for example, in U.S. Pat. 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, and U.S. Pat. US Patent No. 5,312,335, US Patent No. 5,503,627, US Patent No. 5,064,413, US Patent No. 5,520,639, US Patent No. 4,596,556, US Patent No. 4,790,824, US Patent No. 4,941,880, US Patent No. 4,940,460, WO 1997 / 37705 and WO 1997 / 13537 (all of which are incorporated herein by reference). Also suitable is a ballistic powder / particle delivery device that uses compressed gas to accelerate the vaccine in powder form through the outer layer of the skin to the dermis.Furthermore, in the classical Mantoux method of intradermal administration, a conventional syringe can be used.

[0171] Preparations 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, such as saline, buffered media, and the like. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases, and the like.

[0172] kit Further disclosed herein are kits for the vaccine or immunogenic compositions as disclosed herein, which may include a suitable container containing the vaccine composition or multiple containers containing different components of the vaccine composition, optionally together with instructions for use.

[0173] In certain embodiments, the kit may comprise multiple containers, including, for example, a first container comprising one or more influenza virus HA proteins, one or more influenza virus NA proteins, or combinations thereof as disclosed herein, and a second container comprising one or more ribonucleic acid molecules encoding one or more influenza virus HA proteins, one or more influenza virus NA proteins, or combinations thereof as described herein.

[0174] For example, in certain embodiments, disclosed herein is a kit comprising: (i) a first container comprising a first influenza virus HA that is an H1 HA; a second influenza virus HA that is an H3 HA; a third influenza virus HA from B / Victoria lineage; and a fourth influenza virus HA from B / Yamagata lineage; and (ii) a second container comprising one or more ribonucleic acid molecules encoding the first influenza virus NA that is an N1 NA; the second influenza virus NA that is an N2 NA; the third influenza virus NA from B / Victoria lineage; and the fourth influenza virus NA from B / Yamagata lineage. In certain embodiments, the kit may further comprise a third container comprising an optional adjuvant, and in certain embodiments, the first container may comprise an optional adjuvant in addition to the recombinant influenza virus antigen.

[0175] In certain embodiments, the kit may include a single container that includes (i) each of one or more influenza virus HA proteins, one or more influenza virus NA proteins, or a combination thereof as disclosed herein, and (ii) each of one or more ribonucleic acid molecules encoding one or more influenza virus HA proteins, one or more influenza virus NA proteins, or a combination thereof as disclosed herein. For example, in certain embodiments, the kit may include a single container that includes one or more ribonucleic acid molecules encoding a first influenza virus HA that is an H1 HA; a second influenza virus HA that is an H3 HA; a third influenza virus HA from the B / Victoria lineage; a fourth influenza virus HA from the B / Yamagata lineage; and a first influenza virus NA that is an N1 NA; a second influenza virus NA that is an N2 NA; a third influenza virus NA from the B / Victoria lineage; and a fourth influenza virus NA from the B / Yamagata lineage. The single container may also include an optional adjuvant.

[0176] The instructions may instruct that the contents of the first and second containers may be combined prior to administration, or that the contents of the first and second containers are not combined and are administered separately.

[0177] Nucleic acids, cloning and expression systems The present disclosure further provides artificial nucleic acid molecules encoding the disclosed influenza virus HA and NA. The nucleic acid may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence described herein includes DNA molecules having the specified sequence, unless the context requires otherwise, and includes RNA molecules having the specified sequence in which U is replaced with T, or a derivative or analog thereof, such as pseudouridine, particularly N1-methylpseudouridine. Other nucleotide derivatives or modified nucleotides may be incorporated into the disclosed HA and NA encoding nucleic acid molecules.

[0178] The present disclosure also provides constructs in the form of vectors (e.g., plasmids, phagemids, cosmids, transcription or expression cassettes, artificial chromosomes, etc.) that contain a nucleic acid molecule encoding an HA or NA as disclosed herein. The present disclosure further provides host cells that contain one or more constructs as described above.

[0179] Also provided are methods for producing HA or NA encoded by these nucleic acid molecules. HA or NA polypeptides can be produced using recombinant techniques. Recombinant protein production and expression are well known in the art and can be performed using conventional procedures such as those disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Ed. 2012), Cold Spring Harbor Press. For example, expression of HA or NA polypeptides can be achieved by culturing host cells containing artificial nucleic acid molecules encoding HA or NA as disclosed herein under appropriate conditions. For example, expression of recombinant HA or NA polypeptides can be achieved by culturing host cells containing nucleic acid molecules encoding HA or NA as disclosed herein under appropriate conditions. After production by expression, HA or NA can be isolated and / or purified using any suitable technique and then used as needed.

[0180] Systems for cloning and polypeptide expression in a variety of different host cells are well known in the art. Any protein expression system (e.g., stable or transient) compatible with the constructs disclosed herein can be used to produce the HA or NA described herein.

[0181] Suitable vectors can be chosen or constructed to contain appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.

[0182] To express recombinant HA or NA, a nucleic acid encoding HA or NA can be introduced into a host cell. The introduction can use any available technique. For eukaryotic cells, suitable techniques can include calcium phosphate gene transfer, DEAE-dextran, electroporation, liposome-mediated gene transfer, and transduction using retroviruses or other viruses, such as vaccinia, or baculovirus in the case of insect cells. For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and gene transfer using bacteriophage. These techniques are well known in the art. (See, for example, "Current Protocols in Molecular Biology", Ausubel et al. eds., John Wiley & Sons, 2010). DNA transfer can be followed by a selection method (e.g., antibiotic resistance) to select cells containing the vector.

[0183] The host cell may be a plant cell, a yeast cell, or an animal cell. Animal cells include invertebrate cells (e.g., insect cells), non-mammalian vertebrate cells (e.g., birds, reptiles, and amphibians), and mammalian cells. In one embodiment, the host cell is a mammalian cell. Examples of mammalian cells include, but are not limited to, COS-7 cells, HEK293 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO) cells; mouse Sertoli cells; African green monkey kidney cells (VERO); human cervical carcinoma cells (e.g., HeLa); canine kidney cells (e.g., MDCK), and the like. In one embodiment, the host cell is a CHO cell.

[0184] In certain embodiments, the host cell is a plant cell. For example, recombinant HA or NA can be expressed in a microalgae cell, as disclosed in U.S. Patent Application Publication No. 2011 / 0189228, which is hereby incorporated by reference in its entirety.

[0185] In certain embodiments, the host cell is an insect cell. As a further example, recombinant HA or NA can be expressed in insect cells infected with a virus-HA vector, such as a baculovirus vector, as disclosed in U.S. Pat. No. 5,976,552, which is hereby incorporated by reference in its entirety. Baculoviruses are DNA viruses of the Baculoviridae family. These viruses are known to have a narrow host range that is restricted primarily to Lepidopteran insect species (e.g., butterflies and moths). For example, the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) replicates efficiently in susceptible cultured insect cells. AcNPV has a double-stranded closed circular DNA genome of approximately 130,000 base pairs and has been well characterized in terms of host range, molecular biology, and genetics.

[0186] Many baculoviruses, including AcNPV, form large protein crystalline occlusions in the nuclei of infected cells. A single polypeptide called polyhedrin accounts for approximately 95% of the protein mass of these occlusion bodies. The gene for polyhedrin is present as a single copy in the AcNPV viral genome. Since the polyhedrin gene is not required for viral replication in cultured cells, it can be easily modified to express foreign genes. Foreign gene sequences can be inserted just 3' to the polyhedrin promoter sequence of the AcNPV gene so that it is under the transcriptional control of the polyhedrin promoter. Recombinant baculoviruses, including recombinant baculoviruses, encoding recombinant HA or NA proteins can then be replicated in a variety of insect cell lines. Recombinant HA or NA proteins can also be expressed in other expression vectors, including, for example, entomopoxviruses (insect poxviruses), cytoplasmic polyhedrosis viruses (CPVs), and transformation of insect cells with recombinant HA genes.

[0187] Inactivated / Reassortant Influenza Viruses In certain embodiments disclosed herein, the influenza virus HA and / or NA are present in an inactivated influenza virus. Certain licensed influenza vaccines may contain formalin-inactivated whole subunit preparations or chemically resolved subunit preparations from multiple influenza subtypes, including, for example, influenza A subtypes H1N1, influenza A H3N2, influenza B / Victoria, and / or influenza B / Yamagata. The seed viruses for such influenza A and B vaccines may be naturally occurring strains (i.e., wild-type strains) that replicate to high titers in the allantoic cavity of chicken eggs or in cultured cells.

[0188] Alternatively, the strains may be reassortant viruses that have the correct surface antigen genes. Reassortant viruses are those that have the characteristics of each parental strain due to segmentation of the viral genome. When two or more influenza virus strains infect a cell, these viral segments mix to create progeny virions that contain a variety of genes from both parents. Reverse genetics methods used to generate infectious reassortant viruses are well known to those of skill in the art and include, for example, Neumann et al, Generation of influenza A viruses entirely from cloned cDNA, PROC NATL ACAD SCI USA 1999, 96(16):9345-9350; Neumann et al, An improved reverse genetics system for influenza A virus generation and its implications for vaccine production, PROC NATL ACAD SCI USA 2005, 102(46):16825-16829; Zhang et al, A One-Plasmid System To Generate Influenza Virus in Cultured Chicken Cells for Potential Use in Influenza Vaccine, J VIROL 2009, 83(18):9296-9303; Massin et al, Cloning of the Chicken RNA Polymerase I Promoter and Use for Reverse Genetics of Influenza A Viruses in Avian Cells, J VIROL 2005,79(21):13811-13816; Murakami et al,Establishment of Canine RNA Polymerase I-Driven Reverse Genetics for Influenza A Virus: Its Application for H5N1 Vaccine Production,J VIROL 2008,82(3):1605-1609;and / or Neuman et al, 1999; Neumann et al, 2005; Zhang et al, 2009; Massin et al, 2005; Murakami et al, 2008; Koudstaal et al, Suitability of PER.C6 cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics, VACCINE 2009, 27(19):2588 - 2593; Schickli et al, Plasmid-only rescue of influenza A virus vaccine candidates, PHILOS TRANS R SOC LOND BIOL SCI 2001, 356(1416):1965 - 1973; Nicolson et al, Generation of influenza vaccine viruses on Vero cells by reverse genetics: an H5N1 candidate vaccine strains produced under a quality system, VACCINE 2005, 23(22):2943 - 2952; Legastelois et al, Preparation of genetically engineered A / H5N1 and A / H7N1 pandemic vaccine viruses by reverse genetics in a mixture of Vero and chicken embryo cells, INFLUENZA OTHER RESPIR VIRUSES 2007, 1(3):95 - 104;Examples of such methods include, but are not limited to, methods using cells described in Whiteley et al., Generation of candidate human influenza vaccine strains in cell culture - rehearsing the European response to an H7N1 pandemic threat, INFLUENZA OTHER RESPIR VIRUSES 2007, 1(4):157-166;

[0189] Machine Learning Any machine learning algorithm may be used to select one or more machine learning influenza virus HAs and / or NAs. For example, any machine learning algorithm and method disclosed in PCT Publication Nos. WO 2021 / 080990A1 (entitled Systems and Methods for Designing Vaccines) and WO 2021 / 080999A1 (entitled Systems and Methods for Predicting Biological Responses), U.S. Provisional Patent Application No. 63 / 319,692 (entitled Machine-Learning Techniques in Protein Design for Vaccine Generation), and U.S. Provisional Patent Application No. 63 / 319,700 (entitled Machine-Learning Techniques in Protein Design for Vaccine Generation), all of which are incorporated herein by reference in their entirety, are contemplated herein.

