Multivalent influenza vaccines containing recombinant hemagglutinin and neuraminidase and methods of use thereof

Abstract

Disclosed herein is a multivalent vaccine or immunogenic composition comprising one or more recombinant influenza virus hemagglutinins (HA), one or more recombinant influenza virus neuraminidases (NA), and an optional adjuvant. Also disclosed are methods of using the vaccine or immunogenic composition.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of, and relies on the filing date of, U.S. Provisional Patent Application No. 63 / 276,284, filed November 5, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy (created on October 14, 2022) is named 0171_0067_PCT_Sequence_Listing.xml and is 3,450 bytes in size.

[0003] Field of the Disclosure Disclosed herein is a multivalent recombinant influenza vaccine or immunogenic composition for eliciting immunity against both influenza virus hemagglutinin (HA) and influenza virus neuraminidase (NA), as well as methods of using the multivalent recombinant influenza vaccine or immunogenic composition. [Background technology]

[0004] Influenza is caused by a virus that primarily attacks the upper respiratory tract, including the nose, throat, and bronchi, and rarely the lungs. The infection usually lasts for 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 one to two weeks without needing any treatment. However, in the very young, 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 people, the infection can lead to severe complications from underlying diseases, pneumonia, and death, but even healthy adults and older children can be affected as well. The annual seasonal influenza epidemic is thought to cause 3 to 5 million severe illnesses and 250,000 to 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, whereas 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 vaccines containing whole virions or virions treated with lipid-dissolving agents ("split" vaccines), purified glycoproteins expressed in cell culture ("subunit vaccines"), or live attenuated virus vaccines. Other types of vaccines, such as RNA / DNA-based and viral vector-based, are in development. These vaccines provide protection, in part, by inducing the production of antibodies against influenza antigens, such as HA. Antigenic evolution of influenza viruses through mutation (also referred to as antigenic drift) results in changes 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 given group, subtype, and / or strain.

[0007] Therefore, available vaccines can only protect against strains with surface glycoproteins that contain the same or cross-reactive epitopes. To provide a broader antigenic spectrum, conventional vaccines contain components from several different virus strains, for example, from both influenza A and influenza B strains. The selection of strains used in current seasonal influenza vaccines is reviewed annually to account for antigenic drift and to adapt to rapidly evolving virus strains, and is based on the recommendations of the World Health Organization (WHO). This recommendation reflects international epidemiological observations.

[0008] Current influenza virus seeds for vaccine production must be shown to have the appropriate HA antigen for the reassortment procedures used to generate the high-yield virus strains used in production. However, there are currently no requirements or restrictions regarding NA content in influenza vaccines. There is evidence that there is considerable variability in NA levels in vaccines. 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 the NA specific activity of different lots can vary by approximately 40-fold. Kendal et al. also noted a rapid decline in NA activity during 6 months of storage. As a result, the frequency of antibody responses to NA was low (mean seroconversion rate 18%) compared to HA responses (seroconversion rate 64%).

[0009] Moreover, despite growing evidence that NA-specific antibodies correlate with resistance to disease in humans, current vaccine strategies focus almost exclusively on HA antigens, as in the case of the FLUBLOK® tetravalent vaccine containing recombinant HA protein. In addition, available data on immunological responses to NA during influenza infection are limited, especially compared to data on 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 have about 10-fold less NA on the viral surface compared to HA, and established processes for concentrating HA antigens may not be suitable for maintaining NA in an enzymatically active tetrameric structure. Therefore, currently available inactivated influenza virus vaccines may contain NA, but the amount and quality vary widely and are not uniform.

[0010] Furthermore, NA has been described as 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 compared to NA. Id. This immunodominance phenomenon observed in conventional influenza vaccines remains an obstacle to the development of multivalent vaccines that can successfully achieve multivalent immune responses against multiple antigens or epitopes, especially in the case of multivalent vaccines that contain immunodominant proteins such as HA and / or as the valency in the vaccine increases (Woodruff et al., B Cell Competition for Restricted T Cell Help Suppresses Rare-Epitope Responses, CELL REPORTS 2018;25:321-27). Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, the ability to supplement influenza virus HA in a vaccine with one or more influenza virus NA proteins, which may confer enhanced and / or broader protection against circulating influenza strains by eliciting both HA and NA immune responses, is desirable. However, the ability to combine influenza virus HA and influenza virus NA in a vaccine composition to confer enhanced and / or broader protection against circulating influenza strains without antigenic competition can be a challenge, especially in multivalent vaccine compositions. [Means for solving the problem]

[0012] The present disclosure provides a vaccine or immunogenic composition comprising a plurality of recombinant influenza virus proteins, the plurality of recombinant influenza virus proteins comprising or consisting of one or more recombinant influenza virus HAs and one or more recombinant influenza virus NAs.

[0013] In various embodiments, the plurality of recombinant influenza virus proteins comprises 1, 2, 3, 4, 5, 6, 7, 8, or more recombinant influenza virus HA antigens and 1, 2, 3, 4, 5, 6, 7, 8, or more recombinant influenza virus NA antigens. In certain embodiments, the plurality of recombinant influenza virus proteins comprises or consists of 4 influenza virus HAs and 4 influenza virus NAs. In certain embodiments, the plurality of recombinant influenza virus proteins includes a first recombinant influenza virus HA, wherein the first recombinant influenza virus HA is an H1 HA; a second recombinant influenza virus HA, wherein the second recombinant influenza virus HA is an H3 HA; a third recombinant influenza virus HA, wherein the third recombinant influenza virus HA is derived from a B / Victoria lineage; a fourth recombinant influenza virus HA, wherein the fourth recombinant influenza virus HA is derived from a B / Yamagata lineage; a first recombinant influenza virus NA, wherein the first recombinant influenza virus NA is an N1 NA; a second recombinant influenza virus NA, wherein the second recombinant influenza virus NA is an N2 a second recombinant influenza virus NA, which is a NA; a third recombinant influenza virus NA, which is derived from a B / Victoria lineage; and a fourth recombinant influenza virus NA, which is derived from a B / Yamagata lineage.

[0014] In various embodiments of the vaccine or immunogenic composition disclosed herein, each of the first, second, third, and fourth recombinant influenza virus NA is a modified recombinant influenza virus NA. In certain embodiments, the modified recombinant influenza virus NA comprises a modified recombinant tetrameric influenza virus NA comprising four modified recombinant monomeric NA molecules, each of which comprises an influenza virus NA head region but lacks the influenza virus NA cytoplasmic tail, transmembrane region, and all or substantially all of the influenza virus NA stalk region, and the modified monomeric NA molecules form a modified recombinant tetrameric NA when expressed in a host cell. In certain embodiments, each modified recombinant monomeric influenza virus NA comprises a heterologous tetramerization domain, and in certain embodiments, the modified recombinant monomeric influenza virus NA does not comprise a heterologous oligomerization domain.

[0015] In certain embodiments of the present disclosure, the heterologous tetramerization domain is a Staphylothermus marinus Tetrabrachion tetramerization domain, a GCN4 leucine zipper tetramerization domain, a tetramerization domain derived from a paramyxovirus phosphoprotein, or a human vasodilator-stimulated phosphoprotein (VASP) tetramerization domain.

[0016] In one embodiment of the disclosure, each of the recombinant influenza virus HAs is produced in a baculovirus expression system, e.g., in cultured insect cells, and in one embodiment of the disclosure, each of the recombinant influenza virus NAs is produced in Chinese Hamster Ovary (CHO) cells.

[0017] In various embodiments disclosed herein, the vaccine or immunogenic composition does not comprise inactivated or live attenuated influenza virions, and in various embodiments, each of the recombinant influenza virus HAs and / or each of the recombinant influenza virus NAs are derived from a standard of care influenza strain. In certain embodiments, the H1 HA is derived from an H1N1 influenza virus strain, and / or the H3 HA is derived from an H3N2 influenza virus strain, and in certain embodiments, the N1 NA is derived from an H1N1 influenza virus strain, and / or the N2 NA is derived from an H3N2 influenza virus strain. In certain embodiments, 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 the N2 NA is derived from an H3N2 influenza virus strain. In certain embodiments, the H1 HA and N1 NA are derived from the same H1N1 influenza virus strain, and the H3 HA and N2 NA are derived from the same H3N2 influenza virus strain.

[0018] In certain embodiments, the vaccine or immunogenic compositions disclosed herein further comprise an adjuvant, which in certain embodiments comprises a squalene-in-water adjuvant (e.g., AF03) or a liposome-based adjuvant (e.g., SPA14).

[0019] In certain aspects of the disclosure, each of the recombinant influenza virus HAs is present in the vaccine or immunogenic composition in an amount ranging from about 0.1 μg to about 90 μg, and optionally in an amount ranging from about 1 μg to about 60 μg or from about 5 μg to about 45 μg, and in certain aspects, each of the recombinant influenza virus NAs is present in the vaccine or immunogenic composition in an amount ranging from about 0.1 μg to about 90 μg, and optionally in an amount ranging from about 1 μg to about 60 μg or from about 5 μg to about 45 μg. In certain embodiments, the composition is formulated for intramuscular injection.

[0020] In another aspect, disclosed herein is a vaccine comprising an immunogenic composition disclosed herein and a pharmaceutical carrier.

[0021] Also disclosed herein is a method of immunizing a subject against influenza virus, comprising administering to the subject an immunologically effective amount of a vaccine disclosed herein. Also disclosed herein is a vaccine disclosed herein for use in a method of immunizing a subject against influenza virus. Also disclosed herein is an immunogenic composition disclosed herein for manufacturing 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 (e.g., an HA antibody response and / or an NA antibody response) in the subject. In certain embodiments, the subject is a human, and in certain embodiments, the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally, or is prepared for such administration.

[0022] Another aspect of the present disclosure is a method of reducing one or more symptoms of influenza virus infection, comprising administering to a subject a prophylactically effective amount of a vaccine disclosed herein. Also disclosed herein is a vaccine disclosed herein for use in a method of reducing one or more symptoms of influenza virus infection. Also disclosed herein is an immunogenic composition disclosed herein for manufacturing a vaccine for use in a method of reducing one or more symptoms of influenza virus infection.

[0023] Also disclosed herein is a method of enhancing or expanding a protective immune response in a subject, comprising administering to the subject an immunologically effective amount of a vaccine disclosed herein, where the vaccine increases the vaccine efficacy of a standard of care influenza virus vaccine composition by an amount ranging from about 5% to about 100%, e.g., by about 10% to about 25%, or by about 40% to about 80%, or by about 40% to about 60%. Also disclosed herein is a vaccine disclosed herein for use in a method of enhancing or expanding a protective immune response in a subject, comprising administering to the subject an immunologically effective amount of a vaccine disclosed herein, where the vaccine increases the vaccine efficacy of a standard of care influenza virus vaccine composition by an amount ranging from about 5% to about 100%, e.g., by at least about 20%, or by about 40% to about 80%, e.g., by about 40% to about 60%. Also disclosed herein is an immunogenic composition disclosed herein for manufacturing a vaccine for use in a method of enhancing or expanding a protective immune response in a subject, the method comprising administering to the subject an immunologically effective amount of a vaccine disclosed herein, where the vaccine increases vaccine efficacy of a standard of care influenza virus vaccine composition by an amount ranging from about 5% to about 100%, e.g., at least about 20%, or about 40% to about 80%, e.g., about 40% to about 60%. In certain embodiments, the standard of care influenza virus vaccine is an inactivated influenza virus composition comprising an inactivated influenza virus derived 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 derived from an H1N1 strain, an H3N2 strain, a B / Victoria lineage, and a B / Yamagata lineage.