[0190] In certain embodiments, a predictive machine learning model of influenza antigenicity may be constructed to enable prediction of antibody titers in animal models and / or humans. In certain embodiments, the machine learning model may extract feature values ​​from input data of a training set, where features are variables that are considered potentially relevant regardless of whether the input data item has the relevant characteristic. The ordered list of features for the input data may be referred to as a feature vector for the input data. In certain embodiments, the machine learning model applies dimensionality reduction (e.g., via linear discriminant analysis (LDA), principal component analysis (PCA), learned deep features from neural networks, etc.) to reduce the amount of data in the feature vector for the input data to a smaller, more representative set of data. A set of influenza sequences (e.g., target strains) to protect against may then be identified and a selection algorithm constructed.

[0191] In certain embodiments, a system for designing a vaccine is provided. The system includes one or more processors. The system includes computer storage storing executable computer instructions that, when executed by the one or more processors, cause the one or more processors to perform one or more operations. The one or more operations include applying a plurality of driver models configured to generate output data representative of one or more molecular sequences to a first time series data set, the first time series data set representing the one or more molecular sequences and, for each of the one or more molecular sequences, one or more circulations of a pathogenic strain that includes the molecular sequence as a natural antigen. The one or more operations include, for each of the plurality of driver models, training the driver model by: i) receiving output data from the driver model representing one or more predicted molecular sequences based on the received first time series data set; ii) applying a translational model configured to predict a biological response to the molecular sequences for a plurality of translational axes to the output data representing the predicted one or more molecular sequences to generate first translational response data representing one or more first translational responses corresponding to a particular translational axis of the plurality of translational axes based on the one or more predicted molecular sequences in the output data; iii) adjusting one or more parameters of the driver model based on the first translational response data; and iv) repeating steps i-iii a number of times to generate trained translational response data representing one or more trained translational responses corresponding to the particular translational axis. The one or more operations include selecting a set of trained driver models from the plurality of driver models based on the one or more trained translational responses. The one or more operations include applying the trained driver models to the second time series data set to generate, for each trained driver model in the set of trained driver models, trained output data representing one or more predicted molecular sequences for a particular season; applying the translation model to the final output data to generate, for each translational axis of the plurality of translational axes, second translational response data representing one or more second translational responses; and selecting a subset of the trained driver models of the set of trained driver models based on the second translational response data.

[0192] At least one of the plurality of driver models may include a recurrent neural network. At least one of the plurality of driver models includes a long short-term memory recurrent neural network.

[0193] The output data representing one or more predicted molecular sequences based on the received first time series dataset may include output data representing antigens for each of a plurality of pathogenic seasons. The output data representing antigens for each of a plurality of pathogenic seasons may include antigens determined by predicting molecular sequences that will generate a maximized agglutination biological response across all circulating pathogenic strains for a particular season. The output data representing antigens for each of a plurality of pathogenic seasons may include antigens determined by predicting molecular sequences that will generate a response that effectively immunizes against a maximum number of circulating viruses for a particular season.

[0194] The plurality of translational axes may include at least one of a ferret antibody forensics (AF) axis, a ferret hemagglutination inhibition assay (HAI) axis, a mouse AF axis, a mouse HAI axis, a human replica AF axis, a human AF axis, or a human HAI axis. The number of iterations may be based on a predetermined number of iterations. The number of iterations may be based on a predetermined error value. The one or more first translational responses may include at least one of a predicted ferret HAI titer, a predicted ferret AF titer, a predicted mouse AF titer, a predicted mouse HAI titer, a predicted human replica AF titer, a predicted human AF titer, or a predicted human HAI titer.

[0195] Selecting a set of trained driver models of the plurality of driver models may include assigning each driver model of the plurality of driver models to a class of driver models, each class being associated with a particular translational axis of the plurality of translational axes used to train the driver model. Selecting a set of trained driver models of the plurality of driver models may include, for each driver model of the plurality of driver models, comparing a trained translational response of one or more of the driver models to a trained translational response of one or more of at least one other driver models assigned to the same class as the driver model.

[0196] The operations may further include, for each trained driver model of the subset of trained driver models, validating the trained driver model by comparing the second translational response data corresponding to the trained driver model to the observed experimental response data; and, in response to validating the trained driver model, generating a vaccine including one or more molecular sequences represented by the trained output data corresponding to the trained driver model.

[0197] In one aspect, a system is provided. The system includes a computer-readable memory including computer-executable instructions. The system includes at least one processor configured to execute executable logic including at least one machine learning model trained to predict one or more molecular sequences, and when the at least one processor executes the computer-executable instructions, the at least one processor is configured to perform one or more operations. The one or more operations include receiving time series data indicating one or more molecular sequences, and for each of the one or more molecular sequences, receiving one or more circulations of a pathogenic strain that includes the molecular sequence as a natural antigen. The one or more operations include processing the time series data through one or more data structures that store one or more portions of the executable logic included in the machine learning model, and predicting one or more molecular sequences based on the time series data.

[0198] Predicting the one or more molecular sequences based on the time series data may include predicting one or more immunological properties that the predicted one or more molecular sequences will confer for future use. Predicting the one or more molecular sequences based on the time series data may include predicting one or more molecular sequences that generate a maximized agglutinative biological response across all pathogenic strains in the time series data. Predicting the one or more molecular sequences based on the time series data may include predicting one or more molecular sequences that generate a biological response that effectively covers a maximum number of pathogenic strains in the time series data. The predicted one or more molecular sequences may be used to design a vaccine for a pathogenic strain that circulates at a time after one or more circulations of the time series data.

[0199] The machine learning model may include a recurrent neural network.

[0200] In certain embodiments, a data processing system for predicting a biological response is provided. The system includes a computer readable memory including computer executable instructions. The system includes at least one processor configured to execute executable logic including at least one machine learning model trained to predict a biological response, and when the at least one processor executes the computer executable instructions, the at least one processor performs one or more operations. The one or more operations include receiving first sequence data of a first molecular sequence. The one or more operations include receiving second sequence data of a second molecular sequence. The one or more operations include predicting a biological response to the second molecular sequence based at least in part on the received first and second sequence data.

[0201] The one or more operations may include receiving non-human biological response data corresponding to the first molecular sequence and the second molecular sequence. The one or more operations may further include predicting a biological response based at least in part on the non-human biological response data. The one or more operations may include encoding the first sequence data and the second sequence data as amino acid mismatches.

[0202] The first molecular sequence may comprise a candidate antigen, and the second molecular sequence may comprise a known viral strain.

[0203] Predicting the biological response may include predicting a human biological response. Predicting the biological response may include predicting at least one human biological response and at least one non-human biological response. The biological response may include an antibody titer. The machine learning model may include a deep neural network.

[0204] Machine learning techniques may be used to train machine learning models that predict biological responses such that the incidence of false positives and false negatives is reduced. At least some of the described systems and methods may be used to efficiently process inherently sparse data, for example by reducing the dimensionality of the data, when compared to traditional approaches. At least some of the described systems and methods may exploit non-linear relationships in the received data to increase prediction accuracy compared to traditional approaches. At least some of the described systems and methods may be used to simultaneously predict human and non-human biological responses. At least some of the described systems and methods may be used to predict outcomes that are not experimentally observed.

[0205] In certain embodiments, one or more computer systems may be configured to perform certain operations or actions by having software, firmware, hardware, or a combination thereof loaded on the system that causes the system to perform the operation during operation. One or more computer programs may be configured to perform certain operations or actions by including instructions that, when executed by a data processing device, cause the device to perform the operation. One general aspect includes a method for producing a vaccine by using a continuous data algorithm. The method includes receiving a discrete data object that may include a plurality of first discrete values, the discrete data object may include one or more amino acid sequences. The method also includes converting the discrete data object to a continuous data object that may include a plurality of first continuous values. The method also includes applying the continuous data algorithm to the continuous data object to generate a continuous result object that may include a plurality of second continuous values. The method also includes converting the continuous result object to a discrete result object that may include a plurality of second discrete values. The method also includes producing a vaccine that may include at least one of i) a protein defined by the discrete result object, ii) a nucleic acid capable of producing the protein defined by the discrete result object, and iii) a delivery vehicle capable of producing the protein defined by the discrete result object. Other embodiments of this aspect include corresponding computer systems, devices, and computer programs stored on one or more computer storage devices, each configured to perform the operations of the method.

[0206] Implementations may include one or more of the following features: The method, wherein the one or more amino acid sequences may include a first amino acid sequence and a second amino acid sequence, each of the first amino acid sequence and the second amino acid sequence including a respective character or a respective character string. The conversion of the discrete data object to the continuous data object may include: for each first discrete value, generating a weight vector of weight values, each weight value representing a likelihood that the first discrete value represents a particular amino acid; for each weight value of each weight vector, generating a property vector of property values, each property value representing a physicochemical property of a particular amino acid; and combining the weight vector and the property vector to generate the first continuous value of the continuous data object. Each weight vector has 20 weight values, each weight value corresponding to one of the 20 possible amino acids. The conversion of the continuous result object to the discrete result object may include: for each second continuous value, determining a respective single amino acid, the determined single amino acids forming a plurality of second discrete values. The method may further include generating a plurality of candidate discrete result objects; and filtering out at least one discrete result object that identifies an amino acid that failed the manufacturability test from the plurality of candidate discrete result objects. The application of the continuous data algorithm to generate the continuous result objects may include applying a gradient descent method with a loss function that determines a loss value based on a plurality of loss criteria, the loss function may include a first loss criterion based on an immunological reaction given two amino acid sequences; a second loss criterion that modifies the loss value for subsequences not found in the dataset of wild-type sequences or subsequences that are predicted to not fold correctly; and a third loss criterion that modifies the loss value for each weight vector based on the maximum value in the second continuous value. Implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0207] One general aspect includes a system for generating amino acid sequences, which may include a computer memory. The system may also include one or more processors. The system may also include a computer memory storing instructions that, when executed by the processor, cause the processor to perform operations, which may include receiving a discrete data object including a plurality of first discrete values, the discrete data object including one or more amino acid sequences; converting the discrete data object into a continuous data object including a plurality of first continuous values; applying a continuous data algorithm to the continuous data object to generate a continuous result object including a plurality of second continuous values; converting the continuous result object into a discrete result object including a plurality of second discrete values; and manufacturing a vaccine including at least one of i) a protein defined by the discrete result object, ii) a nucleic acid capable of generating the protein defined by the discrete result object, and iii) a delivery vehicle capable of generating the protein defined by the discrete result object. Other embodiments of this aspect include corresponding computer systems, devices, and computer programs stored on one or more computer storage devices, each configured to perform the operations of the method.

[0208] Implementations may include one or more of the following features. In one embodiment, the one or more amino acid sequences may include a first amino acid sequence and a second amino acid sequence, each of the first amino acid sequence and the second amino acid sequence including a respective character or a respective character string. The conversion of the discrete data object to the continuous data object may include generating, for each first discrete value, a weight vector of weight values, each weight value representing a likelihood that the first discrete value represents a particular amino acid; generating, for each weight value of each weight vector, a property vector of property values, each property value representing a physicochemical property of a particular amino acid; and combining the weight vector and the property vector to generate a first continuous value of the continuous data object. Each weight vector has 20 weight values, each weight value corresponding to one of the 20 possible amino acids. The conversion of the continuous result object to the discrete result object may include determining, for each second continuous value, a respective single amino acid, the determined single amino acids forming a plurality of second discrete values. The operations may further include: generating a plurality of candidate discrete result objects; and filtering out from the plurality of candidate discrete result objects at least one discrete result object that identifies an amino acid that failed the manufacturability test. The application of the continuous data algorithm to generate the continuous result objects may include applying a gradient descent method with a loss function that determines a loss value based on a plurality of loss criteria, the loss function may include a first loss criterion based on an immunological reaction given the two amino acid sequences; a second loss criterion that modifies the loss value of a subsequence not found in the data set of wild-type sequences or subsequences that are predicted to not fold correctly; and a third loss criterion that modifies the loss value based on the maximum value in the second continuous value for each weight vector. Implementations of the described techniques may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0209] One general aspect includes a non-transitory computer readable medium storing instructions that, when executed by one or more processors, cause the one or more processors to perform operations that may include: receiving a discrete data object including a plurality of first discrete values, the discrete data object including one or more amino acid sequences; converting the discrete data object into a continuous data object including a plurality of first continuous values; applying a continuous data algorithm to the continuous data object to generate a continuous result object including a plurality of second continuous values; converting the continuous result object into a discrete result object including a plurality of second discrete values; and manufacturing a vaccine comprising at least one of i) a protein defined by the discrete result object, ii) a nucleic acid capable of producing the protein defined by the discrete result object, and iii) a delivery vehicle capable of producing the protein defined by the discrete result object. Other embodiments of this aspect include corresponding computer systems, devices, and computer programs stored on one or more computer storage devices, each configured to perform the operations of the method.

[0210] Implementations may include one or more of the following features: The medium may include a first amino acid sequence and a second amino acid sequence, each of the first amino acid sequence and the second amino acid sequence including a respective character or a respective character string. The conversion of the discrete data object to the continuous data object includes generating, for each first discrete value, a weight vector of weight values, each weight value representing a likelihood that the first discrete value represents a particular amino acid, generating, for each weight value of each weight vector, a property vector of property values, each property value representing a physicochemical property of a particular amino acid; and combining the weight vector and the property vector to generate a first continuous value of the continuous data object. Each weight vector has 20 weight values, each weight value corresponding to one of the 20 possible amino acids. The conversion of the continuous result object to the discrete result object may include determining, for each second continuous value, a respective single amino acid, the determined single amino acids forming a plurality of second discrete values. Implementations of the described techniques may include hardware, a method or process, or computer software on a computer-accessible medium.

[0211] In certain embodiments, algorithms are disclosed herein that can generate influenza antigens for use as vaccines. In one implementation, this can include: 1) generating a reduced dimensional space for all wild-type hemagglutinin sequences through machine learning (e.g., a variational autoencoder architecture) using two stages: a) variable embedding into the reduced space (e.g., the model predicts the mean and variance from the input sequence using embedded coordinates chosen from a normal distribution with predicted mean and variance); b) Then, decode the reduced spatial location “autoencoder” loss function back to the original sequence and reduce it by the similarity of the input and output sequences. 2) Training an immune response prediction model based on the position of antigens (vaccine candidates) and readout strains (target sequences) in reduced dimensional space [input: antigens and readouts embedded by the model from stage 1, output: measures of immune response such as antibody titers]. 3) Sampling candidate vaccine component expressions from the reduced space and ranking the candidate vaccine component expressions by their predictive performance against the target sequence using the model described in step 2 to identify the top candidates. 4) Decoding the top candidate representations [using the model from step 1b] to release hemagglutinin sequences that may or may not have been observed in the original wild-type set.

[0212] A system of one or more computers may be configured to perform a particular operation or action by having software, firmware, hardware, or a combination thereof loaded on the system that causes the system to perform the action during operation. One or more computer programs may be configured to perform a particular operation or action by including instructions that, when executed by a data processing device, cause the device to perform the action. One general aspect includes a dimensionality reduction method for generating amino acid sequences, the method being performed by a system of one or more computers. The method includes receiving one or more data objects defining a plurality of wild-type amino acid sequences. The method also includes generating a plurality of reduced dimensional sequences in a reduced dimensional space from the one or more data objects, each reduced dimensional sequence containing data for at least one respective of the wild-type amino acid sequences, the reduced dimensional space being lower dimensional than the wild-type amino acid sequences, and the plurality of reduced dimensional sequences defining a distribution of values ​​along each dimension of the reduced dimensional space. The method also includes generating a plurality of candidate sequences in the reduced dimensional space using the plurality of reduced dimensional sequences. The method also includes receiving one or more data objects defining a viral amino acid sequence. The method also includes generating at least one reduced dimensional viral sequence in the reduced dimensional space. The method also includes providing each of the candidate sequences and the at least one reduced dimension viral sequence as inputs to a potency predictor. The method also includes receiving a candidate score for each of the candidate sequences as output from the potency predictor. The method also includes selecting at least one candidate sequence from among the candidate sequences. The method also includes generating at least one new amino acid sequence for each of the selected candidate sequences. The method also includes providing the generated at least one amino acid sequence.The method also includes operations where each of the generated amino acid sequences is suitable for producing a respective vaccine, which may include at least one of: i) a protein defined by the generated amino acid sequence, ii) a nucleic acid capable of producing the protein defined by the generated amino acid sequence, and iii) a delivery vehicle capable of producing the protein defined by the generated amino acid sequence. Other embodiments of this aspect include corresponding computer systems, devices, and computer programs stored on one or more computer storage devices, each configured to perform the operations of the method.

[0213] Implementations may include one or more of the following features. The method includes operations in which generating a plurality of reduced dimensional arrays may include creating a representation of a wild-type amino acid sequence using a variational autoencoder that predicts mean and variance values ​​of input data. Each of the reduced dimensional arrays may include a respective set of values, and generating a plurality of candidate sequences in the reduced dimensional space may include sampling a distribution of values ​​of the plurality of reduced dimensional arrays. The potency predictor is configured to receive as input: i) a first sequence in the reduced dimensional space, and ii) a second sequence in the reduced dimensional space; and to provide as output a potency score as a candidate score, the potency score defining a measure of a biological response between the first sequence and the second sequence. Selecting at least one candidate sequence as a selected candidate sequence may include selecting n candidate sequences having the highest candidate scores. The method includes operations in which n is a value of 1 such that a single candidate sequence is selected. The method includes operations in which n is a value greater than 1 such that a plurality of candidate sequences are selected. Selecting at least one candidate sequence as a selected candidate sequence may include selecting candidate sequences having respective candidate scores greater than a threshold value. Each of the generated amino acid sequences is different from any of the wild-type amino acid sequences. At least one of the candidate sequences is within the plurality of reduced dimension sequences. Implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0214] One general aspect includes a system for generating amino acid sequences, which may include a computer memory. The system also includes one or more processors. The system also includes a computer memory that stores instructions that, when executed by the processor, cause the processor to perform operations, including receiving one or more data objects defining a plurality of wild-type amino acid sequences; generating a plurality of reduced-dimensional arrays in a reduced-dimensional space from the one or more data objects, where each reduced-dimensional array contains data for at least one respective wild-type amino acid sequence, the reduced-dimensional space being lower dimensional than the wild-type amino acid sequence, and the plurality of reduced-dimensional arrays defines a distribution of values ​​along each dimension of the reduced-dimensional space; generating a plurality of candidate sequences in the reduced-dimensional space using the plurality of reduced-dimensional arrays; receiving one or more data objects defining a viral amino acid sequence; generating at least one reduced-dimensional viral sequence in the reduced-dimensional space. providing each of the candidate sequences and at least one of the reduced dimensional viral sequence as input to a potency predictor; receiving a candidate score for each of the candidate sequences as output from the potency predictor; selecting at least one candidate sequence from among the candidate sequences; generating at least one new amino acid sequence for each of the selected candidate sequences; and providing the generated at least one amino acid sequence, wherein each of the generated amino acid sequences is suitable for producing a respective vaccine comprising at least one of i) a protein defined by the generated amino acid sequence, ii) a nucleic acid capable of producing the protein defined by the generated amino acid sequence, and iii) a delivery vehicle capable of producing the protein defined by the generated amino acid sequence. Other embodiments of this aspect include corresponding computer systems, devices, and computer programs recorded on one or more computer storage devices, each configured to perform the operations of the method.

[0215] Implementations may include one or more of the following features: The system, where the generation of the multiple reduced dimensional arrays may include creating a representation of the wild-type amino acid sequence using a variational autoencoder that predicts the mean and variance values ​​of the input data. Each of the reduced dimensional arrays may include a respective group of values, and the generation of the multiple candidate sequences in the reduced dimensional space may include sampling a distribution of values ​​of the multiple reduced dimensional arrays. The potency predictor is configured to receive as input: i) a first sequence in the reduced dimensional space, and ii) a second sequence in the reduced dimensional space; and provide as output a potency score as the candidate score, the potency score defining a measure of a biological response between the first sequence and the second sequence. Selecting at least one candidate sequence as the selected candidate sequence may include selecting the n candidate sequences with the highest candidate scores. Implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0216] One general aspect includes a non-transitory computer-readable medium storing instructions that, when executed by one or more processors, cause the one or more processors to perform operations including: receiving one or more data objects defining a plurality of wild-type amino acid sequences; generating a plurality of reduced dimensional arrays in a reduced dimensional space from the one or more data objects, where each reduced dimensional array contains data for at least one respective wild-type amino acid sequence, the reduced dimensional space being lower dimensional than the wild-type amino acid sequence, and the plurality of reduced dimensional arrays defining a distribution of values ​​along each dimension of the reduced dimensional space, and generating a plurality of candidate sequences in the reduced dimensional space using the plurality of reduced dimensional arrays; receiving one or more data objects defining viral amino acid sequences; generating at least one reduced dimensional viral amino acid sequence in the reduced dimensional space. The method includes generating a candidate sequence for each of the candidate sequences; providing each of the candidate sequences and at least one of the reduced dimensional viral sequence as input to a potency predictor; receiving a candidate score for each of the candidate sequences as output from the potency predictor; selecting at least one candidate sequence from among the candidate sequences; generating at least one new amino acid sequence for each of the selected candidate sequences; and providing the generated at least one amino acid sequence, wherein each of the generated amino acid sequences is suitable for producing a respective vaccine comprising at least one of i) a protein defined by the generated amino acid sequence, ii) a nucleic acid capable of producing the protein defined by the generated amino acid sequence, and iii) a delivery vehicle capable of producing the protein defined by the generated amino acid sequence. Other embodiments of this aspect include corresponding computer systems, devices, and computer programs stored on one or more computer storage devices, each configured to perform the operations of the method.