[0024] 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, the human being being 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 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. In certain aspects, the methods or uses disclosed herein comprise administering to the subject two doses of the vaccine or immunogenic composition, 2-6 weeks apart, for example 4 weeks apart. [Brief description of the drawings]

[0025] [Figure 1] 1 is a schematic diagram and partial amino acid sequence of rTET-NA (SEQ ID NO: 2). SEQ ID NO: 2 shows, in order, the CD5 signal sequence, the first linker sequence, the tetrabrachion tetramerization domain, and the second linker sequence. SEQ ID NO: 2 does not include the amino acid sequence of the NA head region. [Diagram 2]FIG. 2A is a schematic showing the experimental design of vaccination in mice, as described in Example 2. FIG. 2B is a plot showing the IC50 of NA inhibition against A / Singapore / INFIMH-16-0019 / 2016 (N2) for mice vaccinated with rTET-NA, live virus-derived NA (LVNA), or monovalent inactivated influenza vaccine (IV), both with or without adjuvant (AF03), as described in Example 2. The circle symbol represents the IC50 NA inhibition value without AF03 added, and the square represents the group with AF03 added. FIG. 2C is a plot showing the IC50 of NA inhibition against A / Michigan / 45 / 2015 (N1) for mice vaccinated with rTET-NA or monovalent inactivated influenza vaccine, both with or without adjuvant (AF03), as described in Example 2. The symbol ◯ represents the IC50 NA inhibitory value without AF03 added, and the symbol ■ represents the group with AF03 added. [Figure 3-1] Figure 3A is a schematic showing the experimental design of vaccination in naive ferrets, with two intramuscular doses of vaccine sample administered on days 0 and 21, with a terminal bleed on day 42, as described in Example 3. Figure 3B is a schematic showing the experimental design of vaccination in pre-immune ferrets (primed intranasally with virus on day 0), as discussed in Example 3. [Figure 3-2]Figure 3C is a plot showing NAI titers against A / Singapore / Infimh / 16 / 2016(N2) in naive ferrets after one or two immunizations with the following doses of rTET-NA, as described in Example 3: diluent only (sham), 5μg+AF03; 45μg+AF03, and 45μg. O symbols represent NAI titers after the first dose and ■ represents NAI titers after the second dose. Figure 3D is a plot showing NAI titers against A / Singapore / Infimh / 16 / 2016(N2) in pre-immune ferrets after single doses of diluent (sham), rTET-NA 1.8μg, 9μg, and 45μg, and IIV 1.8μg and 9μg, as described in Example 3. The open circles represent NAI titers after intranasal viral priming and the closed circles represent NAI titers after a single intramuscular vaccine boost. [Figure 3-3] Figure 3E is a graph showing the boost / prime NAI ratio against A / Singapore / Infimh / 16 / 2016 (N2) in pre-immune ferrets after single doses of diluent (sham), rTET-NA 1.8 μg, 9 μg, and 45 μg, and IIV 1.8 μg and 9 μg, as described in Example 3. Figure 3F is a plot showing NAI titers against A / Michigan / 45 / 2015 (N1) in naive ferrets after one or two immunizations with the following doses of rTET-NA, as described in Example 3: diluent (sham), 5 μg + AF03; 45 μg + AF03, and 45 μg. The circle symbol represents the NAI titer after the first dose, and the square represents the NAI titer after the second dose. [Diagram 3-4]Figure 3G is a plot showing NAI titers against A / Michigan / 45 / 2015(N1) in pre-immunized ferrets after single doses of diluent (sham), rTET-NA 1.8 μg, 9 μg, and 45 μg, and IIV 1.8 μg and 9 μg, as described in Example 3. The circle symbol represents the NAI titer after intranasal viral prime, and the square represents the NAI titer after a single intramuscular vaccine boost. Figure 3H is a graph showing the boost / prime NAI ratio against A / Michigan / 45 / 2015(N1) in pre-immunized ferrets after single doses of diluent (sham), rTET-NA 0.36 μg, 1.8 μg, 9 μg, and 45 μg, and IIV 1.8 μg and 9 μg, as described in Example 3. [Figure 4] 1 is a plot showing NAI titers against A / Perth / 16 / 2009(N2) in naive ferrets after two immunizations with the following doses of rTET-NA: diluent+AF03 (sham), 0.2 μg, 3 μg, 45 μg, 0.2 μg+AF03, 3 μg+AF03, and 45 μg+AF03, as described in Example 4. Also shown are NAI titers following infection with A / Perth / 16 / 2009 H3N2 influenza virus (prior to A / PE / 09 infection). [Diagram 5] FIG. 13 is a graph showing post-challenge weight change (daily and AUC), temperature rise (peak), and viral shedding (AUC) in ferrets prior to immunization with rTET-NA at the following doses: diluent+AF03 (sham), 0.2 μg, 3 μg, 45 μg, 0.2 μg+AF-03, 3 μg+AF03, 45 μg+AF03, as described in Example 4, and following infection with A / Perth / 16 / 2009 H3N2 influenza virus (pre-A / PE / 09 infection). [Figure 6A] FIG. 1 is a graph showing the inverse correlation between disease severity and NAI titers in vaccinated ferrets, as described in Example 4. NAI titers in ferrets with non-severe disease are shown on the left and NAI titers in ferrets with severe disease are shown on the right. [Figure 6B]1 is a graph showing a receiver operating characteristic (ROC) curve model, as discussed in Example 4, showing the area under the curve (AUC) of the ROC curve, where the AUC is significantly higher than chance. [Figure 6C] 1 is a graph showing the inverse correlation between disease severity and NAI titers in vaccinated ferrets on day 42, as described in Example 4. [Figure 7-1] Figure 7A is a schematic showing the experimental design of vaccination in ferrets, as discussed in Example 5. Figure 7B is a chart showing the influenza virus strains used in 4xrNA and 4xrHA vaccine strain selection in ferrets, as discussed in Example 5. [Figure 7-2]Figure 7C, left column, plots showing NAI titers against A / Singapore / Infimh / 16 / 2016(N2) (top row); A / Michigan / 45 / 2015(N1) (second row); B / Colorado / 06 / 2017 (third row); and B / Phuket / 3073 / 2013 (bottom row) after vaccination with (1) a single dose of 45 μg / antigen or 5 μg / antigen + adjuvant of the octavalent (4×rHA+4×rNA) recombinant vaccine composition, (2) a single dose of the tetravalent (4×rNA) recombinant vaccine composition, or (3) a single dose of the tetravalent (4×rHA) recombinant vaccine composition, as described in Example 5. The right column is a plot showing NAI titers against A / Singapore / Infimh / 16 / 2016 (N2) (top row); A / Michigan / 45 / 2015 (N1) (second row); B / Colorado / 06 / 2017 (third row); and B / Phuket / 3073 / 2013 (bottom row) following vaccination with (1) a booster dose of 45 μg / antigen or 5 μg / antigen + adjuvant of the octavalent (4×rHA + 4×rNA) recombinant vaccine composition, (2) a booster dose of the tetravalent (4×rNA) recombinant vaccine composition, or (3) a booster dose of the tetravalent (4×rHA) recombinant vaccine, as described in Example 5. Open squares represent NAI titers after administration of the octavalent recombinant composition, filled squares represent NAI titers after administration of the tetravalent (4x rNA) recombinant composition, and triangles represent NAI titers after administration of the tetravalent (4x rHA) recombinant composition. [Figure 8-1] FIG. 8A is a plot showing HAI titers against A / Singapore / Infimh / 16 / 2016 H3N2 virus after vaccination with either 45 μg / antigen or 5 μg / antigen+adjuvant of (1) tetravalent rNA (filled squares); (2) tetravalent rHA (triangles); or (3) octavalent rHA+rNA (open squares), as described in Example 5. [Figure 8-2]Figure 8B is a plot showing IgG titers measured by antibody forensics against the H3 rHA bead panel after vaccination with 45 μg / antigen of (1) octavalent rHA+rNA (Y-axis) or (2) tetravalent rHA (X-axis), as described in Example 6. Figure 8C is a plot showing IgG titers measured by antibody forensics against the H3 rHA bead panel after vaccination with 5 μg / antigen+adjuvant of (1) octavalent rHA+rNA (Y-axis) or (2) tetravalent rHA (X-axis), as described in Example 6. [Figure 8-3] Figure 8D is a plot showing HAI titers against A / Michigan / 45 / 2015 H1N1 virus after vaccination with either 45 μg / antigen or 5 μg / antigen + adjuvant of (1) tetravalent rNA (filled black squares); (2) tetravalent rHA (triangles); or (3) octavalent rHA+rNA (open squares), as described in Example 5. [Figure 8-4] Figure 8E is a plot showing IgG titers measured by antibody forensics against the H1 rHA bead panel after vaccination with 45 μg / antigen of (1) octavalent rHA+rNA (Y-axis) or (2) tetravalent rHA (X-axis), as described in Example 6. Figure 8F is a plot showing IgG titers measured by antibody forensics against the H1 rHA bead panel after vaccination with 5 μg / antigen+adjuvant of (1) octavalent rHA+rNA (Y-axis) or (2) tetravalent rHA (X-axis), as described in Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] 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. 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.

[0027] HA and NA have completely different evolutionary patterns. For example, the rate of silent nucleotide substitution has been shown to be higher than the rate of coding nucleotide substitution in all influenza virus genes, including the HA gene (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 internal proteins. The higher rate of coding nucleotide change in the HA gene compared to other genes is considered 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. have studied the evolutionary rates of epidemiologically important HA and NA antigens isolated from humans over a 10-year period and determined 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 has been 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 puts selective pressure on HA to maintain itself in partially immune populations. Therefore, because NA undergoes slower antigenic drift 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.

[0028] Because influenza viruses naturally contain about 10 times less NA than HA on the viral surface, and because established processes for concentrating HA antigens may not be suitable for maintaining NA in its enzymatically active, tetrameric conformation, the amount of NA detectable in a vaccine composition (e.g., an inactivated virus vaccine) may vary greatly. Thus, the addition of recombinant NA to the vaccine or immunogenic composition disclosed herein may allow for better control of the amount of NA contained in the vaccine or immunogenic composition. The addition of a stable recombinantly produced NA to an HA antigen (e.g., a recombinantly produced HA antigen) may allow for a better balance of both HA and NA immune responses in subjects administered the vaccine or immunogenic composition compared to currently available vaccines, which may allow for enhanced and / or broader protection against circulating influenza strains.

[0029] Thus, disclosed herein are multivalent vaccines or immunogenic compositions comprising multiple recombinant influenza virus proteins (e.g., multiple recombinant influenza virus HAs, and multiple recombinant influenza virus NAs).

[0030] definition In order to make this disclosure more readily understandable, certain terms are first defined below. Further definitions of the following terms and other terms may be found throughout this specification. 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.

[0031] 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. So, 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.

[0032] 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 element having the same name, in order to distinguish between the claim elements.

[0033] 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 the antigenic component of a vaccine.

[0034] 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) 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 understand that a particular antigen may elicit an immune response in one or some members of a target organism (e.g., mice, ferrets, rabbits, primates, humans), but not in all members of the target organism. 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 elicit 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, whether or not such interactions occur in vivo. In some embodiments, the antigen reacts with specific humoral or cellular immune products, including those elicited by heterologous immunogens. Antigens include NA and HA forms as described herein.

[0035] Approximately: As used herein, the term "approximately" or "about" as applied to one or more subject values ​​refers to a value similar to a stated reference value. In some embodiments, the term "approximately" or "about" refers to a value that falls within a range of 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 a possible value).

[0036] 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 includes 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.

[0037] Epitope: As used herein, the term "epitope" includes a moiety that is specifically recognized in whole or in part by an immunoglobulin (e.g., an antibody or T-cell 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).

[0038] 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, 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.

[0039] H1: As used herein, "H1" refers to influenza virus subtype 1 hemagglutinin (HA). Influenza A viruses are divided into Group 1 and Group 2. Group 1 and Group 2 are further divided into subtypes, which refers to classification of the virus based on the sequence of two proteins on the surface of the virus, HA and neuraminidase (NA). Currently, 18 HA subtypes (H1-H18) are recognized. Therefore, H1 is different from other HA subtypes, including H2-H18.

[0040] H3: As used herein, "H3" refers to influenza virus subtype 3 HA. As such, H3 is distinct from other HA subtypes, including H1, H2, and H4-H18.

[0041] 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 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.

[0042] Immunogen: As used herein, the term "immunogen" or "immunogenic" refers to a compound, composition, or substance that, under appropriate conditions, can stimulate 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, "immunize" means to elicit a protective immune response in a subject against an infectious disease (e.g., influenza).

[0043] Immunologically effective amount: As used herein, the term "immunologically effective amount" means an amount sufficient to immunize a subject.

[0044] 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.

[0045] 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 involve the construction of 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 a selection algorithm built. Examples of machine learning algorithms and methods can be found, for example, in PCT application 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 that has been identified or designed by machine learning, and "machine learning influenza virus NA" refers to an influenza virus NA that has been 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.

[0046] Modified: As used herein, the term "modified" refers to any protein or nucleic acid that has a different amino acid sequence or nucleic acid sequence compared to the wild type of the protein or nucleic acid. For example, a modified influenza NA or HA refers to an influenza NA or HA that has an amino acid sequence or nucleic acid sequence that is different from the wild type NA protein or nucleic acid sequence. A modified influenza NA or HA can include one or more amino acid deletions and / or substitutions relative to the wild type influenza NA or HA.

[0047] Monomeric influenza virus neuraminidase: Wild-type influenza virus neuraminidase (NA) is a tetramer of four identical monomers. Each NA monomer of wild-type influenza NA consists of four distinct structural domains: an enzyme 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 an influenza virus NA head region, but can include a heterologous tetramerization domain or a portion thereof, and / or can lack at least a portion of one or more of the cytoplasmic tail, the transmembrane region, and the stalk region.

[0048] N1: As used herein, "N1" refers to influenza virus subtype 1 neuraminidase (NA). Influenza A viruses are divided into Group 1 and Group 2. Group 1 and Group 2 are further divided into subtypes, which refers to classification of viruses based on the sequences of two proteins, HA and neuraminidase (NA), on the surface of the virus. Currently, eleven NA subtypes (N1-N11) are recognized. Therefore, N1 is different from other NA subtypes, including N2-N11. N2: As used herein, "N2" refers to influenza virus subtype 2 neuraminidase (NA). Therefore, N2 is different from other NA subtypes, including N1 and N3-N11.

[0049] Influenza B strains are divided into two lineages: B / Yamagata and B / Victoria.

[0050] 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.

[0051] Prevention: The term "prevention," as used herein, refers to the prevention of, avoidance of disease onset, delay in onset, and / or reduction in frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., infection with 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.

[0052] Recombinant: As used herein, the term "recombinant" is intended to refer to a polypeptide that has been designed, engineered, prepared, expressed, created, or isolated by recombinant means (e.g., HA and / or NA polypeptides described herein), e.g., a polypeptide that has been expressed using a recombinant expression vector transfected into a host cell, a polypeptide that has been isolated from a recombinant combinatorial polypeptide library, or a polypeptide that has been prepared, expressed, created, 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 computer designed. 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 (e.g., two epitopes from two separate HA polypeptides or two separate NA polypeptides) that do not naturally occur in the same polypeptide. The recombinant HA is a rHA and the recombinant NA is a rNA.

[0053] 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.

[0054] 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.

[0055] 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 being 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 being 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 may 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.).

[0056] The percentage identity is obtained by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (eg, the number of positions in the reference sequence) and multiplying this result by 100.