[0217] Implementations may include one or more of the following features: A medium in which generating a plurality of reduced dimensional arrays may include creating a representation of a wild-type amino acid sequence using a variational autoencoder that predicts mean and variance values ​​of input data. Each of the reduced dimensional arrays may include a set of respective values, and generating a plurality of candidate sequences in the reduced dimensional space may include sampling a distribution of values ​​of the plurality of reduced dimensional arrays. The potency predictor is configured to receive as input: i) a first sequence in the reduced dimensional space and ii) a second sequence in the reduced dimensional space; and to provide as output a potency score as a candidate score, the potency score defining a measure of a biological response between the first sequence and the second sequence. Implementations of the described technology may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0218] These and other aspects, features and implementations may be expressed as methods, apparatus, systems, components, program products, methods of doing business, means or steps for performing a function, and in other manners, and will become apparent from the following description, including the claims.

[0219] Implementations of the present disclosure may provide the following advantages: Compared to conventional techniques, vaccines may be designed for future pathogenic seasons to confer more protection in terms of biological response to at least one pathogenic strain of the future pathogenic season; Compared to conventional approaches, vaccines may be designed for future pathogenic seasons to confer more protection in terms of effective coverage of multiple pathogenic strains of the future pathogenic season (i.e., eliciting effective immunological responses against many pathogenic strains in the future pathogenic season); Unlike conventional techniques, rarely observed strains that may confer "more protection" because they cross-react with more strains than frequently observed strains may be evaluated and their vaccination effectiveness may be predicted.

[0220] How to use The present disclosure provides a method of administering a vaccine described herein to a subject. The method can be used to vaccinate a subject against influenza virus. In some embodiments, the vaccination method comprises administering to a subject in need of vaccination any of the vaccines described herein, including, for example, a vaccine comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof, as described herein, in an amount effective to vaccinate the subject against influenza virus. Similarly, the present disclosure provides any of the vaccines described herein for use in vaccinating a subject against influenza virus, including, for example, a vaccine comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof, for use in vaccinating a subject against influenza virus. Also disclosed herein is an immunogenic composition comprising: (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof as described herein, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof as described herein, for the manufacture of a vaccine for use in vaccinating a subject against influenza virus.

[0221] The present disclosure also provides a method of immunizing a subject against influenza virus comprising administering to the subject an immunologically effective amount of any of the vaccines described herein, including, for example, a vaccine comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof. Similarly, the present disclosure provides any of the vaccines described herein for use in immunizing a subject against influenza virus, including, for example, a vaccine comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof. Also disclosed herein is an immunogenic composition comprising: (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof as described herein, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof as described herein, for the manufacture of a vaccine for use in immunizing a subject against influenza virus.

[0222] In some embodiments, the method or use prevents influenza virus infection or disease in a subject. 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.

[0223] The immunization methods (or related uses) provided herein may broadly induce a neutralizing immune response against one or more influenza viruses. Thus, in various embodiments, the compositions described herein may provide broad cross-protection against various types of influenza viruses. In some embodiments, the compositions provide cross-protection against avian, swine, seasonal and / or pandemic influenza viruses. In some embodiments, the immunization methods (or related uses) may induce an improved immune response against one or more seasonal influenza strains (e.g., standard of care strains). For example, the improved immune response may be an improved humoral immune response. In some embodiments, the immunization methods (or related uses) may induce an improved immune response against one or more pandemic influenza strains. In some embodiments, the immunization methods (or related uses) may induce an improved immune response against one or more swine influenza strains. In some embodiments, the immunization methods (or related uses) may induce an improved immune response against one or more avian influenza strains.

[0224] In certain embodiments, provided herein are methods of enhancing or broadening a protective immune response in a subject, comprising administering to the subject an immunologically effective amount of any of the vaccines disclosed herein. Similarly, the disclosure provides any of the vaccines described herein for use in enhancing or broadening a protective immune response in a subject, including, for example, a vaccine comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof. Also disclosed herein are immunogenic compositions as described herein for the manufacture of a vaccine for use in enhancing or broadening a protective immune response in a subject. In certain embodiments, the vaccines disclosed herein improve the vaccine efficacy of a standard of care influenza virus vaccine composition by an amount in the range of about 5% to about 100%, e.g., about 10% to about 25%, about 20% to about 100%, about 15% to about 75%, about 15% to about 50%, about 20% to about 75%, about 20% to about 50%, or about 40% to about 80%, such as about 40% to about 60% or about 60% to about 80%. In certain embodiments, the vaccines disclosed herein have a vaccine efficacy that is at least 5% greater than the vaccine efficacy of a standard of care influenza virus vaccine, e.g., a vaccine efficacy that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% greater than the vaccine efficacy of a standard of care influenza virus vaccine. In some embodiments, the vaccines disclosed herein have a vaccine efficacy that is at least equal to the vaccine efficacy of a standard of care influenza virus vaccine.

[0225] In certain embodiments, the standard of care influenza virus vaccine can be an inactivated influenza vaccine (IIV), such as a trivalent or quadrivalent IIV. Typically, the standard of care, inactivated influenza virus vaccine composition includes inactivated influenza viruses from H1N1, H3N2, B / Victoria, and B / Yamagata lineages. In certain embodiments, the standard of care influenza virus vaccine can include a recombinant influenza virus HA, such as a trivalent or quadrivalent vaccine composition including a recombinant influenza virus HA. Typically, the standard of care, recombinant HA vaccine composition includes recombinant HA from H1N1, H3N2, B / Victoria, and B / Yamagata lineages. Vaccine efficacy can be expressed as the percentage of disease reduction between a vaccinated population and a non-vaccinated population or a population administered a different vaccine. In certain embodiments, vaccine efficacy may be calculated by subtracting the rate of disease cases in the vaccinated population from the rate of disease cases in the non-vaccinated population and dividing by the rate of disease cases in the non-vaccinated population according to the following formula: [(rate of disease in non-vaccinated population)-(rate of disease in vaccinated population) / (rate of disease in non-vaccinated population) x 100].

[0226] Also provided are methods of preventing influenza virus disease in a subject, comprising administering to the subject any of the vaccines described herein, including, for example, a vaccine comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof, in an amount effective to prevent influenza virus disease in the subject. Similarly, the present disclosure provides any of the vaccines described herein for use in preventing influenza virus disease in a subject, including, for example, a vaccine comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof. Also disclosed herein are immunogenic compositions comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof as described herein, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof, for the manufacture of a vaccine for use in preventing influenza virus disease in a subject.

[0227] Also provided are methods of inducing an immune response against influenza virus HA and influenza virus NA in a subject, comprising administering to the subject any of the vaccines described herein, including, for example, a vaccine comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or combinations thereof. Similarly, the present disclosure provides any of the vaccines described herein for use in inducing an immune response against influenza virus HA and influenza virus NA in a subject, including, for example, a vaccine composition comprising (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or combinations thereof. Also disclosed herein is an immunogenic composition comprising: (i) one or more influenza virus HAs, one or more influenza virus NAs, or a combination thereof as described herein, and (ii) one or more ribonucleic acid molecules encoding influenza virus HAs, influenza virus NAs, or a combination thereof as described herein, for the manufacture of a vaccine for use in inducing an immune response against influenza virus HA and influenza virus NA in a subject.

[0228] A vaccine comprising an HA, NA and / or ribonucleic acid molecule as described herein and an optional adjuvant may be administered prior to or after the onset of one or more symptoms of influenza infection. That is, in some embodiments, the vaccine described herein may be administered prophylactically to prevent influenza infection or ameliorate symptoms of possible influenza infection. In some embodiments, a subject is at risk for influenza virus infection if the subject comes into contact with other individuals or livestock (e.g., pigs) known or suspected to be infected with seasonal or pandemic influenza virus, and / or if the subject is present in an area where influenza infection is known or considered to be epidemic or endemic. In some embodiments, the vaccine is administered to a subject suffering from influenza infection, or the subject is exhibiting one or more symptoms commonly associated with influenza infection. In some embodiments, the subject is known or believed to have been exposed to influenza virus. In some embodiments, the subject is at risk or susceptible to influenza infection if the subject is known or believed to have been exposed to influenza virus. In some embodiments, a subject is known to be or is believed to be exposed to influenza virus if the subject has been in contact with other individuals or livestock (e.g., pigs) known or suspected to be infected with pandemic influenza virus, and / or if the subject is or has been present in an area where influenza infection is known or believed to be epidemic or endemic. The vaccines disclosed herein can be used to treat or prevent disease caused by either or both seasonal and pandemic influenza strains.

[0229] Vaccines according to the present disclosure may be administered in any amount or dose appropriate to achieve the desired outcome. In some embodiments, the desired outcome is the induction of a sustained adaptive immune response against a broad range of influenza strains, including both seasonal and pandemic strains. In some embodiments, the desired outcome is a reduction in the intensity, severity and / or frequency and / or delay in onset of one or more symptoms of influenza infection. The required dose may vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition used and its method of administration.

[0230] In various embodiments, the vaccines described herein are administered to a subject, which may be any member of the animal kingdom. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human subject is a bird (e.g., a chicken or a bird), a reptile, an amphibian, a fish, an insect, and / or a worm. In some embodiments, the non-human subject is a mammal (e.g., a ferret, a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig).

[0231] In some embodiments, the vaccines described herein are administered to a human subject. In certain 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 old 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 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 elderly person (at least 60 years old or at least 65 years old). In some embodiments, the human subject is a non-elderly adult (at least 18 years old and less than 65 years old or at least 18 years old and less than 60 years old).

[0232] Generally, the methods and uses of the vaccines described herein include a single administration to a subject (i.e., no booster administration). However, in some embodiments, the methods and uses of the vaccines described herein include a prime-boost vaccination strategy. Prime-boost vaccination involves administering a prime vaccine and then, after a period of time, administering a boost vaccine to a subject. The immune response is "primed" upon administration of the prime vaccine and "boosted" upon administration of the boost vaccine. The prime vaccine may include a vaccine comprising influenza virus HA, influenza virus NA and / or ribonucleic acid molecules as described herein and an optional adjuvant. Similarly, the boost vaccine may include a vaccine comprising influenza virus HA, influenza virus NA and / or ribonucleic acid molecules as described herein and an optional adjuvant. The prime vaccine may be the same as the boost vaccine, but need not be the same. The boost vaccine is generally administered several weeks or months after administration of the prime 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 later. In certain embodiments, the recipient of the prime-boost vaccination is a naive subject, typically a naive infant or child.

[0233] The vaccine may be administered using any suitable route of administration, including, for example, parenteral delivery, as discussed above.

[0234] Generally, influenza virus HA, influenza virus NA and / or ribonucleic acid molecule as described herein and optional adjuvant are administered together as components of the same vaccine composition. However, influenza virus HA, influenza virus NA and / or ribonucleic acid molecule as described herein need not be administered as part of the same vaccine composition. That is, if desired, influenza virus HA, influenza virus NA, ribonucleic acid molecule and / or optional adjuvant as described herein can be administered separately to a subject. For example, a first vaccine comprising at least four influenza virus HA proteins, such as four recombinant influenza virus HAs, can be administered to a subject separately from a second vaccine comprising one or more ribonucleic acids encoding one or more, such as four, influenza virus NA proteins. When the first and second vaccines are administered separately, the first and second vaccines can be administered to a subject at different sites.

[0235] The present disclosure will be better understood with reference to the following examples.

[0236] Representative embodiments of the present disclosure 1. (i) one or more influenza virus proteins selected from one or more influenza virus hemagglutinin (HA) proteins, one or more influenza virus neuraminidase (NA) proteins, or a combination thereof; (ii) one or more ribonucleic acid molecules encoding one or more influenza virus proteins selected from one or more influenza virus HA proteins, one or more influenza virus NA proteins, or a combination thereof; An immunogenic composition comprising: 2. The immunogenic composition of embodiment 1, wherein the one or more influenza virus proteins in (i) are recombinant influenza virus proteins. 3. The immunogenic composition according to embodiment 1 or 2, wherein the one or more influenza virus proteins in (i) are present in an inactivated influenza virus (IIV). 4. The immunogenic composition according to any one of embodiments 1 to 3, wherein said one or more types of ribonucleic acid molecules are mRNA molecules. 5. The immunogenic composition according to any one of embodiments 1 to 4, wherein said immunogenic composition comprises not more than eight influenza virus proteins in (i) and ribonucleic acid molecules in (ii) encoding not more than eight influenza virus proteins. 6. An immunogenic composition according to any one of embodiments 1 to 5, (i) the one or more types of influenza virus proteins include 1 to 8 types of influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA derived from the B / Victoria lineage, influenza virus HA derived from the B / Yamagata lineage, influenza virus N1 NA, influenza virus N2 NA, influenza virus NA derived from the B / Victoria lineage, or influenza virus NA derived from the B / Yamagata lineage; An immunogenic composition, wherein the one or more ribonucleic acid molecules of (ii) encode one to eight types of influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA derived from the B / Victoria lineage, influenza virus HA derived from the B / Yamagata lineage, influenza virus N1 NA, influenza virus N2 NA, influenza virus NA derived from the B / Victoria lineage, or influenza virus NA derived from the B / Yamagata lineage. 7. The immunogenic composition according to any one of embodiments 1 to 6, comprising not more than four influenza virus proteins in (i) and a ribonucleic acid molecule in (ii) encoding not more than four influenza virus proteins. 8. The immunogenic composition according to any one of embodiments 1 to 7, which is an octavalent immunogenic composition. 9. The immunogenic composition according to any one of embodiments 1 to 8, wherein the one or more influenza virus proteins in (i) comprise four recombinant influenza virus HA proteins; and the one or more ribonucleic acid molecules encode four influenza virus NA proteins. 10. The immunogenic composition according to any one of embodiments 1 to 8, wherein the one or more influenza virus proteins in (i) include four recombinant influenza virus NA proteins; and the one or more ribonucleic acid molecules encode four influenza virus HA proteins. 11. The immunogenic composition according to any one of embodiments 1 to 6, which is a 16-valent immunogenic composition. 12. An immunogenic composition according to any one of embodiments 1 to 6, (a) the one or more influenza virus proteins are a first influenza virus HA protein, wherein said first influenza virus HA protein is H1 HA; a second influenza virus HA protein, wherein said second influenza virus HA protein is H3 HA; A third influenza virus HA protein from the B / Victoria influenza virus lineage; and A fourth influenza virus HA protein from the B / Yamagata influenza virus lineage Includes; (b) said one or more ribonucleic acid molecules a first influenza virus NA protein, said first influenza virus NA protein being N1 NA; a second influenza virus NA protein, said second influenza virus NA protein being N2 NA; A third influenza virus NA protein from the B / Victoria influenza virus lineage; and A fourth influenza virus NA protein from the B / Yamagata influenza virus lineage An immunogenic composition encoding the 13. The immunogenic composition of embodiment 12, wherein the H1 HA is from an H1N1 influenza virus strain, the H3 HA is from an H3N2 influenza virus strain, the N1 NA is from an H1N1 influenza virus strain, and / or the N2 NA is from an H3N2 influenza virus strain. 14. The immunogenic composition of embodiment 13, wherein the H1 HA and the N1 NA are from the same H1N1 influenza virus strain, and / or the H3 HA and N2 NA are from the same H3N2 influenza virus strain. 15. The immunogenic composition of any one of embodiments 12 to 14, wherein each of the first, second, third and fourth influenza virus HA proteins is a recombinant influenza virus HA. 16. The immunogenic composition of any one of embodiments 1-9 or 11-15, wherein said one or more ribonucleic acid molecules encode four full-length influenza virus NA proteins. 17. An immunogenic composition according to any one of embodiments 1 to 6, (a) the one or more influenza virus proteins are a first influenza virus NA protein, said first influenza virus NA protein being N1 NA; a second influenza virus NA protein, said second influenza virus NA protein being N2 NA; A third influenza virus NA protein from the B / Victoria influenza virus lineage; A fourth influenza virus NA protein from the B / Yamagata influenza virus lineage Includes; (b) said one or more ribonucleic acid molecules a first influenza virus HA protein, wherein said first influenza virus HA protein is H1 HA; a second influenza virus HA protein, wherein said second influenza virus HA protein is H3 HA; A third influenza virus HA protein from the B / Victoria influenza virus lineage; and A fourth influenza virus HA protein from the B / Yamagata influenza virus lineage An immunogenic composition encoding the 18. The immunogenic composition of embodiment 17, wherein the N1 NA is from an H1N1 influenza virus strain, the N2 NA is from an H3N2 influenza virus strain, the H1 HA is from an H1N1 influenza virus strain, and / or the H3 HA is from an H3N2 influenza virus strain. 19. The immunogenic composition of embodiment 18, wherein the H1 HA and the N1 NA are from the same H1N1 influenza virus strain, and / or the H3 HA and the N2 NA are from the same H3N2 influenza virus strain. 20. The immunogenic composition of any one of embodiments 17 to 19, wherein each of the first, second, third and fourth influenza virus NA proteins is a recombinant influenza virus NA. 21. The immunogenic composition of embodiment 20, wherein each of the first, second, third and fourth influenza virus NA proteins is a modified recombinant tetrameric influenza virus NA comprising four modified recombinant monomeric NA molecules, each of the modified recombinant monomeric NA molecules comprising the head region and heterologous tetramerization domain of the influenza virus NA, but lacking all or substantially all of the cytoplasmic tail, transmembrane region and stalk region of the influenza virus NA, and wherein the four modified recombinant monomeric NA molecules form a modified recombinant tetrameric NA when expressed in a host cell. 22. The immunogenic composition of embodiment 21, wherein the heterologous tetramerization domain is a Staphylothermus marinus tetrabrachion tetramerization domain, a GCN4 leucine zipper tetramerization domain, a tetramerization domain from a paramyxovirus phosphoprotein, or a human vasodilator-stimulated phosphoprotein (VASP) tetramerization domain. 23. The second influenza virus NA protein is a modified recombinant tetrameric N2 NA comprising four modified recombinant monomeric influenza virus N2s, each of which is Contains the head region of influenza virus N2, The immunogenic composition of embodiment 20, wherein each of the modified recombinant monomeric influenza virus N2 does not contain all or substantially all of the cytoplasmic tail, transmembrane region, and stalk region of the influenza virus N2, and each of the modified recombinant monomeric influenza virus N2 does not contain a heterologous oligomerization domain. 24. The immunogenic composition according to embodiment 23, wherein the modified recombinant monomeric influenza virus N2 lacks amino acids 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83 or 1-84 of the influenza virus N2. 25. The immunogenic composition according to any one of embodiments 1 to 24, wherein at least one of the one or more influenza virus proteins comprises an influenza virus HA protein and / or an influenza virus NA protein having a molecular sequence identified or designed from a machine learning model, and / or at least one of the one or more ribonucleic acid molecules encodes one or more influenza virus proteins having a molecular sequence identified or designed from a machine learning model. 26. The immunogenic composition of any one of embodiments 1 to 25, further comprising an adjuvant. 27. The immunogenic composition of embodiment 26, wherein the adjuvant comprises a squalene-in-water adjuvant or a liposome-based adjuvant. 28. The immunogenic composition according to any one of the preceding embodiments, wherein said one or more ribonucleic acid molecules comprise at least one chemically modified nucleotide. 29. The immunogenic composition of embodiment 28, wherein the at least one chemically modified nucleotide comprises pseudouridine, optionally N1-methylpseudouridine, 2'-fluororibonucleotides, 2'-methoxyribonucleotides and / or phosphorothioate linkages. 30. The immunogenic composition of any one of embodiments 1 to 29, wherein the one or more influenza virus HA proteins are recombinant influenza virus HA proteins produced by a baculovirus expression system in cultured insect cells. 31. The immunogenic composition of any one of embodiments 1-30, wherein one or more of the influenza virus NA proteins is a recombinant influenza virus NA protein produced by Chinese Hamster Ovary (CHO) cells. 32. The immunogenic composition of any one of embodiments 1 to 31, wherein the one or more ribonucleic acid molecules are encapsulated in a lipid nanoparticle (LNP). 33. An immunogenic composition according to any one of embodiments 1 to 32, wherein said one or more types of ribonucleic acid molecules are encapsulated in LNPs and do not further comprise an adjuvant. 34. An immunogenic composition according to any one of embodiments 1 to 33, comprising at least two types of ribonucleic acid molecules encapsulated in the same LNP. 35. An immunogenic composition according to any one of the preceding embodiments, comprising at least four types of ribonucleic acid molecules encapsulated in the same LNP. 36. The immunogenic composition according to any one of the preceding embodiments, wherein said influenza virus protein in (i) and / or said ribonucleic acid molecule in (ii) are derived from a standard of care influenza strain. 37. The immunogenic composition of any one of embodiments 32 to 36, wherein the LNP comprises a cationic lipid, a polyethylene glycol-conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid. 38. The LNP: cationic lipids at a molar ratio of 35% to 45%, PEGylated lipids at molar ratios between 0.25% and 2.75%, Cholesterol-based lipids at a molar ratio of 25% to 35%, and Helper lipids at a molar ratio of 25% to 35% 38. The immunogenic composition of embodiment 37, comprising: 39. The LNP: cationic lipid at a molar ratio of 40%, PEGylated lipid at a molar ratio of 1.5%, Cholesterol-based lipids at a molar ratio of 28.5% and 30% molar ratio of helper lipid The immunogenic composition of embodiment 38, comprising: 40. The immunogenic composition according to any one of embodiments 37 to 39, wherein the cationic lipid is selected from the group comprising OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10 and GL-HEPES-E3-E12-DS-3-E14. 41. The immunogenic composition of any one of embodiments 37 to 40, wherein the cationic lipid is cKK-E10. 42. The immunogenic composition according to any one of embodiments 37 to 41, wherein the PEGylated lipid is dimyristoyl-PEG2000. 43. The immunogenic composition of any one of embodiments 37-42, wherein the cholesterol-based lipid is cholesterol. 44. The immunogenic composition of any one of embodiments 37 to 43, wherein the helper lipid is dioleoyl-SN-glycero-3-phosphoethanolamine. 45. The immunogenic composition of any one of embodiments 37 to 44, wherein the LNP comprises: cKK-E10 at a molar ratio of 40%; dimyristoyl-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and dioleoyl-SN-glycero-3-phosphoethanolamine at a molar ratio of 30%. 46. ​​The immunogenic composition of any one of embodiments 32 to 37, wherein the LNP comprises (i) ALC-0315 as the cationic lipid, (ii) N,N-ditetradecylacetamide-polyethylene glycol as the PEGylated lipid, (iii) DSPC as the helper lipid, and (iv) cholesterol. 47. The immunogenic composition according to any one of embodiments 32 to 37 or 46, wherein the LNP comprises: (i) ALC-0315 as the cationic lipid in a molar ratio of about 25% to about 65%, (ii) N,N-ditetradecylacetamide-polyethylene glycol as the PEGylated lipid in a molar ratio of about 0.5% to about 2.6%, (iii) DSPC as the helper lipid in a molar ratio of about 5% to about 15%, and (iv) cholesterol in a molar ratio of about 20% to about 60%, such as i) ALC-0315 as the cationic lipid in a molar ratio of about 46.3%, (ii) ALC-0159 as the PEGylated lipid in a molar ratio of about 1.6%, iii) DSPC as the helper lipid in a molar ratio of about 9.4%, and (iv) cholesterol in a molar ratio of about 42.7%. 48. An immunogenic composition according to any one of embodiments 1 to 47, wherein each of the influenza virus proteins in (i) is present in the immunogenic composition in an amount ranging from about 0.1 μg to about 90 μg, optionally from about 1 μg to about 60 μg or from about 5 μg to about 45 μg. 49. The immunogenic composition according to any one of embodiments 1 to 48, wherein each of the ribonucleic acid molecules is present in the immunogenic composition in an amount ranging from about 0.1 μg to about 150 μg, optionally from about 1 μg to about 60 μg or from about 5 μg to about 45 μg. 50. An immunogenic composition according to any one of embodiments 1 to 49, formulated for intramuscular injection. 51. A vaccine comprising the immunogenic composition according to any one of claims 1 to 50 and a pharmaceutical carrier. 52. A method for immunizing a subject against influenza virus, comprising administering to the subject an immunologically effective amount of the vaccine of embodiment 51. 53. The method of embodiment 52, wherein the method prevents influenza virus infection in the subject. 54. The method of embodiment 52 or 53, which generates a protective immune response in the subject. 55. The method of embodiment 54, wherein the protective immune response comprises an HA antibody response and / or an NA antibody response. 56. The method of any one of claims 52 to 55, wherein the subject is a human. 57. The method of any one of embodiments 52-56, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally. 58. The method of any one of embodiments 52-57, for treating or preventing a disease caused by either or both seasonal and pandemic influenza strains. 59. The method of any one of embodiments 52-58, wherein the subject is a human and the human is 6 months or older, under 18 years of age, at least 6 months and under 18 years of age, at least 18 years of age and under 65 years of age, at least 6 months of age and under 5 years of age, at least 5 years of age and under 65 years of age, at least 60 years of age, or at least 65 years of age. 60. A method for reducing one or more symptoms of influenza virus infection, comprising administering to a subject a prophylactically effective amount of the vaccine of embodiment 51. 61. A method for enhancing or broadening a protective immune response in a subject, comprising administering to the subject an immunologically effective amount of a vaccine described in embodiment 51, wherein the vaccine improves the vaccine efficacy of a standard of care influenza virus vaccine composition by an amount ranging from about 5% to about 100%, such as at least about 20%. 62. The method of embodiment 61, wherein the standard of care influenza virus vaccine composition is an inactivated influenza virus composition comprising inactivated influenza viruses from H1N1 strains, H3N2 strains, B / Victoria lineage and B / Yamagata lineage. 63. The method of embodiment 61, wherein the standard of care influenza virus vaccine composition comprises recombinant influenza virus HA from H1N1 strains, H3N2 strains, B / Victoria lineage and B / Yamagata lineage. 64. The method of any one of embodiments 52-63, comprising administering to the subject two doses of the vaccine, spaced 2 to 6 weeks apart, optionally 4 weeks apart. EXAMPLES