[0057] 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, and in some embodiments, for consecutive nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0058] 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.

[0059] 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.

[0060] 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."

[0061] Tetrameric NA molecule: As used herein, the term "tetrameric NA molecule" refers to a compound comprising four NA monomer polypeptide units. In some embodiments, each monomeric NA molecule in a given tetrameric NA compound includes 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 truncated, absent, or modified compared to a reference wild-type monomeric NA molecule.

[0062] Tetramerization domain: As used herein, the term "tetramerization domain" refers to an amino acid sequence that encodes a domain that causes 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 tetramerization domain or a heterologous tetramerization domain. Exemplary tetramerization domains include, but are not limited to, sequences from Tetrabrachion, GCN4 leucine zipper, or vasodilator-stimulated phosphoprotein (VASP).

[0063] 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 disease associated with infection) or reduces the symptoms of infection (e.g., infection with influenza virus). Vaccines can induce both prophylactic (preventative) and therapeutic responses. Methods of administration vary depending on the vaccine, but may include inoculation, ingestion, inhalation, or other forms of administration. Inoculation may be delivered by any of a number of routes, including parenterally, such as intravenously, subcutaneously, intraperitoneally, intradermally, or intramuscularly. Vaccines may be administered with adjuvants to enhance the immune response.

[0064] Vaccinate: 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 may 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.

[0065] Vaccine efficacy: As used herein, the term "vaccine efficacy" or "vaccine effectiveness" refers to a measurement of the percentage reduction in evidence of disease in subjects receiving a vaccine. For example, 50% vaccine efficacy indicates a 50% reduction in the number of disease cases in a group of vaccinated subjects compared to a group of unvaccinated subjects or a group of subjects receiving a different vaccine.

[0066] 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.

[0067] Influenza virus nomenclature 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 the three main 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 Group 1 and Group 2. These groups are further divided into subtypes, which refers to classification of the virus based on the sequences of two proteins, HA and NA, on the surface of the virus. Currently, there are 18 recognized HA subtypes (H1-H18) and 11 recognized NA subtypes (N1-N11). Group 1 includes N1, N4, N5, and N8, and H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18. Group 2 includes 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., Bat-derived influenza-like viruses H17N10 and H18N11, 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).

[0068] Influenza A subtypes may be further divided into different genetic "clades" and "subclades." For example, A subtype A(H1N1) includes clade 6B.1 and subclade 6B.1A. A subtype A(H3N2) includes clades 3C.2A and 3C.3A, and subclades 3C.2A1, 3C.2A2, 3C2A3, and 3C.2A4. Similarly, B subtype Victoria includes clade V1A and subclades V1A.1, V1A.2, and V1A.3, while B subtype Yamagata includes 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.

[0069] 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 that contains a heterologous tetramerization domain that forms tetrameric NA when expressed in cells. HA refers to hemagglutinin or parts thereof.

[0070] 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 is attached to sugar moieties on glycoproteins or glycolipids expressed on the surface of cells. Binding of HA to sialic acid on the cell surface triggers 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.

[0071] HA is thought to mediate influenza virus attachment to host cells and virus-cell membrane fusion during viral penetration into the cell. Antigenic variation in the HA molecule is responsible for frequent influenza epidemics and limited control of infection by immunization.

[0072] 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 conformation and antigenic properties. Furthermore, HA0 is more stable and resistant to denaturation and proteolysis.

[0073] Isolation, propagation, and purification of influenza virus strains for cloning the desired HA gene can be performed by any method known in the art, for example, by the method disclosed in U.S. Pat. No. 5,762,939, incorporated herein by reference.

[0074] The recombinant HA antigen can be expressed in a suitable host cell. For example, recombinant HA can be expressed in microalgae cells, as disclosed in U.S. Patent Application Publication No. 2011 / 0189228, the entirety of which is incorporated herein by reference. Alternatively, recombinant HA can be expressed in insect cells. Other suitable host cells (e.g., mammalian cells, plant cells, or yeast cells) can be used to express recombinant HA.

[0075] In certain embodiments, recombinant HA is expressed in insect cells infected with a virus-HA vector (e.g., a baculovirus vector), for example, as disclosed in U.S. Pat. No. 5,976,552, the entirety of which is incorporated herein by reference. Baculovirus / insect cell cultures derived from recombinant HA are known to confer protective immunity against influenza. Baculoviruses are DNA viruses of the family Baculoviridae. These viruses are known to have a narrow host range, 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.

[0076] 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. The polyhedrin gene is not required for viral replication in cultured cells, so 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 they are under the transcriptional control of the polyhedrin promoter. Recombinant baculoviruses, including those encoding recombinant HA proteins, can then be replicated in a variety of insect cell lines. Recombinant HA 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.

[0077] The primary 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 post-translationally forms trimers and subsequently accumulates in the cytoplasmic membrane.

[0078] The rHA0 may be selectively extracted from the peripheral membrane with a non-denaturing non-ionic detergent or using other methods known in the art for purifying recombinant proteins from cells (e.g., insect cells) (e.g., affinity or gel chromatography, antigen binding, DEAE ion exchange, or lentil lectin affinity chromatography). The purified rHA0 may 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%.

[0079] The recombinant influenza virus HA disclosed herein may be formulated and packaged alone or in combination with other recombinant influenza virus HA antigens and / or recombinant influenza virus HAs described below. In certain embodiments, the recombinant influenza virus HA is formulated with 1, 2, 3, 4, 5, 6, or 7 additional recombinant influenza virus HA antigens, and in certain embodiments, the recombinant influenza virus HA is formulated with 1, 2, 3, 4, 5, 6, or 7 additional recombinant influenza virus NA antigens. In certain embodiments, the recombinant influenza virus HA is formulated with three additional recombinant influenza virus HA antigens to produce a tetravalent vaccine or immunogenic composition. In certain embodiments, the recombinant influenza virus HA is formulated with three additional recombinant influenza virus HA antigens and four additional recombinant influenza virus NA antigens to produce an octavalent vaccine or immunogenic composition.

[0080] The recombinant influenza virus HA present in the vaccine or immunogenic composition disclosed herein may include recombinant influenza virus HA from a standard of care influenza virus strain, and / or any combination of machine learning influenza virus HAs disclosed herein. For example, in certain embodiments, the recombinant influenza virus HA may be a wild type influenza HA, a modified influenza HA, an HA from a seasonal influenza virus strain or a pandemic influenza virus strain, and / or any other form of influenza HA known in the art. In certain embodiments, disclosed herein is a recombinant influenza virus HA 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 of the B / Victoria lineage, or an HA from a standard of care influenza virus of the B / Yamagata lineage.

[0081] In certain embodiments disclosed herein, the recombinant influenza virus HA is derived from a pandemic strain or a strain with pandemic potential (e.g., H1, H2, H3, H5, H7, H9, and / or H10).

[0082] In certain embodiments disclosed herein, the recombinant influenza virus HA is one or more machine learning recombinant influenza virus HAs having molecular sequences identified or designed from a machine learning model. In certain embodiments, the machine learning recombinant influenza virus HA can be selected from one or more of H1 HA, H3 HA, HA from B / Victoria lineage, HA from B / Yamagata lineage, or combinations thereof.

[0083] Any machine learning algorithm may be used to select one or more machine learning influenza virus HAs, for example, contemplated herein are any of the machine learning algorithms and methods disclosed in PCT Publication Nos. WO 2021 / 080990A1 (entitled Systems and Methods for Designing Vaccines), WO 2021 / 080999A1 (entitled Systems and Methods for Predicting Biological Responses), U.S. Provisional Application No. 63 / 319,692 (entitled Machine-Learning Techniques in Protein Design for Vaccine Generation), and U.S. Provisional 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 entireties.

[0084] 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 the 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 of 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.

[0085] Hemagglutinin activity can be measured using techniques known in the art, such as the hemagglutinin inhibition assay (HAI). HAI applies a process of hemagglutination, called hemagglutination, in which sialic acid receptors on the surface of red blood cells (RBCs) bind to the hemagglutinin glycoprotein 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 the 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).

[0086] Another approach to measuring HA antibody responses is to measure a potentially larger set of antibodies that may be elicited by the human or animal immune response and not necessarily affect hemagglutination in an HAI assay. A common approach for this is to utilize enzyme-linked immunosorbent assay (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 a substrate for an exogenous enzyme conjugated to an antibody from the antisera, or to another antibody that itself binds to the antibody of the antisera. The catalysis of the substrate produces a product that is easily detectable. There are many variations of this type of in vitro assay. One such variation is called antibody forensics (AF), a multiplex bead array technology that allows a single serum sample to be measured simultaneously against many antigens. These measurements characterize concentration and total antibody recognition, as 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 measurements that are proportionally higher or lower 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, generally do not correlate linearly.

[0087] Another method for measuring HA antibody responses includes virus neutralization assays (e.g., microneutralization assays), in which antibody titers are measured in permissive cell cultures after incubation of virus with serial dilutions of antibody / serum samples by the reduction in plaques, foci, and / or fluorescent signal, depending on the specific neutralization assay technique.

[0088] Neuraminidase (NA) Neuraminidase (NA) is the second major influenza surface protein, along with HA. NA removes sialic acid from cellular glycoproteins and glycolipids, as well as from newly synthesized HA and NA on nascent virions. Removal of sialic acid by NA prevents viral particle aggregation and promotes efficient release of viral particles from the surface of infected cells. It also prevents virus binding via HA to already infected dying cells, promoting further spread of viral infection. When NA is present in an immunogenic form in conventional vaccines or on intact virions, it is a minor component and therefore continues to compete for antigen with the immunodominant HA. Due to competitive mechanisms, immunogenic responses to NA appear to be partially suppressed in favor of the more frequent 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 are generally overshadowed by neutralizing HA antibodies.

[0089] A wild-type influenza virus neuraminidase The compositions and methods disclosed herein may, in certain embodiments, involve the use of tetrameric NA polypeptides that contain four copies of a wild-type monomeric NA molecule. 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 typically about 55-72 kDa, depending on the influenza subtype; the molecular weight of the tetramer is typically about 240-260 kDa, depending on the influenza subtype. Each monomer consists of four distinct structural domains: an enzyme head region, a stalk region, a transmembrane region, and a cytoplasmic tail. The largest domain is the head region, which is tethered to the viral membrane by a stalk region that connects to the transmembrane region and ultimately to the N-terminal cytoplasmic domain.

[0090] The stalk regions between various influenza A virus subtypes, such as 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 regulate the distance of the enzyme head domain and affect the ability of NA to access sialic acids on cell surface receptors, with shorter stalk domains correlating with reduced 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 the variability among the stalk domains of the various subtypes, the stalk domains of NA also share several structural features, such as at least one cysteine ​​residue and a potential glycosylation site. Cysteine ​​residues may participate in the formation of disulfide bonds between NA monomers to help form stabilized NA tetramers, and glycosylation sites may contribute to tetramer stabilization (McAuley et al., 2019). For example, the conserved cysteine ​​residue at amino acid position 78 of N2NA is thought to play a role in the tetramer assembly mechanism (Shtyrya et al., Influenza virus neuraminidase: structure and function, ACTA NATURAE 2009;1(2):26-32).

[0091] The enzyme 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 these 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).

[0092] Although the amino acid sequence and length of NA can vary significantly between various influenza A virus NA subtypes, such as N1 and N2, and in particular between the NA stalk regions of various influenza A virus NA subtypes, the length of the amino acid sequence of N2 from various influenza strains is typically about 469 amino acids, with some strains having insertions or deletions of about 1 or 2 (or more) amino acids, typically in the head region. When referring to a particular amino acid residue in wild-type N2, the numbering of the particular amino acid residue is based on N2 numbering, as understood in the art. The N-terminal cytoplasmic tail typically corresponds to amino acids 1-6 of the wild-type N2 sequence, and the transmembrane domain typically corresponds to amino acids 7-35 of the wild-type N2 sequence. For example, in the wild-type NA sequence of the A / PERTH / 16 / 2009 strain (SEQ ID NO: 1), the cytoplasmic region corresponds to amino acids 1-6 of SEQ ID NO: 1, and the transmembrane region corresponds to amino acids 7-35 of SEQ ID NO: 1. The length of the N2 stalk region is typically about 46 amino acids long, beginning at about amino acid 36 and ending at about amino acid 82 of the wild-type N2 sequence. For example, in the wild-type N2 sequence of the A / PERTH / 16 / 2009 strain (SEQ ID NO: 1), the stalk region corresponds to amino acid 36 to about 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 elucidated by x-ray crystallography.

[0093] b. Recombinant and / or modified influenza virus neuraminidase The methods and compositions of the present disclosure involve the use of recombinant NA. In certain embodiments, the recombinant NA comprises four copies of a modified monomeric NA molecule that forms 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 influenza virus NA, but lacks at least a portion of one or more of the cytoplasmic tail, transmembrane region, and stalk region of influenza virus NA.

[0094] For example, the modified monomeric NA may contain 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, for example, a tetramerization domain disclosed in US Patent Application Publication No. 2013 / 0034578, which is incorporated herein by reference in its entirety. See also, 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., 2010, J. Virology, 84(19):10366-74; Prevato et al., 2015, PLos ONE, 10(8):e0135474. In other embodiments, the heterologous tetramerization domain is a peptide found at the C-terminus of the lamprey VLR-B antibody described in WO 2016 / 097769 (i.e., the domain designated "C-TERM" in Figure 11C of WO 2008 / 016854, which is incorporated by reference in its entirety), e.g., SEQ ID NO: 1 or SEQ ID NO: 2 of WO 2016 / 097769, which is incorporated by reference in its entirety.