[0237] The following examples should be considered illustrative, and not limiting, of the scope of the above disclosure.

[0238] Animal studies were performed in accordance with the Public Health Service (PHS) guidelines for the Humane Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals, and with approved animal protocols from the Sanofi Institutional animal Care and Use Committee (IACUC). All animals were housed under specific pathogen-free conditions with food and water available ad libitum.

[0239] Influenza virus: Reassortant H6 viruses used in enzyme-linked lectin assays (ELLA) were generated by reverse genetics, with each reassortant expressing the targeted NA antigen, HA from A / mallard / Sweden / 81 / 2002 H6N1 and an internal gene ("PR8") from A / Puerto Rico / 8 / 1934 H1N1. HA and NA segments, including non-coding regions, were generated by custom gene synthesis (Geneart AG), and the PR8 segment was derived from a virus isolate. All segments were cloned into bidirectional transcription plasmids derived from pUC57 (Genscript) with the integration of polymerase (Pol) I and Pol II promoters. Briefly, 293FT cells (Thermo Fisher Scientific) were transfected with a total of eight plasmids representing each influenza virus segment using Lipofectamine 2000 CD (Thermo Fisher Scientific). After 24 hours, MDCK-ATL cells (ATCC) were added to the transfected cells in the presence of TPCK-treated trypsin (Sigma) to allow influenza virus growth. Seven days after MDCK addition, the cell culture supernatant containing influenza virus was harvested and passaged in 8-10 day old embryonated chicken eggs (Charles River Laboratories, Inc.). The inoculated eggs were incubated at 37°C for 48 hours, then cooled to 4°C for 12 hours, harvested, and clarified by low speed centrifugation (3,000 rpm, 20 min). Virus titers were determined by plaque assay on MDCK cells.

[0240] Egg-grown stocks of A / Michigan / 45 / 2015 (H1N1), A / Singapore / INFIMH-16-0019 / 2016 (H3N2), B / Colorado / 06 / 2017 (Victoria lineage), B / Maryland / 15 / 2016 (Victoria lineage), and B / Phuket / 3073 / 2013 (Yamagata lineage) included in the HAI testing were provided by Sanofi Paster Global Clinical Immunology (Swiftwater, PA). Wild-type influenza A / Perth / 16 / 2009 (H3N2) used in the ferret studies was provided by IIT Research Institute (Chicago, IL). All viruses were stored at <-65°C until use.

[0241] Vaccine antigen: Constructs were designed for expression of recombinant, soluble influenza NA. Both tetrameric and monomeric NA construct designs include an N-terminal CD5 secretion signal peptide, an optional 6HIS tag (for purification) and a globular neuraminidase head domain. The tetrameric design (rTET-NA) also contains a tetrabrachion domain between the HIS tag and the globular head for multimerization. The defined amino acid sequence was used to assemble a codon-optimized synthetic gene from oligonucleotides and / or PCR products and insert the fragment into pcDNA3.4-TOPO (ThermoFisher). Plasmid DNA was purified from transformed bacteria and adjusted to achieve the appropriate concentration for gene transfer. Protein expression was performed in CHO-S cells using the ExpiCHO™ Expression System Max Titer Protocol (ThermoFisher). A clarification step was performed to separate the secreted protein from the cells. NA protein was purified from host cell proteins by affinity (HisTrap™ HP column-GE Healthcare) followed by anion exchange chromatography (HiTrap™ Q HP-GE HealthCare), dialysis into 10 mM phosphate buffered saline (pH 7.2) and 0.2 μm sterile filtration. NA vaccine preparations were made according to current good research practices (cGRPs).

[0242] Enzyme-linked lectin assay (ELLA) assessment of NAI responses: NAI antibody responses were measured against H6 reassortant viruses containing NA from the strain of interest by ELLA as previously described in Couzens, An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera, J. Virological Methods 2014, 210:7-14. Briefly, H6 reassortant viruses containing NA from the strain of interest were titrated in fetuin-coated 96-well plates to determine the standard amount of virus that provides 70% of the maximum NA enzyme activity. Titration of NAI antibodies present in the serum was achieved by performing two-fold serial dilutions of heat-inactivated serum. A total of 50 μL of diluted serum was then added to 50 μL of diluted virus corresponding to 70% of the maximum NA enzyme activity in the fetuin-coated plate. The serum-virus mixture was incubated overnight at 37°C. Plates were washed four times, incubated with horseradish peroxidase-(HRP-) conjugated peanut agglutinin (PNA) and washed again before color development by addition of o-phenylenediamine dihydrochloride (OPD). Low or no signal compared to virus control indicates inhibition of NA activity due to the presence of NA-specific antibodies. NAI titers were fitted using a nonlinear four-parameter logistic (4PL) curve using GraphPad Prism software and the 50% maximal inhibitory concentration (IC 50 ) was calculated.

[0243] Hemagglutinin inhibition (HAI) assay: Prior to the HAI assay, sera were treated with receptor-destroying enzyme (RDE; Denka Seiken, Co., Japan) to inactivate nonspecific inhibitors. RDE-treated sera were serially diluted (2-fold dilutions) in v-bottom microtiter plates. Equal amounts of each virus from the HAI readout panel were added to each well (4 hemagglutination units (HAU) per well). For this example, unless otherwise indicated, the homologous virus panel included A / Michigan / 45 / 2015 (H1N1), A / Singapore / INFIMH-16-0019 / 2016 (H3N2), B / Colorado / 06 / 2017 or B / Maryland / 15 / 2016 (Victoria lineage) and B / Phuket / 3073 / 2013 (Yamagata lineage) viruses grown in eggs. Plates were covered and incubated at room temperature for 20 minutes (or 45-60 minutes), followed by the addition of a 1% mixture of chicken red blood cells (CRBCs) or a 0.5% mixture of turkey red blood cells (TRBCs) (Lampire Biologicals) in PBS. The plates were mixed by vortexing, covered and the RBCs were allowed to settle at room temperature for approximately 30 minutes to an hour. The HAI titer was determined by the reciprocal dilution of the last well containing non-agglutinated RBCs.

[0244] HINT mNT Influenza Protocol: Neutralizing titers against influenza strains were measured as described in Jorquera, PA et al, Insights into the antigenic advancement of influenza A (H3N2) viruses, 2011-2018, Sci. Reports 9, 2676 (2019). Briefly, serial two-fold dilutions of RDE-treated serum, 1:20 to 1:2,560, were mixed with an equal volume of virus, approximately 1000 focus forming units (FFU), and incubated at 37°C for 60 min. After incubation, MDCK-SIAT1 cell suspensions were added to the virus:serum mixture and incubated for approximately 22 h. Monolayers were fixed with methanol and prepared for staining. The wells were then incubated with anti-influenza monoclonal antibody against nucleoprotein (NP), followed by ALEXA FLUOR® 488-conjugated secondary antibody, the cells were washed, and the plates were scanned on CTL ImmunoSpot® Cell Imaging v2. The counts from the plates were transferred to Graphpad Prism software to calculate the neutralization titer that achieved a 50% reduction in foci from a sigmoid curve. This assay measures the inhibition of virus entry compared to trypsin-free, serum-free virus input control wells. The counts are individual infected cells, and this assay is suitable for all live virus subtypes, including H1, H3, BVic, and BYam.

[0245] For the following examples, recombinant HA protein was obtained from Protein Sciences. Briefly, purified HA protein was produced in a continuous insect cell line (EXPRESSF+®) derived from Sf9 cells and grown in serum-free medium. IIV was prepared from influenza virus grown in embryonated chicken eggs, inactivated with formaldehyde, concentrated, purified by zonal centrifugation on a sucrose gradient, separated with Triton® X-100, further purified, and then suspended in sodium phosphate-buffered isotonic sodium chloride solution. The preparation was sterile filtered using a 0.2 μm syringe filter. Live influenza virus-derived neuraminidase (LVNA) was isolated from influenza virus grown in embryonated chicken eggs. Virus was purified by sucrose gradient ultracentrifugation, NA was extracted by detergent solubilization, further purified by column chromatography, and suspended in sodium phosphate-buffered isotonic sodium chloride solution. The preparation was sterile filtered using a 0.2 μm syringe filter.

[0246] Example 1 - Evaluation of multivalent HA and NA immunogenicity in mice Mice were injected with a prime vaccine on day 0 and the same dose of a booster vaccine on day 21. Blood was collected on days 1, 20, 22 and 35.

[0247] For monovalent compositions containing mRNA encoding HA antigens, mRNA encoding each of the following was used individually: H1, H3, HA from the B / Victoria lineage, and HA from the B / Yamagata lineage (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013). Tetravalent vaccine compositions containing mRNAs encoding each of N1, N2, NA from the B / Victoria lineage, and NA from the B / Yamagata lineage (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013) were prepared and administered as a quadrivalent vaccine or in combination with a quadrivalent rHA vaccine composition to create a hybrid octavalent vaccine. See Table 1 below. Additionally, quadrivalent vaccine compositions containing mRNAs encoding each of H1, H3, HA from the B / Victoria lineage, and HA from the B / Yamagata lineage were prepared and administered as a quadrivalent vaccine, as shown in Table 1 below. Each mRNA for both the monovalent and quadrivalent compositions was added in an amount of 0.4 μg / strain. As a control, an inactivated influenza vaccine (IIV) containing the 2018 / 2019 standard of care influenza strain was used (QIV(2018 / 2019)).

[0248] For recombinant antigens, a tetravalent vaccine composition was used containing rHA with each of H1, H3, HA from the B / Victoria lineage, and HA from the B / Yamagata lineage (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013), as shown below in Table 1. Each recombinant HA was added to the composition in an amount of either 0.1 μg / strain or 1 μg / strain, and with or without adjuvant (AF03), as shown below in Table 1.

[0249] For each group, n=6 mice. HAI titers were measured against the following influenza virus strains: A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013 (grown in eggs). Lipid nanoparticle diluent was used as a negative control. The results are reported in Table 1 below.

[0250] [Table 1]

[0251] No interference was observed between the tetravalent and octavalent responses. As shown above, the octavalent hybrid combination of tetravalent NA mRNA and tetravalent recombinant HA (1 μg / strain and AF03) (column 12) showed a significant improvement in HAI over tetravalent recombinant HA (1 μg / strain and -AF03) alone (column 7). Thus, the combination of tetravalent NA vaccine and tetravalent rHA unexpectedly enhanced the HAI response. The octavalent hybrid combination was within 4-fold of the tetravalent recombinant HA against all four influenza strains evaluated.

[0252] Similarly, NAI titers were similarly assessed in mice using four influenza virus strains: A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013. The results are shown in Table 2 below.

[0253] [Table 2]

[0254] As shown in Table 2, vaccination with an octavalent vaccine combination of four NA-encoding mRNAs and four recombinant HAs demonstrated NAI titers comparable to those observed with the tetravalent NA mRNAs. Thus, the data from Tables 1 and 2 reveal that the hybrid octavalent vaccine was able to induce robust HA and NA immune responses and that the presence of immunodominant HAs from four different influenza strains does not appear to suppress or interfere with anti-NA immune responses. For example, compare column 6 with columns 9 and 10 in Table 2. Furthermore, when compared to the standard of care IIV vaccine (from 2018 / 2019), the NA titers induced by the octavalent vaccine (0.1 μg / strain) against H1N1, H3N2, B Victoria and B Yamagata strains were elevated.

[0255] Example 2 - Evaluation of multivalent HA and NA immunogenicity in ferrets Ferrets used to evaluate the immunogenicity of the multivalent hybrid vaccine were administered the following vaccines: (1) a mixture of four mRNAs encoding NA antigens (N1, N2, BvNA, and ByNA) (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013); (2) a mixture of four mRNAs encoding HA antigens (H1, H3, BvHA, and ByHA) (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013); The mice were vaccinated twice, 21 days apart, with either (1) a mixture of four recombinant HA antigens (derived from A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013), (2) a mixture of four recombinant HA antigens (H1, H3, BvHA, and ByHA), or (3) a mixture of four mRNAs encoding NA antigens (N1, N2, BvNA, and ByNA) combined with a mixture of four recombinant HA antigens (H1, H3, BvHA, and ByHA). Each HA contained HA from one of four strains: A / Michigan / 45 / 2015 (H1); A / Singapore / Infimh-16-0019 / 2016 (H3); B / Maryland / 15 / 2016 (B / Victoria lineage); and B / Phuket / 3073 / 2013 (B / Yamagata lineage). All antigens were administered in a 1:1 ratio without adjuvant.

[0256] All ferrets were bled under sedation at baseline, 1 day or immediately prior to booster, at the time of booster vaccination, and 2 weeks after challenge if required. Serum samples (stored at -20°C until required) were tested by ELLA to assess NAI activity. Additionally, a HINT mNT assay was performed to assess antibody responses to hemagglutinin antigens after polyvalent vaccination.

[0257] n=6 ferrets for each group. HINT titers were measured against the following influenza virus strains: A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Iowa / 06 / 2017; and B / Phuket / 3037 / 2013 (the A / Singapore / Infimh160019 / 2016 readout strain was grown in eggs, the other three strains were cell-grown viruses). Results are reported in Table 3 below. Ferrets were primed with vaccine on day 0 and boosted with the same dose of vaccine on day 21. Blood was collected on days -7, 1, 20, 22, and 42.

[0258] [Table 3]

[0259] As shown above, the octavalent hybrid combination of tetravalent NA mRNA and tetravalent recombinant HA showed a significant increase in titers compared to tetravalent recombinant HA, with more than four-fold increase observed against A / Singapore / Infimh160019 / 2016 (330.2v.90), B / Iowa / 06 / 2017 (108.3v.11.7) and B / Phuket / 3037 / 2013 (275.2v.77.9). Indeed, the octavalent hybrid combination of tetravalent NA mRNA and tetravalent recombinant HA showed synergy when compared to the HAI titers observed when tetravalent NA mRNA and tetravalent recombinant HA were administered individually.

[0260] Similarly, NA titers were similarly evaluated in ferrets using four strains of influenza virus: A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013. The results are shown below in Table 4 (day 20) and Table 5 (day 42).

[0261] [Table 4]

[0262] [Table 5]

[0263] As shown in Table 4 above, after a single prime dose, vaccination with an octavalent hybrid combination of tetravalent NA mRNA and tetravalent recombinant HA induced similar NAI titers as vaccination with tetravalent NA mRNA. As shown in Table 5, booster administration of an octavalent hybrid combination of tetravalent NA mRNA and tetravalent recombinant HA demonstrated an increase in overall NAI titers (37.4) over a single prime dose (35.4). Thus, the data from Tables 3-5 indicate that the hybrid octavalent vaccine was capable of inducing robust HA and NA immune responses and that the presence of immunodominant HAs from four different influenza strains does not appear to suppress or interfere with anti-NA immune responses.

[0264] Example 3 - Evaluation of broad NAI immunogenicity using images of multivalent HA and NA vaccines in ferrets To generate the hybrid octavalent vaccine, the NAs for each of N1, N2, B / Victoria NA, and B / Yamagata NA (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013) were determined as shown in Table 6 below. The tetravalent vaccine composition containing mRNA was combined with a tetravalent vaccine composition containing rHA for each of H1, H3, B / Victoria HA and B / Yamagata HA (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013). Ferrets were injected with the prime vaccine on day 0 and the same dose of boost vaccine on day 21. Control ferrets received either PBS or the recombinant HA tetravalent vaccine without NA mRNA on days 0 and 21. Blood was collected on days 1, 20, 22 and 42 for all groups. NAI titers were measured against the following influenza virus strains: A / Singapore / Infimh160019 / 2016; A / Hatay / 4990 / 2016; A / Sweden / 3 / 2017; A / Louisiana / 13 / 2017; A / Townsville 51 / 2016; A / Aksaray / 4048 / 2016; A / Perth / 16 / 2009; and A / Ohio 13 / 2017. The results are reported in Table 6 below.

[0265] [Table 6]

[0266] As shown above, a broad range of NAI responses across strains was evident following administration of the hybrid vaccine combination.