[0095] In certain embodiments, the modified monomeric influenza virus NA comprises a signal peptide, a heterologous tetramerization 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 the secretion of the tetrameric NA.

[0096] The wild-type NA protein is a membrane-bound protein that contains a transmembrane domain. To make a soluble NA protein, it is possible to delete the transmembrane domain and add a signal peptide. 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 in 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. Thus, the modified monomeric NA may contain a signal peptide after translation to target the modified monomeric NA to the secretory pathway, but the signal peptide is removed by post-translational processing, so that 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.

[0097] In certain embodiments, for example, the tetrameric NA comprises four copies of a modified monomeric influenza virus NA, which comprises the head region of an influenza virus NA and a heterologous tetramerization domain.

[0098] In certain embodiments, the cytoplasmic tail, transmembrane region, and all or substantially all of the stalk region of the influenza virus NA may be replaced with a signal peptide and a heterologous tetramerization domain. A modified NA containing a heterologous tetramerization domain may lack the entire NA stalk region or 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 containing 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 containing 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 heterologous tetramerization domain construct may contain about 8 of the most C-terminal amino acids of the NA stalk region.

[0099] In certain embodiments, the heterologous tetramerization domain is a Staphylothermus marinus tetrabrachion tetramerization domain, a GCN4 leucine zipper tetramerization domain, a tetramerization domain derived from a paramyxovirus phosphoprotein, or a human vasodilator-stimulated phosphoprotein (VASP) tetramerization domain.

[0100] As a further example, as disclosed in PCT Application No. PCT / US2022 / 039980, which is incorporated herein 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 produced detectable amounts of soluble tetrameric NA, the majority of the N2 strains tested produced detectable amounts of soluble tetrameric NA, indicating that the truncated stalk design strategy is broadly applicable to NA proteins from various N2 influenza strains. Depending on the N2 strain used, this modified monomeric NA design strategy can produce primarily tetrameric NA or a mixture of monomeric NA and tetramers when expressed in a host cell. Therefore, certain N2 strains and certain stalk-deleted variants of certain N2 strains produce soluble tetrameric NAs at higher yields when expressed in cells. In either case, it may be desirable to purify the tetrameric NAs produced when such modified NA constructs are expressed in host cells.

[0101] 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 the cytoplasmic tail, transmembrane region, and substantially all of the 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, with the most C-terminal amino acids of the stalk region typically referring to amino acids 70-82 of N2. In certain embodiments, the cytoplasmic tail, transmembrane region, and the entire stalk region (e.g., amino acids 1-82) are removed from modified N2.

[0102] In some embodiments, for example, the tetrameric NA comprises four copies of a modified influenza virus subtype 2 neuraminidase, 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, and the tetrameric NA does not comprise 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 with a signal peptide. The signal peptide is usually cleaved during post-translational processing, and therefore the secreted NA polypeptide typically does not comprise the signal peptide. In some of these embodiments, for example, amino acid 1 to at least amino acids 70-82 of wild-type N2 influenza virus NA are replaced with a signal peptide. These modified N2 constructs, in which the cytoplasmic domain, the transmembrane domain, and all or substantially all of the stalk region are replaced with a signal peptide and which form tetrameric NA when expressed in cells, are also described in further detail in International PCT Application No. PCT / US2022 / 039980, which is incorporated by reference in its entirety.

[0103] The tetrameric NA molecules formed by these modified monomeric NAs are generally substantially soluble in a fluid sample and are typically 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.

[0104] Neuraminidase activity can be measured using techniques known in the art, such as, for example, the MUNANA assay, the ELLA assay, or the NA-Star® assay (ThermoFisher Scientific, Waltham, Mass.). The MUNANA assay uses 2'-(4-methylumbelliferyl)-alpha-DN-acetylneuraminic acid (MUNANA) as a substrate. Any enzymatically active neuraminidase in the sample cleaves the MUNANA substrate, releasing the fluorescent compound 4-methylumbelliferone (4-MU). 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).

[0105] 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 hour with shaking; stop the reaction by adding alkaline pH solution (0.2 M Na2CO3); measure the fluorescence intensity using an excitation wavelength of 355 nm and an emission wavelength of 460 nm, respectively; calculate the enzyme activity against a 4MU reference. If necessary, an equivalent assay can be used to measure neuraminidase enzyme activity.

[0106] The recombinant influenza virus NA present in the vaccine or immunogenic composition disclosed herein may include recombinant influenza virus NA derived from a standard of care influenza virus strain, and / or any combination of machine learning influenza virus NA disclosed herein. For example, in certain embodiments, the recombinant influenza virus NA may be a wild type influenza NA, a non-wild type influenza NA, an NA derived from a seasonal influenza virus strain or a pandemic influenza virus strain, and / or any other form of influenza NA known in the art. In certain embodiments, disclosed herein is a recombinant influenza virus NA, the NA being selected from an N1 NA derived from a standard of care influenza virus, an N2 NA derived from a standard of care influenza virus, an NA derived from a standard of care influenza virus strain of the B / Victoria lineage, or an NA derived from a standard of care influenza virus of the B / Yamagata lineage.

[0107] In certain embodiments disclosed herein, the recombinant influenza virus NA is derived from a pandemic strain or a strain with pandemic potential, including, for example, N1, N2, N7, and / or N9.

[0108] In certain embodiments disclosed herein, one or more recombinant influenza virus NAs are identified or designed using a machine learning model ("recombinant machine learning influenza virus NA"). In certain embodiments, the machine learning recombinant influenza virus NAs may be selected from one or more of N1 NA, N2 NA, NA from B / Victoria lineage, NA of B / Yamagata lineage, or combinations thereof.

[0109] As disclosed above with respect to machine learning recombinant influenza virus HA, any machine learning algorithm may be used to select one or more machine learning recombinant influenza virus NAs, for example, contemplated herein are any of the machine learning algorithms and methods disclosed in PCT Publication No. 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.

[0110] Vaccine or immunogenic composition In certain aspects, disclosed herein are vaccines or immunogenic compositions comprising multiple recombinant influenza virus proteins, including one or more (e.g., two, three, or four) recombinant influenza virus HAs and one or more (e.g., two, three, or four) recombinant influenza virus NAs. In certain embodiments, the one or more (e.g., two, three, or four) recombinant influenza virus HAs are selected from H1 HA, H3 HA, HA from B / Victoria lineage, HA from B / Yamagata lineage, or combinations thereof. In certain embodiments, the one or more (e.g., two, three, or four) recombinant influenza virus NAs comprising a heterologous tetramerization domain are selected from N1 NA, N2 NA, NA from B / Victoria lineage, NA from B / Yamagata lineage, or combinations thereof. In certain embodiments, the one or more recombinant influenza virus NAs that lack the cytoplasmic tail, transmembrane region, and all or substantially all of the stalk region of influenza virus neuraminidase and do not contain a heterologous tetramerization domain are N2 NAs.

[0111] In certain aspects, disclosed herein is a vaccine or immunogenic composition comprising a plurality of recombinant influenza virus proteins, the plurality of recombinant influenza virus proteins being: (1) a first recombinant influenza virus hemagglutinin (HA), the first recombinant influenza virus HA being an H1 HA; (2) a second recombinant influenza virus HA, the second recombinant influenza virus HA being an H3 HA; (3) a third recombinant influenza virus HA, the third recombinant influenza virus HA being derived from a B / Victoria lineage; (4) a fourth recombinant influenza virus HA, the fourth recombinant influenza virus HA being derived from a B / Yamagata lineage; and (5) a first recombinant influenza virus neuraminidase (NA), the first recombinant influenza virus NA being an N1 (5) a first recombinant influenza virus NA, the second recombinant influenza virus NA being an N2 NA; (6) a second recombinant influenza virus NA, the second recombinant influenza virus NA being an N2 NA; (7) a third recombinant influenza virus NA, the third recombinant influenza virus NA being derived from a B / Victoria lineage; and (8) a fourth recombinant influenza virus NA, the fourth recombinant influenza virus NA being derived from a B / Yamagata lineage. In various embodiments, one or more of the recombinant NAs are modified as described herein.

[0112] Further disclosed herein is a vaccine or immunogenic composition comprising a plurality of recombinant influenza virus proteins, the plurality of recombinant influenza virus proteins being: (1) a first recombinant influenza virus hemagglutinin (HA), the first recombinant influenza virus HA being an H1 HA; (2) a second recombinant influenza virus HA, the second recombinant influenza virus HA being an H3 HA; (3) a third recombinant influenza virus HA, the third recombinant influenza virus HA being derived from a B / Victoria lineage; (4) a fourth recombinant influenza virus HA, the fourth recombinant influenza virus HA being derived from a B / Yamagata lineage; and (5) a first recombinant influenza virus neuraminidase (NA), the first recombinant influenza virus NA being an N1 (5) a first recombinant influenza virus NA, the second recombinant influenza virus NA being an N2 NA; (6) a second recombinant influenza virus NA, the second recombinant influenza virus NA being an N2 NA; (7) a third recombinant influenza virus NA, the third recombinant influenza virus NA being derived from a B / Victoria lineage; and (8) a fourth recombinant influenza virus NA, the fourth recombinant influenza virus NA being derived from a B / Yamagata lineage. In various embodiments, one or more of the recombinant NAs are modified as described herein.

[0113] In certain embodiments, one or more (e.g., 1, 2, 3, or 4) of the recombinant influenza virus HAs in a vaccine or immunogenic composition are derived from a standard therapeutic influenza strain, and in certain embodiments, each of the recombinant influenza virus HAs in a vaccine or immunogenic composition are derived from a standard therapeutic influenza strain. In certain embodiments, one or more (e.g., 1, 2, 3, or 4) of the recombinant influenza virus NAs in a vaccine or immunogenic composition are derived from a standard therapeutic influenza strain, and in certain embodiments, each of the recombinant influenza virus NAs in a vaccine or immunogenic composition are derived from a standard therapeutic influenza strain.

[0114] In certain embodiments, the vaccine or immunogenic composition comprises an H1 HA from an H1N1 influenza virus strain. In certain embodiments, the vaccine or immunogenic composition comprises an H3 HA from an H3N2 influenza virus strain. In certain embodiments, the vaccine or immunogenic composition comprises an N1 NA from an H1N1 influenza virus strain. In certain embodiments, the vaccine or immunogenic composition comprises an N2 NA from an N3N2 influenza virus strain. In certain embodiments, the vaccine or immunogenic composition comprises an H1 HA and an N1 NA from the same H1N1 influenza virus strain, and in certain embodiments, the vaccine or immunogenic composition comprises an H3 HA and an N2 NA from the same H3N2 influenza virus strain. In certain embodiments, the vaccine or immunogenic composition comprises an H1 HA and an N1 NA from different H1N1 influenza virus strains, and in certain embodiments, the vaccine or immunogenic composition comprises an H3 HA and an N2 NA from different H3N2 influenza virus strains.

[0115] In certain embodiments, the vaccine or immunogenic composition comprises an H1 HA from an H1N1 influenza virus strain, an H3 HA from an H3N2 influenza virus strain, an N1 NA from an H1N1 influenza virus strain, and an N2 NA from an H3N2 influenza virus strain. In certain embodiments, the vaccine or immunogenic composition comprises an H1 HA and an N1 NA from the same H1N1 influenza virus strain, and in certain embodiments, the vaccine or immunogenic composition comprises an H3 HA and an N2 NA from the same H3N2 influenza virus strain. In certain embodiments, the vaccine or immunogenic composition comprises an H1 HA and an N1 NA from different H1N1 influenza virus strains, and in certain embodiments, the vaccine or immunogenic composition comprises an H3 HA and an N2 NA from different H3N2 influenza virus strains.

[0116] One or more of the recombinant influenza virus HAs and one or more of the recombinant influenza virus NAs in a multivalent vaccine or immunogenic composition may be formulated and packaged alone or in combination with other recombinant HA and / or NA antigens. In certain embodiments, the recombinant influenza virus HA is formulated with one, two, or three additional recombinant influenza virus HA antigens (e.g., one, two, or three additional recombinant antigens from a standard of care influenza virus strain). In certain embodiments, the recombinant influenza virus HA is formulated with three additional recombinant influenza virus HA antigens to produce a quadrivalent vaccine or immunogenic composition.

[0117] In certain embodiments, the recombinant influenza virus NA is formulated with one, two, or three additional recombinant influenza virus NA antigens (e.g., one, two, or three additional recombinant antigens from a standard of care influenza virus strain). In certain embodiments, the recombinant influenza virus NA is formulated with three additional recombinant influenza virus NA antigens to generate a quadrivalent vaccine or immunogenic composition.

[0118] In certain embodiments, one or more (e.g., one, two, or three) recombinant influenza virus NAs are formulated with one or more (e.g., one, two, three, or four) recombinant influenza virus HAs. In certain embodiments, the vaccine or immunogenic composition may comprise four recombinant influenza virus HA antigens and four recombinant influenza virus NA antigens to produce an octavalent vaccine or immunogenic composition. In certain embodiments, the four recombinant influenza virus HA antigens and the four recombinant influenza virus NA antigens may each be derived from a standard of care influenza virus strain. In certain embodiments, the octavalent vaccine or immunogenic composition comprising four recombinant influenza virus HA antigens and four recombinant influenza virus NA antigens further comprises one or more machine learning influenza virus HAs and / or one or more machine learning influenza virus NAs.

[0119] In certain embodiments, the recombinant influenza virus H1 HA, recombinant influenza virus H3 HA, recombinant influenza virus HA derived from the B / Victoria lineage, recombinant influenza virus HA derived from the B / Yamagata lineage, recombinant influenza virus N1 NA, recombinant influenza virus N2 NA, recombinant influenza virus NA derived from the B / Victoria lineage, and / or recombinant influenza virus NA derived from the B / Yamagata lineage have molecular sequences that have been identified or designed from a machine learning model.

[0120] In certain embodiments, the vaccine or immunogenic composition is a pentavalent vaccine or immunogenic composition comprising one or more recombinant HAs and one or more recombinant NAs. In certain embodiments, the vaccine or immunogenic composition is a hexavalent vaccine or immunogenic composition comprising one or more recombinant HAs and one or more recombinant NAs. In certain embodiments, the vaccine or immunogenic composition is a heptavalent vaccine or immunogenic composition comprising one or more recombinant HAs and one or more recombinant NAs. In certain embodiments, the vaccine or immunogenic composition is an octavalent vaccine or immunogenic composition comprising one or more recombinant HAs and one or more recombinant NAs (e.g., four recombinant HAs and four recombinant NAs). In certain embodiments, the vaccine or immunogenic composition is a multivalent vaccine or immunogenic composition comprising more than eight different HA and NA molecules.

[0121] Each recombinant HA may be present in the compositions disclosed herein in an amount effective to elicit an immune response in a subject to which the composition is administered. In certain embodiments, each recombinant HA may be present in the vaccine or immunogenic compositions disclosed herein in an amount ranging from about 0.1 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 40 μg to about 50 μg, from about 42 μg to about 47 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, or from 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.

[0122] Each recombinant HA 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 recombinant NA may be present in the vaccine or immunogenic composition disclosed herein in an amount ranging from about 0.1 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.

[0123] In certain embodiments, the total amount of recombinant influenza HA and NA present in a vaccine or immunogenic composition disclosed herein can range from about 150 μg to about 400 μg, about 150 μg to about 300 μg, about 200 μg to about 300 μg, about 200 μg to about 250 μg, or about 225 μg to about 245 μg. In certain embodiments, the total amount of recombinant influenza HA and NA present in a vaccine or immunogenic composition disclosed herein is less than about 500 μg, 400 μg, 350 μg, 300 μg, 250 μg, 200 μg, or 150 μg. In certain embodiments, the total amount of recombinant influenza HA and NA present in the vaccine or immunogenic composition disclosed herein is about 500 μg, about 400 μg, about 350 μg, about 300 μg, about 290 μg, about 280 μg, about 270 μg, about 260 μg, about 250 μg, about 240 μg, about 230 μg, about 220 μg, about 210 μg, about 200 μg, about 190 μg, about 180 μg, about 170 μg, about 160 μg, or about 150 μg.

[0124] 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 include suspensions of minerals (alum, aluminum salts (e.g., aluminum hydroxide / oxyhydroxide (AlOOH), aluminum phosphate (AlPO4), aluminum hydroxyphosphate sulfate (AAHS), and / or aluminum potassium sulfate)) to which the antigen is adsorbed; or water-in-oil emulsions in which an antigen solution is emulsified in mineral oil (e.g., Freund's incomplete adjuvant), which may also include 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. Exemplary 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.

[0125] 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 a size of less than 90% of the population by volume of the oil droplets of less than 200 nm.

[0126] 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 one particular embodiment, the polyoxyethylene alkyl ether surfactant is polyoxyethylene (12) cetostearyl ether.

[0127] 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.

[0128] In certain embodiments, the adjuvant further comprises at least one alditol, including but not limited to glycerol, erythritol, xylitol, sorbitol, and mannitol.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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 incorporated herein by reference in its entirety. In certain embodiments, the adjuvant comprises a liposome-based adjuvant, such as SPA14, as described in WO 2022 / 090359, which is incorporated herein by reference in its entirety. SPA14 is a liposome-based adjuvant that comprises a Toll-like receptor 4 (TLR4) agonist (E6020) and a saponin (QS21).

[0133] In addition to recombinant HA, recombinant NA, and optional adjuvants, the vaccine or immunogenic composition may also 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, which contains 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 (e.g., wetting or emulsifying agents, pharma- ceutically acceptable salts for adjusting osmotic pressure, preservatives, stabilizers, buffers, sugars, amino acids, and pH buffers, such as, for example, sodium acetate or sorbitan monolaurate).

[0134] 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.

[0135] 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, 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 therapeutic agents 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 or immunogenic 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 by reference herein. 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, which is incorporated by reference herein, and functional equivalents thereof. Also suitable are jet injection devices that deliver liquid vaccines to the dermis by means of a liquid jet injector or by means of 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 5,31 The method is described in WO 2006 / 02335, WO 2006 / 02335, WO 2005 ...

[0136] Preparations for parenteral administration typically include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include 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, and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those 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, etc.

[0137] kit Further disclosed herein are kits for the vaccines or immunogenic compositions disclosed herein, which may include a suitable container containing the vaccine or immunogenic composition, or multiple containers containing various components of the vaccine or immunogenic composition, optionally together with instructions for use.

[0138] In certain embodiments, the kit may include multiple containers, for example, a first container containing one or more recombinant influenza virus HAs disclosed herein and a second container containing one or more recombinant influenza virus NAs disclosed herein. For example, in certain embodiments, disclosed herein is a recombinant influenza virus HA comprising: (1) a first container comprising a first recombinant influenza virus HA, wherein the first recombinant influenza virus HA is an H1 HA; a second recombinant influenza virus HA, wherein the second recombinant influenza virus HA is an H3 HA; a third recombinant influenza virus HA, wherein the third recombinant influenza virus HA is derived from a B / Victoria lineage; and a fourth recombinant influenza virus HA, wherein the fourth recombinant influenza virus HA is derived from a B / Yamagata lineage; and (2) a first recombinant influenza virus NA, wherein the first recombinant influenza virus NA is an N1 NA; a second recombinant influenza virus NA, wherein the second recombinant influenza virus NA is an N2 NA. a second recombinant influenza virus NA, the third recombinant influenza virus NA being derived from a B / Victoria lineage; and a second container containing a fourth recombinant influenza virus NA, the fourth recombinant influenza virus NA being derived from a B / Yamagata lineage. In certain embodiments, the kit may further include a third container comprising an optional adjuvant, and in certain embodiments, the first and / or second container may comprise an optional adjuvant in addition to the recombinant influenza virus antigen.

[0139] In certain embodiments, the kit may include a single container containing one or more each of the recombinant influenza virus HAs disclosed herein and one or more each of the recombinant influenza virus NAs disclosed herein, and an optional adjuvant, which in certain embodiments may be in a separate container.

[0140] The instructions may indicate that the contents of the first and second containers are to be combined prior to administration, or that the contents of the first and second containers are to be administered separately without being combined.

[0141] Nucleic acids, cloning and expression systems The present disclosure further provides an artificial nucleic acid molecule encoding the recombinant HA and NA of the present disclosure. 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 a DNA molecule having the specified sequence, and includes an RNA molecule having the specified sequence in which U is replaced with T or its derivative, such as pseudouridine, unless the context requires otherwise. Other nucleotide derivatives or modified nucleotides may be incorporated into the artificial nucleic acid molecule encoding the HA and NA of the present disclosure.

[0142] 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 the artificial nucleic acid molecules encoding the HA or NA disclosed herein. The present disclosure further provides host cells that contain one or more of the above constructs.

[0143] Also provided are methods for producing recombinant HA or NA polypeptides using recombinant techniques known in the art and described above. Recombinant protein production and expression can be carried out using conventional procedures known in the art and 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.

[0144] Systems for cloning and polypeptide expression in a variety of different host cells are known in the art. Any protein expression system (e.g., stable or transient) that is compatible with the constructs disclosed in this application can be used to produce the HA or NA described herein.

[0145] Suitable vectors can be selected or constructed to contain appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate.

[0146] To express the recombinant HA and / or recombinant NA disclosed herein, a nucleic acid encoding an HA or a nucleic acid encoding an NA can be introduced into a host cell. This introduction can use any available technique. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia, or baculovirus for insect cells). For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using bacteriophages. These techniques are known in the art. (See, for example, "Current Protocols in Molecular Biology", Ausubel et al. eds., John Wiley & Sons, 2010). DNA introduction can be followed by a selection method (e.g., antibiotic resistance) to select for cells containing the vector.

[0147] 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-76); 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. In one embodiment, the host cell is an insect cell.

[0148] Machine Learning Any machine learning algorithm may be used to select one or more machine learning influenza virus HAs and / or NAs, for example, contemplated herein are any of the machine learning algorithms and methods 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 Application Nos. 63 / 319,692 (entitled Machine-Learning Techniques in Protein Design for Vaccine Generation), and 63 / 319,700 (entitled Machine-Learning Techniques in Protein Design for Vaccine Generation), which are incorporated herein by reference in their entireties.

[0149] 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 the input data of a training set, where the 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 of 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 to construct a selection algorithm.

[0150] In certain embodiments, a system for designing a vaccine is provided. The system includes one or more processors. The system includes computer storage that stores 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 to a first time series data set configured to generate output data representing one or more molecular sequences, the first time series data set representing 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 biological responses to 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 multiple 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: for each trained driver model of the set of trained driver models, applying the trained driver model to the second time series dataset to generate 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 second translation response data representing one or more second translation responses, for each translational axis of the plurality of translational axes; and selecting a subset of the trained driver models of the set of trained driver models based on the second translation response data.

[0151] 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.

[0152] The output data representing one or more predicted molecular sequences based on the received first time series data set 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 pathogenic strains in circulation 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 viruses in circulation for a particular season.

[0153] 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.

[0154] Selecting a set of trained driver models from 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 from the plurality of driver models may include, for each driver model of the plurality of driver models, comparing one or more trained translational responses of the driver model to one or more trained translational responses of at least one other driver model assigned to the same class as the driver model.

[0155] 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 with 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.

[0156] 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 is executing 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 the one or more molecular sequences based on the time series data.

[0157] Predicting 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 impart to future use. Predicting one or more molecular sequences based on the time series data may include predicting one or more molecular sequences that generate a maximized aggregate biological response across all pathogenic strains in the time series data. Predicting 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.

[0158] The machine learning model appears to include a recurrent neural network.

[0159] 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 is executing 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.

[0160] 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.

[0161] The first molecular sequence may comprise a candidate antigen, and the second molecular sequence may comprise a known viral strain.

[0162] 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.

[0163] Machine learning methods 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 conventional 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 conventional 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.

[0164] In certain embodiments, a system of one or more computers may be configured to perform a particular operation or behavior 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 a particular operation or behavior by including instructions that, when executed by a data processing device, cause the device to perform the operation. One general aspect includes a method of 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 manufacturing 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, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the operations of the method.

[0165] Implementations may include one or more of the following features: 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 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 determining, for each second continuous value, 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 from the plurality of candidate discrete result objects at least one discrete result object that identifies an amino acid that failed the manufacturability test. Applying 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.

[0166] 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 that stores 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 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, apparatus, and computer programs stored on one or more computer storage devices, each configured to perform the operations of the methods.

[0167] Implementations may include one or more of the following features. In one embodiment, the system includes a first amino acid sequence and a second amino acid sequence, each of the first and second amino acid sequences 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. 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.

[0168] 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 to 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 to 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, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the operations of the method.

[0169] 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 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 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 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.

[0170] In certain embodiments, disclosed herein are algorithms that can generate influenza antigens for use as vaccines. In one implementation, this can include: 1) generating a reduced dimensional space of all wild-type hemagglutinin sequences by machine learning (e.g., a variational autoencoder architecture) using the following two steps: a) variably embedding into a 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) The reduced spatial location “autoencoder” loss function is then used to decode the original sequence and reduce it by the similarity of the input and output sequences.

[0171] 2) Train an immune response prediction model based on the position of antigens (vaccine candidates) and readout strains (target sequences) in the reduced dimensional space [input: antigens and readouts embedded by the model from step 1, output: measures of immune response such as antibody titers].

[0172] 3) Sample candidate vaccine component representations from the reduced space and rank the candidate vaccine component representations by their predictive performance against the target sequence using the model described in step 2 to identify the top candidates.

[0173] 4) Decode 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.

[0174] 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 arrays in a reduced dimensional space from the one or more data objects, each reduced dimensional array including 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. The method also includes generating a plurality of candidate sequences in the reduced dimensional space using the plurality of reduced dimensional arrays. 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 input 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 operating each of the generated amino acid sequences suitable for producing a respective vaccine.The vaccine 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, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the operations of the method.

[0175] 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 group 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 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 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 techniques may include hardware, methods or processes, or computer software on a computer accessible medium.

[0176] 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 includes 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 viral amino acid sequences; and generating at least one reduced-dimensional viral candidate 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 and 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, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the operations of the method.

[0177] 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 to 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 techniques may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0178] 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 includes 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; 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 a plurality of candidate sequences in the reduced dimensional space using the plurality of reduced dimensional arrays; generating three reduced-dimension viral sequences; providing each of the candidate sequences and at least one of the reduced-dimension viral sequences as input to a potency predictor; receiving a candidate score for each of the candidate sequences as output from the potency predictor and 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, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the operations of the method.

[0179] Implementations may include one or more of the following features: A medium in which generating the 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 group of values, and generating the 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 the candidate score, the potency score defining a measure of a biological response between the first sequence and the second sequence. Implementations of the described approaches may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0180] 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.

[0181] Implementations of the present disclosure may provide the following advantages: Compared to conventional approaches, vaccines may be designed for future pathogenic seasons and 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 and confer more protection in terms of the breadth of coverage that can effectively cover 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 approaches, rarely observed strains may confer "more protection" because they cross-react with more strains than frequently observed strains.