[0267] Assessment of NA heterologous breadth was performed via Multiplex Serology ELISA chips, where a ferret serum pool from day 42 was evaluated at one dilution for binding to 16 TetNA and TET-HA. The N1 / NB and N2 heterologous panels were selected by amino acid distance from the homologous strain (N1 A / Michigan / 45 / 2015 or N2 A / Singapore / Infimh / 160019 / 2016) as shown in Table 7 below.

[0268] [Table 7]

[0269] Binding to the N1 heterologous panel and NB homologous strains at a 1:4000 dilution is shown in Figure 1A and Figure 1B, respectively. Binding to the N2 homologous panel at a 1:1000 dilution is shown in Figure 1C. As shown in Figure 1C, N2 heterologous binding levels decrease as the heterologous strains become more distant (i.e., more amino acid differences), with the most distant strain (i.e., A / Michigan / 84 / 2916 at a distance of 63 amino acids) showing the lowest N2 binding.

[0270] Example 4 - Images of multivalent HA and NA vaccines in a pre-immune ferret model After confirmation of flu-negative HAI status, the next viral imprinted strain [1 × 10 5Pre-immunized ferrets were infected intranasally on day 0 with: A / NewCaledonia / 20 / 1999; A / Perth / 16 / 2009; B / HongKong330 / 2001; and B / Florida / 4 / 2006. On day 21, rH was measured with each of H1, H3, B / Victoria HA, and B / Yamagata HA (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013) as shown in Table 8 below. Ferrets were administered immunizations of a hybrid octavalent vaccine composition containing NA mRNA for each of N1, N2, B / Victoria NA, and B / Yamagata NA (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013) in combination with a quadrivalent vaccine composition containing A. Control ferrets were administered either PBS, a recombinant HA quadrivalent vaccine without NA mRNA (35 μg / strain), or an IIV HA quadrivalent vaccine (15 μg / strain). Blood was collected on day 20 to establish baseline titers (after virus intranasal priming) and again on day 42 to measure ELLA antibody responses after immunization. The results are shown in Table 8 below, and the mean IC50 ratios for each group are shown in Table 9 below.

[0271] [Table 8]

[0272] [Table 9]

[0273] As shown in Tables 8 and 9 above, the hybrid octavalent vaccine composition induced strong ELLA responses against most of the SOC2018 / 2019 strains.

[0274] In addition, HA neutralization titers were measured using the HINT mNT protocol, and the results are shown in Table 10 below.

[0275] [Table 10]

[0276] As shown in Table 10, similar HINT titers were observed in all groups after immunization on days 20 to 42, indicating a HINT response to A / Singapore / Infimh160019 / 2016.

[0277] It should also be noted that, as used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Optionally or optionally means that the subsequently described event or circumstance may or may not occur, and the description means that the event or circumstance may or may not occur. For example, the phrase "a composition may optionally include a combination" means that the composition may or may not include a combination of different molecules, such that the description includes both the combination and the absence of the combination (i.e., the individual members of the combination). Ranges may be expressed herein as from about one particular value and / or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by using the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that each endpoint of a range has meaning both in relation to the other endpoint, and independently of the other endpoint. All references cited in this disclosure are hereby incorporated herein in their entirety.

Claims

1. (i) one or more influenza virus proteins selected from one or more influenza virus hemagglutinin (HA) proteins, one or more influenza virus neuraminidase (NA) proteins, or a combination thereof; (ii) one or more ribonucleic acid molecules encoding one or more influenza virus proteins selected from one or more influenza virus HA proteins, one or more influenza virus NA proteins, or a combination thereof; An immunogenic composition comprising:

2. 2. The immunogenic composition of claim 1, wherein the one or more influenza virus proteins in (i) are recombinant influenza virus proteins or are present in an inactivated influenza virus (IIV), and the one or more ribonucleic acid molecules are mRNA molecules.

3. (i) the one or more influenza virus proteins comprise 1 to 4 influenza virus proteins selected from influenza virus H1 HA, influenza virus H3 HA, influenza virus HA from B / Victoria lineage, and influenza virus HA from B / Yamagata lineage; The immunogenic composition according to claim 1, wherein the one or more ribonucleic acid molecules in (ii) encode one to four influenza virus proteins selected from influenza virus N1 NA, influenza virus N2 NA, influenza virus NA derived from the B / Victoria lineage, or influenza virus NA derived from the B / Yamagata lineage.

4. The immunogenic composition of claim 1, which is an octavalent, hexavalent, or 16-valent immunogenic composition.

5. The H1 HA is derived from an H1N1 influenza virus strain, the H3 HA is derived from an H3N2 influenza virus strain, the N1 NA is derived from an H1N1 influenza virus strain, and / or the N2 NA is derived from an H3N2 influenza virus strain. derived from a strain of the virus, or 4. The immunogenic composition of claim 3, wherein the H1 HA and the N1 NA are derived from the same H1N1 influenza virus strain, and / or the H3 HA and N2 NA are derived from the same H3N2 influenza virus strain.

6. 2. The immunogenic composition of claim 1, wherein at least one of the one or more influenza virus proteins comprises an influenza virus HA protein and / or an influenza virus NA protein having a molecular sequence identified or designed from a machine learning model, and / or at least one of the one or more ribonucleic acid molecules encodes one or more influenza virus proteins having a molecular sequence identified or designed from a machine learning model.

7. 10. The immunogenic composition of claim 1, further comprising an adjuvant, optionally comprising a squalene-in-water adjuvant or a liposome-based adjuvant.

8. 2. The immunogenic composition of claim 1, wherein said one or more ribonucleic acid molecules comprise at least one chemically modified nucleotide, optionally wherein said at least one chemically modified nucleotide comprises pseudouridine, optionally N1-methylpseudouridine, 2'-fluororibonucleotides, 2'-methoxyribonucleotides and / or phosphorothioate linkages.

9. the one or more influenza virus HA proteins are recombinant influenza virus HA proteins produced in cultured insect cells using a baculovirus expression system; and / or 2. The immunogenic composition of claim 1, wherein one or more of the influenza virus NA proteins is a recombinant influenza virus NA produced in Chinese hamster ovary (CHO) cells.

10. The immunogenic composition of claim 1, wherein the one or more types of ribonucleic acid molecules are encapsulated in a lipid nanoparticle (LNP), optionally comprising at least two types of ribonucleic acid molecules encapsulated in the same LNP, and optionally comprising at least four types of ribonucleic acid molecules encapsulated in the same LNP.

11. 2. The immunogenic composition of claim 1, wherein the influenza virus protein in (i) and / or the ribonucleic acid molecule in (ii) are derived from a standard of care influenza strain.

12. the LNPs comprise a cationic lipid, a polyethylene glycol-conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid, and optionally (i) the LNP is a molar ratio of 35% to 45% cationic lipid; PEGylated lipid at a molar ratio of 0.25% to 2.75%; Cholesterol-based lipids at a molar ratio of 25% to 35% and Helper lipids at a molar ratio of 25% to 35% Includes; and / or (ii) the LNP is 40% molar ratio of cationic lipid, PEGylated lipid at a molar ratio of 1.5%, Cholesterol-based lipids at a molar ratio of 28.5% and 30% molar ratio of helper lipid and / or (iii) the cationic lipid is selected from the group comprising OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10 and GL-HEPES-E3-E12-DS-3-E14; and / or (iv) the cationic lipid is cKK-E10; and / or (v) the PEGylated lipid is dimyristoyl-PEG2000; and / or (vi) the cholesterol-based lipid is cholesterol; and / or (vii) the helper lipid is dioleoyl-SN-glycero-3-phosphoethanolamine; and / or (viii) the LNP is cKK-E10 at a molar ratio of 40%; Dimyristoyl-PEG2000 at a molar ratio of 1.5%; Cholesterol at a molar ratio of 28.5%; and 30% molar ratio of dioleoyl-SN-glycero-3-phosphoethanolamine Includes; and / or (ix) the LNPs comprise (i) ALC-0315 as a cationic lipid, (ii) N,N-ditetradecylacetamide-polyethylene glycol as a PEGylated lipid, (iii) DSPC as a helper lipid, and (iv) cholesterol; and / or (x) the LNPs comprise (i) ALC-0315 as the cationic lipid at a molar ratio of about 25% to about 65%, (ii) N,N-ditetradecylacetamide-polyethylene glycol as the PEGylated lipid at a molar ratio of about 0.5% to about 2.6%, (iii) DSPC as the helper lipid at a molar ratio of about 5% to about 15%, and (iv) cholesterol at a molar ratio of about 20% to about 60%, such as i) ALC-0315 as the cationic lipid at a molar ratio of about 46.3%, (ii) ALC-0159 as the PEGylated lipid at a molar ratio of about 1.6%, iii) DSPC as the helper lipid at a molar ratio of about 9.4%, and (iv) cholesterol at a molar ratio of about 42.7%. The immunogenic composition of claim 10.

13. each of the influenza virus proteins in (i) is present in the immunogenic composition in an amount ranging from about 0.1 μg to about 90 μg, optionally in an amount such as from about 1 μg to about 60 μg or from about 5 μg to about 45 μg; and / or 13. The immunogenic composition of claim 12, wherein each of said ribonucleic acid molecules is present in said immunogenic composition in an amount ranging from about 0.1 μg to about 150 μg, optionally from about 1 μg to about 60 μg or from about 5 μg to about 45 μg.

14. A vaccine comprising the immunogenic composition of any one of claims 1 to 13 and a pharmaceutical carrier.

15. 15. The vaccine of claim 14 for use in a method of immunizing a subject against influenza virus.

16. The method comprises: (i) preventing influenza virus infection in said subject; and / or (ii) generating a protective immune response in the subject, optionally wherein the protective immune response comprises an HA antibody response and / or an NA antibody response; and / or (iii) treating or preventing disease caused by either or both seasonal and pandemic influenza strains; The vaccine of claim 15.

17. 16. The vaccine of claim 15, wherein the subject is a human and the human is 6 months or older, under 18 years of age, at least 6 months and under 18 years of age, at least 18 years of age and under 65 years of age, at least 6 months of age and under 5 years of age, at least 5 years of age and under 65 years of age, at least 60 years of age or at least 65 years of age.

18. 15. The vaccine of claim 14 for use in a method for reducing one or more symptoms of influenza virus infection, the method comprising administering to a subject a prophylactically effective amount of the vaccine of claim 14.

19. 15. The vaccine of claim 14 for use in a method of enhancing or broadening a protective immune response in a subject, said method comprising administering to said subject an immunologically effective amount of the vaccine of claim 14, wherein said vaccine improves vaccine efficacy of a standard of care influenza virus vaccine composition by an amount in the range of about 5% to about 100%, such as at least about 20%, and optionally said method comprises administering to said subject two doses of the vaccine, spaced 2 to 6 weeks apart, optionally 4 weeks apart.

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