[0182] 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 thereof a vaccine comprising one or more recombinant influenza virus HAs described herein, one or more recombinant influenza virus NAs described herein, and an optional adjuvant in an amount effective to vaccinate the subject against influenza virus. Similarly, the present disclosure provides a vaccine comprising one or more influenza virus HAs described herein, one or more NAs described herein, and an optional adjuvant for use in vaccinating a subject against influenza virus. Also disclosed herein is an immunogenic composition comprising a vaccine comprising one or more recombinant influenza virus HAs described herein, one or more recombinant influenza virus NAs described herein, and an optional adjuvant for producing a vaccine for use in vaccinating a subject against influenza virus.

[0183] The present disclosure also provides a method of immunizing a subject against influenza virus, comprising administering to the subject an immunologically effective amount of a vaccine comprising one or more recombinant influenza virus HAs described herein, one or more recombinant influenza virus NAs described herein, and an optional adjuvant. Similarly, the present disclosure provides a vaccine comprising one or more recombinant influenza virus HAs described herein, one or more recombinant influenza virus NAs described herein, and an optional adjuvant for use in immunizing a subject against influenza virus. Also disclosed herein is an immunogenic composition comprising one or more recombinant influenza virus HAs described herein, one or more recombinant influenza virus NAs described herein, and an optional adjuvant for producing a vaccine for use in immunizing a subject against influenza virus.

[0184] In some embodiments, the method or use prevents infection or disease by influenza virus 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.

[0185] The immunization methods (or related uses) provided herein may induce a broad 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.

[0186] In certain embodiments, provided herein is a method of enhancing or expanding a protective immune response in a subject, comprising administering to the subject an immunologically effective amount of a vaccine disclosed herein. Also disclosed herein is any of the vaccines described herein for use in enhancing or expanding a protective immune response in a subject, for example, a vaccine comprising a plurality of recombinant influenza virus proteins, the plurality of recombinant influenza virus proteins comprising or consisting of one or more recombinant influenza virus HAs and one or more recombinant influenza virus NAs. Also disclosed herein is an immunogenic composition as described herein for manufacturing a vaccine for use in enhancing or expanding a protective immune response in a subject. In certain embodiments, the vaccines disclosed herein increase the vaccine efficacy of a standard of care influenza virus vaccine composition by an amount ranging from 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%, e.g., 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., 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.

[0187] In certain embodiments, the standard of care influenza virus vaccine may be an inactivated influenza vaccine (IIV), such as a trivalent or quadrivalent IIV. Typically, a standard of care inactivated influenza virus vaccine composition includes inactivated influenza viruses derived from H1N1, H3N2, B / Victoria, and B / Yamagata lineages. In certain embodiments, the standard of care influenza virus vaccine may include a recombinant influenza virus HA, such as a trivalent or quadrivalent vaccine composition including a recombinant influenza virus HA. Typically, a standard of care recombinant HA vaccine composition includes rHA derived from H1N1, H3N2, B / Victoria, and B / Yamagata lineages. Vaccine efficacy may be expressed as a 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 incidence rate of the vaccinated population from the incidence rate of the unvaccinated population and dividing by the incidence rate of the unvaccinated population according to the following formula: [(incidence rate of the unvaccinated population)-(incidence rate of the vaccinated population) / (incidence rate of the unvaccinated population) x 100].

[0188] Also provided is a method of preventing influenza virus disease in a subject, comprising administering to the subject a vaccine comprising one or more recombinant influenza virus HAs described herein, one or more recombinant influenza virus NAs described herein, and an optional adjuvant in an amount effective to prevent influenza virus disease in the subject. Similarly, the present disclosure provides a vaccine comprising one or more recombinant influenza virus HAs described herein, one or more recombinant influenza viruses described herein, and an optional adjuvant for use in preventing influenza virus disease in a subject. Also disclosed herein is an immunogenic composition comprising one or more recombinant influenza virus HAs described herein, one or more recombinant influenza virus NAs described herein, and an optional adjuvant for the manufacture of a vaccine for use in preventing influenza virus disease in a subject.

[0189] Also provided is a method of eliciting an immune response against influenza virus HA and influenza virus NA in a subject, comprising administering to the subject a vaccine comprising one or more recombinant influenza virus HAs as described herein, one or more recombinant influenza virus NAs as described herein, and an optional adjuvant. Similarly, the present disclosure provides a vaccine comprising one or more recombinant influenza virus HAs as described herein, one or more recombinant influenza virus NAs as described herein, and an optional adjuvant for use in eliciting an immune response against influenza virus HA and influenza virus NA in a subject. Also provided herein is an immunogenic composition comprising one or more recombinant influenza virus HAs as described herein, one or more recombinant influenza virus NAs as described herein, and an optional adjuvant for the manufacture of a vaccine for use in eliciting an immune response against influenza virus HA and influenza virus NA in a subject.

[0190] A vaccine comprising one or more recombinant influenza virus HAs described herein, one or more recombinant influenza virus NAs described herein, and an optional adjuvant may be administered before or after the onset of one or more symptoms of influenza infection. That is, in some embodiments, a vaccine described herein may be administered prophylactically to prevent influenza infection or to ameliorate symptoms of potential influenza infection. In some embodiments, a subject is at risk of infection with influenza virus when 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 when 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 a patient exhibiting one or more symptoms commonly associated with influenza infection. In some embodiments, the subject is known to have been exposed or is believed to have been exposed to influenza virus. In some embodiments, a 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 or believed to have been exposed to influenza virus if the subject is 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 present or has been in an area where influenza infection is known or believed to be epidemic or endemic. The vaccines disclosed herein may be used to treat or prevent disease caused by seasonal or pandemic influenza strains, or both.

[0191] Vaccines according to the present disclosure may be administered in any amount or dose appropriate to achieve the desired result. In some embodiments, the desired result 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 result is a reduction in the intensity, severity, and / or frequency of, and / or a delay in the 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 mode of administration.

[0192] In various embodiments, the vaccine or immunogenic compositions described herein are administered to a subject, which can be any member of the animal kingdom. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human subject is an avian (e.g., a chicken or bird, a reptile, an amphibian, a fish, an insect, and / or a nematode. 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).

[0193] In some embodiments, a vaccine or immunogenic composition described herein is 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).

[0194] Typically, the vaccine methods and uses described herein include administration of a single dose (i.e., no booster dose) to a subject. However, in some embodiments, the vaccine methods and uses described herein include a prime-boost vaccination strategy. Prime-boost vaccination includes administering a priming vaccine, followed by administration of a boosting vaccine to a subject after a period of time. An immune response is "primed" upon administration of the priming vaccine, and "boosted" upon administration of the boosting vaccine. The priming vaccine may include a vaccine comprising one or more recombinant influenza virus HAs, one or more recombinant influenza virus NAs, and an optional adjuvant. Similarly, the boosting vaccine may include a vaccine comprising one or more recombinant influenza virus HAs, one or more recombinant influenza virus NAs, and an optional adjuvant. The priming vaccine may be, but need not be, the same as the boosting vaccine. Administration of the boosting vaccine generally occurs several weeks to several months after administration of the priming 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 after administration of the priming composition. In certain embodiments, the recipient of the prime-boost vaccination is a naive subject, typically a naive infant or child.

[0195] The vaccine may be administered using any suitable route of administration, including, for example, parenteral delivery, as described above.

[0196] Typically, one or more recombinant influenza virus HAs described herein, one or more recombinant influenza virus NAs described herein, and optional adjuvants are administered together as components of the same vaccine. However, one or more recombinant influenza virus HAs, one or more recombinant influenza virus NAs, and / or optional adjuvants do not have to be administered as part of the same vaccine. That is, if desired, one or more recombinant influenza virus HAs, one or more recombinant influenza virus NAs, and / or optional adjuvants may be administered separately to a subject. For example, a first vaccine comprising one or more recombinant influenza virus HAs may be administered to a subject separately from a second vaccine comprising one or more recombinant influenza virus NAs. When the first vaccine and the second vaccine are administered separately, the first vaccine and the second vaccine may be administered to a subject at different sites.

[0197] Representative embodiments of the present disclosure 1. An immunogenic composition comprising a plurality of recombinant influenza virus proteins, the plurality of recombinant influenza virus proteins comprising: a first recombinant influenza virus hemagglutinin (HA), wherein said first recombinant influenza virus HA is an H1 HA; a second recombinant influenza virus HA, wherein said second recombinant influenza virus HA is an H3 HA; a third recombinant influenza virus HA, wherein said third recombinant influenza virus HA is derived from a B / Victoria lineage; a fourth recombinant influenza virus HA, wherein said fourth recombinant influenza virus HA is derived from a B / Yamagata lineage; a first recombinant influenza virus neuraminidase (NA), wherein said first recombinant influenza virus NA is an N1 NA; a second recombinant influenza virus NA, wherein said second recombinant influenza virus NA is N2 NA; a third recombinant influenza virus NA, said third recombinant influenza virus NA being derived from the B / Victoria lineage; and A fourth recombinant influenza virus NA, the fourth recombinant influenza virus NA being derived from the B / Yamagata lineage. An immunogenic composition comprising:

[0198] 2. The immunogenic composition of embodiment 1, wherein each of the first, second, third, and fourth recombinant influenza virus NAs is a modified recombinant influenza virus NA.

[0199] 3. The immunogenic composition of embodiment 2, wherein the modified recombinant influenza virus NA comprises a modified recombinant tetrameric influenza virus NA comprising four modified monomeric NA molecules, each of which comprises a head region of the NA of the influenza virus but lacks the cytoplasmic tail, the transmembrane region, and all or substantially all of the stalk region of the NA of the influenza virus, and wherein the modified monomeric NA molecules form a modified recombinant tetrameric NA when expressed in a host cell.

[0200] 4. The immunogenic composition of embodiment 3, wherein each modified recombinant monomeric influenza virus NA comprises a heterologous tetramerization domain.

[0201] 5. The immunogenic composition of embodiment 3, wherein each modified recombinant monomeric influenza virus NA does not contain a heterologous oligomerization domain.

[0202] 6. The immunogenic composition of embodiment 4, wherein the heterologous tetramerization domain is a Staphylothermus marinus tetrabrachion tetramerization domain, a GCN4 leucine zipper tetramerization domain, a tetramerization domain derived from a paramyxovirus phosphoprotein, or a human vasodilator-stimulated phosphoprotein (VASP) tetramerization domain.

[0203] 7. The immunogenic composition of any one of embodiments 1 to 6, wherein each of the recombinant influenza virus HAs is produced in a baculovirus expression system in cultured insect cells.

[0204] 8. An immunogenic composition according to any one of embodiments 1 to 7, wherein each of the recombinant influenza virus NAs is produced in Chinese Hamster Ovary (CHO) cells.

[0205] 9. The immunogenic composition of any one of embodiments 1 to 8, wherein the immunogenic composition does not comprise inactivated influenza virions or live attenuated influenza virions.

[0206] 10. An immunogenic composition according to any one of embodiments 1 to 9, wherein each of the recombinant influenza virus HAs and / or each of the recombinant influenza virus NAs is derived from a standard of care influenza strain.

[0207] 11. An immunogenic composition according to any one of embodiments 1 to 10, wherein the H1 HA is derived from an H1N1 influenza virus strain and / or the H3 HA is derived from an H3N2 influenza virus strain.

[0208] 12. An immunogenic composition according to any one of embodiments 1 to 11, wherein the N1 NA is derived from an H1N1 influenza virus strain and / or the N2 NA is derived from an H3N2 influenza virus strain.

[0209] 13. The immunogenic composition according to any one of embodiments 1 to 12, wherein 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 the N2 NA is derived from an H3N2 influenza virus strain.

[0210] 14. The immunogenic composition of embodiment 13, wherein the H1 HA and N1 NA are derived from the same H1N1 influenza virus strain, and the H3 HA and N2 NA are derived from the same H3N2 influenza virus strain.

[0211] 15. The recombinant influenza virus proteins include a first recombinant influenza virus HA, wherein said first recombinant influenza virus HA is an H1 HA; a second recombinant influenza virus HA, wherein said second recombinant influenza virus HA is an H3 HA; a third recombinant influenza virus HA, wherein said third recombinant influenza virus HA is derived from the B / Victoria lineage; a fourth recombinant influenza virus HA, wherein said fourth recombinant influenza virus HA is derived from a B / Yamagata lineage; a first recombinant influenza virus NA, wherein said first recombinant influenza virus NA is an N1 NA; a second recombinant influenza virus NA, wherein said second recombinant influenza virus NA is N2 NA; a third recombinant influenza virus NA, said third recombinant influenza virus NA being derived from the B / Victoria lineage; and A fourth recombinant influenza virus NA, wherein the fourth recombinant influenza virus NA is derived from the B / Yamagata lineage. The immunogenic composition according to any one of embodiments 1 to 14, comprising:

[0212] 16. An immunogenic composition described in any one of embodiments 1 to 15, wherein the composition further comprises an adjuvant.

[0213] 17. The immunogenic composition of embodiment 16, wherein the adjuvant comprises a squalene-in-water adjuvant or a liposome-based adjuvant.

[0214] 18. The immunogenic composition of embodiment 17, wherein the squalene adjuvant in water comprises AF03.

[0215] 19. The immunogenic composition of embodiment 17, wherein the liposome-based adjuvant comprises SPA14.

[0216] 20. An immunogenic composition described in any one of embodiments 1 to 19, wherein each of the recombinant influenza virus HAs is present in the composition in an amount ranging from about 0.1 μg to about 90 μg, and optionally, in an amount ranging from about 1 μg to about 60 μg or from 5 μg to about 45 μg.

[0217] 21. An immunogenic composition according to any one of embodiments 1 to 20, wherein each of the recombinant influenza virus NAs is present in the composition in an amount ranging from about 0.1 μg to about 90 μg, and optionally, in an amount ranging from about 1 μg to about 60 μg or from about 5 μg to about 45 μg.

[0218] 22. An immunogenic composition described in any one of embodiments 1 to 21, wherein the composition is formulated for intramuscular injection.

[0219] 23. A vaccine comprising the immunogenic composition of any one of claims 1 to 22 and a pharmaceutical carrier.

[0220] 24. A method of immunizing a subject against influenza virus, comprising administering to the subject an immunologically effective amount of the vaccine of claim 23.

[0221] 25. The method of embodiment 24, wherein the method prevents influenza virus infection in the subject.

[0222] 26. The method of embodiment 24 or 25, wherein the method generates a protective immune response in the subject.

[0223] 27. The method of embodiment 26, wherein the protective immune response comprises an HA antibody response and / or an NA antibody response.

[0224] 28. The method of any one of embodiments 24 to 27, wherein the subject is a human.

[0225] 29. The method of any one of embodiments 24-28, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0226] 30. The method of any one of embodiments 24-29, wherein the method treats or prevents disease caused by either or both seasonal and pandemic influenza strains.

[0227] 31. The method of any one of embodiments 24-30, wherein the subject is a human, and the human is at least 6 months old, under 18 years old, at least 6 months old and under 18 years old, at least 18 years old and under 65 years old, at least 6 months old and under 5 years old, at least 5 years old and under 65 years old, at least 60 years old, or at least 65 years old.

[0228] 32. A method for reducing one or more symptoms of influenza virus infection, comprising administering to a subject a prophylactically effective amount of the vaccine described in embodiment 23.

[0229] 33. A method for enhancing or expanding a protective immune response in a subject, comprising administering to the subject an immunologically effective amount of the vaccine described in embodiment 23, wherein the vaccine increases the vaccine efficacy of a standard of care influenza virus vaccine composition by an amount in the range of about 5% to about 100%, for example, by at least about 20%, or by about 40% to about 80%, for example, by an amount of about 40% to about 60%.

[0230] 34. The method of embodiment 33, wherein the standard of care influenza virus vaccine composition is an inactivated influenza virus composition comprising inactivated influenza viruses derived from H1N1 strains, H3N2 strains, B / Victoria lineage, and B / Yamagata lineage.

[0231] 35. The method of embodiment 33, wherein the standard of care influenza virus vaccine composition comprises recombinant influenza virus HA derived from H1N1 strains, H3N2 strains, B / Victoria lineage, and B / Yamagata lineage.

[0232] 36. The method of any one of embodiments 24-35, comprising administering to the subject two doses of the vaccine, 2 to 6 weeks apart, optionally 4 weeks apart.

[0233] The present disclosure will be better understood with reference to the following examples. EXAMPLES

[0234] The following examples should be considered as illustrative, and not limiting, of the scope of the above disclosure.

[0235] 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 were conducted with animal protocols approved by 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.

[0236] Influenza Virus: Reassortant H6 viruses used in the enzyme-linked lectin assay (ELLA) were generated by reverse genetics, with each reassortant expressing the targeted NA antigen, HA from A / mallard / Sweden / 81 / 2002 H6N1, and internal genes from A / Puerto Rico / 8 / 1934 H1N1 ("PR8"). HA and NA segments, including non-coding regions, were generated by custom gene synthesis (Geneart AG), and PR8 segments were obtained from virus isolates. All segments were cloned into bidirectional transcription plasmids derived from pUC57 (Genscript) with 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, the transfected cells were added to MDCK-ATL cells (ATCC) in the presence of TPCK-treated trypsin (Sigma) to allow the growth of influenza virus. Cell culture supernatants containing influenza virus were harvested 7 days after MDCK addition 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). Viral titers were determined by plaque assay with MDCK cells.

[0237] 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 studies were provided by Sanofi Paster Global Clinical Immunology (Swiftwater, PA). Wild-type influenza A / Perth / 16 / 2009 (H3N2) used in the ferret study was provided by IIT Research Institute (Chicago, IL). All viruses were stored below −65°C until use.

[0238] Vaccine antigen: Constructs were designed to express recombinant soluble influenza NA. Both tetrameric and monomeric NA construct designs contain 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. A codon-optimized synthetic gene was constructed from oligonucleotides and / or PCR products using the defined amino acid sequence, and the fragment was inserted into pcDNA3.4-TOPO (ThermoFisher). The plasmid DNA was purified from transformed bacteria and adjusted to the appropriate concentration for transfection. 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 manufactured in accordance with current good research practices (cGRPs).

[0239] Enzyme-linked lectin assay (ELLA) evaluation of NAI responses: NAI antibody responses were measured against H6 reassortant viruses containing NA from the strains of interest by ELLA as previously described by 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 strains of interest were titrated in fetuin-coated 96-well plates to determine the standard viral load that provided 70% of the maximum NA enzyme activity. Titration of NAI antibodies present in serum was performed 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 fetuin-coated plates. The serum-virus mixture was incubated overnight at 37°C. The plates were washed four times, incubated with horseradish peroxidase (HRP)-conjugated peanut agglutinin (PNA), washed again, and then developed by adding o-phenylenediamine dihydrochloride (OPD). Low or no signal relative to the virus control indicates inhibition of NA activity due to the presence of NA-specific antibodies. NAI titers were fitted with a nonlinear four-parameter logistic (4PL) curve using GraphPad Prism software to determine the 50% maximal inhibitory concentration (IC 50 ) was calculated.

[0240] 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). In this example, unless otherwise indicated, the homologous virus panel included egg-grown 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. The plate was covered and incubated for 20 minutes (or 45-60 minutes) at room temperature, followed by the addition of a 1% mixture of chicken erythrocytes (red blood cells; CRBCs) or a 0.5% mixture of turkey red blood cells (TRBCs) (Lampire Biologicals) in PBS. The plate was mixed by vortexing, covered, and the RBCs were allowed to sit at room temperature for approximately 30 minutes to 1 hour. The HAI titer was determined by the reciprocal dilution of the last well containing non-agglutinated RBCs.

[0241] Antibody forensic assay: Antibody forensic assay (AF) was used to measure strain-specific rHA antibodies in ferret sera using fluorescent dye-based magnetic bead arrays (MagPlex® Microspheres). The intensity of antibody binding to strain-specific rHA was expressed in normalized mean fluorescence intensity units (nMFI) calculated by multiplying the raw fluorescence intensity signal by serum dilution. rHA bound to magnetic beads was selected based on antigenic data published in the Francis Crick Institute Annual and Interim Reports on the Composition of Influenza Vaccines. In addition to the 2018-2019 Northern Hemisphere recommended strains, the rH3 panel included strains from the 2013 to 2016 seasons, and the H1 panel encompassed strains from the 2009 to 2016 seasons. Individual ferret sera were analyzed and the resulting antibody forensic data of 40 H3 and 18 H1 strains were evaluated.

[0242] HINT mNT Influenza Protocol: Neutralizing titers against influenza strains were measured as per the reference 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 from 1:20 to 1:2,560 were mixed with an equal amount of virus, approximately 1000 focus forming units (FFU), and incubated at 37°C for 60 minutes. After incubation, MDCK-SIAT1 cell suspensions were added to the virus:serum mixture and incubated for approximately 22 hours. Monolayers were fixed with methanol and prepared for staining. Wells were then incubated with an anti-influenza monoclonal antibody against nucleoprotein (NP), followed by incubation with an ALEXA FLUOR® 488-conjugated secondary antibody. Cells were washed and plates were scanned with CTL IMMUNOSPOT® Cell Imaging v2. Counts from plates were transferred to Graphpad Prism software and neutralization titers were calculated from sigmoidal curves to achieve 50% foci reduction. This assay measures inhibition of viral entry compared to virus input control wells without trypsin and without serum. Counts are individual infected cells and this assay is suitable for all live virus subtypes including H1, H3, BVic, and BYam.

[0243] The resulting solvent is a specific rHA inhibitor:H3 AFパネル:A / URUGUAY / 716 / 2007, A / VICTORIA / 361 / 2011, A / HONGKONG / 4801 / 2014, A / SINGAPORE / INFIMH-16-0019 / 2016, A / BRISBA NE / 1059 / 2017、A / ETHIOPIA / 1877 / 2017、A / KENYA / 105 / 2017、A / MISSOURI / 37 / 2017、A / MIYAZAKI / 89 / 2017、A / OSORNO / 60580 / 20 17、A / SAPPORO / 46 / 2017、A / SHANGHAIXUHUI / 1373 / 2017、A / SYDNEY / 1093 / 2017、A / SYDNEY / 1013 / 2017、A / AKSARAY / 4048 / 2016、A / ALBANIA / 7165 / 2016、A / ANKARA / 4110 / 2016、A / BRITAIN / 2836 / 2016、A / BRISBANE / 1009 / 2016、A / CALIFORNIA / 168 / 2016、A / CH IBA / 33 / 2016、A / CHRISTCHURCH / 513 / 2016、A / GUANGXIQIXIN / 328 / 2016、A / HAWAII / 67 / 2016、A / JORDAN / J16420301NT / 2016、A / K AWASAKI / 142 / 2016、A / KHMELNITSKY / 719 / 2016、A / LAOS / F2884 / 2016、A / LINKOU / 0051 / 2016、A / LISBOA / NIEVA063 / 2016、A / MART INIQUE / 531 / 2016、A / MARYLAND / 24 / 2016、A / MEKNES / 168 / 2016、A / MICHIGAN / 84 / 2016、A / PORTUGAL / MS68 / 2016、A / SAUDI ARABIA / 192150 / 2016、A / SHANDONGLAICHENG / 1763 / 2016、A / SINGAPORE / GP2366 / 2016、A / TASMANIA / 97 / 2016、A / TOWNSVILLE / 51 / 2016.

[0244] H1 AF panel: A / CALIFORNIA / 07 / 2009, A / BAYERN / 69 / 2009, A / HONGKONG / 34079 / 2009, A / HONGKONG / 33597 / 2009, A / L VIV / N6 / 2009, A / MONTPELLIER / 2051 / 2009, A / CHRISTCHURCH / 16 / 2010, A / ANKARA / TR40 / 2011, A / ASTRAKHAN / 1 / 2011, A / HONGKONG / 3934 / 2011, A / GOTEBORG / 1 / 2011, A / MEXICO / 2208 / 2011, A / HONG / KONG / 5659 / 2012, A / STOC KHOLM / 25 / 2012, A / ISRAEL / Q504 / 2015, A / MICHIGAN / 45 / 2015, A / HUNGARY / 12 / 2016, A / BRATISLAVA / 342 / 2016.

[0245] In 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, fractionated with Triton® X-100, further purified, and then suspended in 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, and 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 - Expression, purification, and characterization of rTET-NA rTET-NA constructs derived from the NA of all four subtypes present in currently circulating seasonal influenza viruses (A / Michigan / 45 / 2015 N1; A / Singapore / 2016 N2; B / Colorado / 06 / 2017 Victoria lineage; and B / Phuket / 3073 / 2013 Yamagata lineage) were expressed in CHO-S cells and purified to near homogeneity for further characterization. Schematic and partial amino acid sequences of rTET-NA constructs (e.g., from A / Singapore / 2016 N2) are shown in FIG. 1. Unless otherwise indicated, a heterologous tetrabrachion tetramerization sequence is present in all rTET-NA constructs disclosed in the Examples below, regardless of the influenza virus strain from which the NA head region was derived. However, as known in the art, any suitable heterologous tetramerization domain may be used in place of the tetrabrachion tetramerization sequence. In the rTET-NA construct, the cytoplasmic domain, transmembrane region, and all or substantially all of the stalk region of wild-type influenza neuraminidase are replaced with a "secretion signal" peptide, a heterologous tetrabrachion tetramerization domain, and an optional histidine tag, which can be used to facilitate purification of the rTET-NA.

[0247] Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) demonstrated efficient tetramerization of rTET-NA, confirming that all rTET-NA was formed as a tetramer with a molecular weight (~260 kDa) that was ~4-fold higher than that of the control (ectodomain) monomer (~74 kDa) (data not shown). Size-exclusion chromatography used a TSK-GEL G4000 PWXL (7.8 mm x 30 cm) column (Tosoh Bioscience) and the mobile phase was phosphate-buffered saline (pH 7.4) containing 0.02% sodium azide. All detectors were connected in series downstream of the column. UV peak areas on the chromatograms were integrated using Empower® (Waters Corporation, Milford, MA) software. Purity of the sample was calculated based on the ratio of specific peak area / total peak area. The molecular weight (MW) of proteins in the peaks was determined using the light scattering signal from a concentration detector (RI or UV) with ASTRA (Wyatt Technologies, Santa Barbara, Calif.) software.

[0248] rTET-NA enzyme activity was demonstrated with a 2'-(4-methylumbelliferyl)-α-dN-acetylneuraminic acid (MUNANA) assay. Two-fold serial rTET-NA dilutions were prepared in a 96-well plate using buffer (33.3 mM 2-[N-morpholino]ethanesulfonic acid [MES, pH 6.5], 4 mM CaCl2, 50 mM BSA), mixed with MUNANA substrate (100 μM) and incubated at 37°C for 1 h with shaking. The reaction was stopped by the addition of alkaline solution (0.2 M Na2CO3). The fluorescence intensity (RFU, relative fluorescence units) from the mixture of rTET-NA and MUNANA substrate was measured using an excitation wavelength of 355 nm and an emission wavelength of 460 nm, respectively. A standard curve was constructed using 4-methylumbelliferone (4-MU) diluted in enzyme buffer at various concentrations; rTET-NA enzyme activity was determined against a 4MU standard and results were expressed in μM / 60 min for total NA activity and nmoles / min / μg for specific NA activity.

[0249] In addition, rTET was shown to bind to oseltamivir-phosphate. In contrast, an enzymatically inactive monomeric N2 ectodomain variant did not bind to oseltamivir-phosphate. SEC-MALS analysis was also performed on rTET-NAs derived from various influenza subtypes and 34 different N2 strains, and tetramerization of rTET-NAs and binding to oseltamivir-phosphate were demonstrated in all cases. Oseltamivir binding assays were performed using the following conditions: Oseltamivir-phosphate-biotin conjugate (5-10 μg / ml in 1xKB buffer (1% BSA and 0.02% Tween in PBS)) was captured on the surface of a streptavidin-coated biosensor; the biosensor was immersed in wells containing serial 2-fold dilutions of recombinant NA samples (0.16-10 μg / ml in 1xKB); the binding kinetics of recombinant NA to oseltamivir-phosphate was measured using biolayer interferometry (BLI) technology on an Octet instrument (ForteBio, Molecular Devices, LLC).

[0250] Example 2 - Evaluation of rTET-NA immunogenicity in mice A mouse model was used to evaluate the immunogenicity of rTET-NA. Six to eight week old female BALB / c mice (8 per group) were vaccinated twice with either N2 rTET-NA from A / Singapore / INFIMH-16-0019 / 2016, N1 rTET-NA from A / Michigan / 45 / 2015, 0.2 μg or 1 μg monovalent inactivated influenza vaccine (IIV), or 0.2 μg live virus-derived NA (LVNA), with or without AF03 (squalene in water) adjuvant (all doses were 50 μL). The first dose was administered intramuscularly on day 0 and a booster dose was administered intramuscularly on day 21, as shown in Figure 2A. Two weeks after the final dose (day 35), NAI antibody titers in serum were measured. Serum pools from two animals were generated (stored at -20°C until needed) giving a total of four samples per group. Sera were tested in the ELLA to assess NAI activity or by ELISA to obtain NA-binding antibodies.

[0251] It was demonstrated that rTET-NA has immunogenicity comparable to other NA-containing virus preparations, and this immunogenicity was significantly enhanced with AF03 adjuvant. See Figures 2B and 2C. As shown in Figure 2C, the immunogenicity of rTET-NA was higher than that of IIV for the N1 subtype, A / Michigan45 / 2015 strain, which was also tested in mice. Without wishing to be bound by theory, this subtype-specific difference may be due to the latter's split inactivation process, which results in reduced enzymatic activity and loss of immunogenicity compared to N2.

[0252] Example 3 - Evaluation of rTET-NA immunogenicity in ferrets We assessed the immunogenicity of rTET-NA in influenza-naïve, pre-immune ferrets, which are widely used in influenza research based on their shared lung physiology and susceptibility to influenza with humans.

[0253] Naive outbred male and female Fitch ferrets (6 per group) aged 17-21 weeks were vaccinated intramuscularly twice, 21 days apart, with either 5μg+AF03, 45μg+AF03, or 45μg of N2 rTET-NA from A / Singapore / INFIMH-16-0019 / 2016 (Figure 3C) or 5μg+AF03, 45μg+AF03, or 45μg of N1 rTET-NA from A / Michigan / 45 / 2015 (Figure 3F) (500μL / dose) (study outline shown in Figure 3A). In the preimmune ferret model, ferrets were first primed by intranasal administration of influenza virus (1,000μL / dose, evenly divided between nostrils) on day 0, as shown in Figure 3B. Three weeks after the prime (day 21), ferrets were vaccinated with N2 rTET-NA (1.8 μg, 9 μg, or 45 μg), IIV (1.8 μg or 9 μg), or vaccine diluent (sham) from A / Singapore / INFIMH-16-0019 / 2016 (FIG. 3D), or N1 rTET-NA (0.36 μg, 1.8 μg, 9 μg, or 45 μg), IIV (1.8 μg or 9 μg), or vaccine diluent (sham) from A / Michigan / 45 / 2015 (FIG. 3G). NAI antibody levels were measured in serum 3 weeks after the prime and boost.

[0254] All ferrets were bled under sedation at baseline, 1 day or immediately prior to the booster, and 3 weeks after the booster. Serum samples (stored at -20°C until needed) were tested in the ELLA to assess NAI activity.

[0255] rTET-NA was demonstrated to be highly immunogenic in naive ferrets and as a booster vaccine in pre-immunized ferrets (Figures 3C-3H). rTET-NA was highly immunogenic in naive ferrets after a single dose and could be further boosted with a second dose (Figures 3C and 3F). Similar to the mouse model, NAI titers were enhanced with AF03 compared to the non-adjuvanted formulation. The addition of adjuvant is dose-sparing (e.g., 5μg+AF03 is more immunogenic compared to 45μg without adjuvant). Vaccination of pre-immune ferrets with a single dose of unadjuvanted A / Singapore / INFIMH-16-0019 / 2016 N2 rTET-NA or A / Michigan / 45 / 2015 N1 rTET-NA resulted in a comparable or superior boost in NAI responses compared to matched IIV doses (Figures 3D and 3G).

[0256] Similar to naive mice, rTET-N1 was more immunogenic than IIV in the naive ferret model at both doses tested, demonstrating an extended advantage of the rNA platform against the N1 subtype (Figure 3G). In the pre-immune ferret model, the boost in NAI responses following infection (NAI boost / prime ratio) was also greater for N1 compared to N2 at the two highest doses evaluated (9 μg and 45 μg), but not at the lower dose evaluated (1.8 μg). See Figures 3E and 3H.

[0257] Example 4 - Influenza virus challenge study in ferrets For influenza virus challenge studies, naive outbred male Fitch ferrets (Triple F farms, Sayre, PA) aged 17-21 weeks (16 per group) were initially vaccinated twice, 21 days apart, with identical doses (0.2 μg, 3 μg, or 45 μg) of A / Perth / 09 N2 TET-NA (500 μL / dose, intramuscular) with or without AF03 adjuvant. Three weeks after the booster vaccination, ferrets were given 107 PFU of A / Perth / 09 H3N2 wild-type influenza A challenge virus (1,000 μL / dose, divided evenly between nostrils) was administered intranasally. The animals were monitored daily for clinical signs and changes in body weight and twice daily for body temperature for 14 days after virus administration.

[0258] Nasal washes were collected from all challenged animals on days 1, 3, 5, and 7 post-challenge and samples were stored at -65°C or below for virus evaluation. Ferrets were anesthetized with a mixture of ketamine (25 mg / kg) and xylazine (2 mg / kg) and 0.5 mL of sterile PBS containing penicillin (100 U / mL), streptomycin (100 μg / mL), and gentamicin (50 μg) was injected into each nostril, collected, and stored at -65°C or below. Virus in nasal wash specimens was analyzed using standard 50% tissue culture infectious dose (TCID 50 The antibodies were titrated using a nasal wash centrifugation assay. The nasal washes were thawed and then clarified by centrifugation. The resulting supernatants were serially diluted 10-fold and then transferred to individual wells of a 96-well plate containing a monolayer of Madin-Darby Canine Kidney Cells (MDCK) cells for titration.

[0259] Lung sections (left and right cranial lobes, and left and right caudal lobes) and nasal turbinates were harvested for virus titers on days 1, 3, 6, and 14 after viral challenge. Tissue sections were weighed and then rapidly frozen in ethanol / dry ice or liquid nitrogen, processed, and titrated at standard TCID 2010 as described above. 50The samples were stored at -65°C or below for virus titration by assay. Selected ferrets (1-2) from each group were euthanized and necropsied on days 1, 3, 6, and 14 postchallenge. Lungs and nasal turbinates were harvested from necropsied ferrets for virus titer and histopathological analysis. Lungs and nasal turbinates were fixed in 10% neutral buffered formalin. Necropsies scheduled for histopathology were overseen by a certified veterinary pathologist. Fixed lung lobes and NT sections were embedded in paraffin, processed by routine histological methods, stained with hematoxylin and eosin, randomized, and graded for the presence and severity of pathology by a certified veterinary pathologist.

[0260] As shown above for A / Singapore / 2016, rTET-NA based on A / Perth / 16 / 2009 N2 induced dose-dependent NAI titers that were boosted by AF03. The highest rTET-NA dose evaluated with AF03 (45 μg) induced superior titers compared to infection at challenge alone (Fig. 4).

[0261] As shown in Figure 5, rTET-NA vaccination prevented disease severity following homologous H3N2 challenge in ferrets by reducing the intensity and duration of clinical symptoms such as weight loss, fever, and overall viral shedding. NA-mediated protection was characterized by a dose- and adjuvant-dependent reduction in overall weight loss and peak temperature rise similar to those observed before infection only at high rTET-NA doses with AF03. Only a minor effect on total viral shedding was observed and did not appear to follow a dose-dependent pattern (Figure 5).

[0262] Analysis of individual ferret data showed that the onset of symptoms in the sham AF03 group was variable, not only in terms of symptom intensity but also in timing: overall peak symptoms were observed 2 days after challenge, but some animals developed delayed symptoms or a second peak of symptoms 2 weeks after challenge, making longitudinal analysis difficult.

[0263] Although rTET-NA appeared to be less efficient at preventing viral shedding than previous infections, this result was expected, as infection confers both anti-NA and anti-HA immunity in addition to T cell immunity against conserved epitopes.

[0264] As shown in Figure 6A-C, post-vaccination NAI titers were inversely correlated with disease severity and had predictive power to be used as a correlate of protection. Animals exhibiting severe disease (n=21) had a mean Log2 NAI titer of 5.2 ± 2.80 at day 42, whereas animals exhibiting non-severe disease (n=55) had a mean Log3 NAI titer of 7.7 ± 3.00 at day 42 (p-value = 0.0014) (Figure 6A).

[0265] The association of anti-NA antibody responses with disease severity was examined by developing a disease scoring system based on peak symptoms and overall viral shedding. Ferrets were classified as severe and non-severe based on their total severity score, and the distribution of NAI titers according to this binary disease classification was analyzed. Ferrets with severe symptoms exhibited significantly lower NAI titers than ferrets classified as non-severe, although some non-responding ferrets were also protected (Figure 6C). To further evaluate NAI as a protective biomarker, a receiver operating characteristic (ROC) analysis was developed. The area under the curve (AUC) of the ROC curve was significantly higher than chance (0.73 vs. 0.50, p-value = 0.0002), confirming that NAI titers can be used to accurately predict disease severity in ferrets (Figure 6B). ROC analysis was also used to determine the NAI titer threshold required for protection.

[0266] Example 5 - Evaluation of immunogenicity of multivalent HA and NA 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. When AF03 adjuvant was used, it was mixed with the antigen in a 1:1 ratio. As shown in Table 1 below, a tetravalent vaccine composition was used that included rTET-NA having N1, N2, NA from B / Victoria lineage, and NA from B / Yamagata lineage (specifically, from A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013 strains), and a tetravalent vaccine composition was used that included rHA having H1, H3, HA from B / Victoria lineage, and HA from B / Yamagata lineage (specifically, from A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013 strains). For each group, n=6 mice. HAI titers on day 35 were measured for the following influenza virus strains: A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013. The results are reported in Table 1 below.

[0267] [Table 1]

[0268] Similarly, NAI titers were similarly assessed in mice with the following 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 in Table 2 below.

[0269] [Table 2]

[0270] As shown in Tables 1 and 2, vaccination with an octavalent vaccine containing four rTET-NAs and four rHAs resulted in HAIs within 4-fold of the tetravalent rHA for three of the four strains and more than 4-fold for B / Maryland / 15 / 2016. For example, this octavalent vaccine showed improved HAIs for B / Maryland / 15 / 2016 at a dose of 1 μg / strain compared to vaccination with tetravalent rHA alone (140 vs. 23.3). Similarly, vaccination with an octavalent vaccine containing four rTET-NAs and four rHAs resulted in NAIs within 4-fold of the tetravalent rHA for three of the four strains, but a reduction in NAI for A / Michigan / 45 / 2015, which was less than 4-fold.

[0271] Example 6 - Evaluation of immunogenicity of multivalent HA and NA in ferrets Part 1 As shown in Figure 7A, naive ferrets used to evaluate the immunogenicity of the multivalent vaccine were vaccinated twice, 21 days apart, with an octavalent vaccine composition containing a mixture of four rTET-NA antigens and / or four recombinant HA antigens with or without adjuvant. This experiment also evaluated monovalent rTET-NA (i.e., rTET-NA derived only from A / Singapore / Infimh-16-0019 / 2016). The complete study design is shown in Table 3.

[0272] [Table 3]

[0273] Each rTET-NA antigen contains an NA head domain from one of the standard of care strains included in the tetravalent 2018-19 seasonal influenza vaccine (A / Singapore / INFIMH-16-0019 / 2016 (N2), A / Michigan / 45 / 2015 (N1), B / Colorado / 06 / 2017 (B / Victoria lineage), and B / Phuket / 3073 / 2013 (B / Yamagata lineage). Similarly, each recombinant HA contains an HA from one of the following 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). Figure 7B.

[0274] All ferrets were bled under sedation at baseline, 1 day or just before the booster, at the time of booster vaccination, and 3 weeks after the booster (day 42). Serum samples (stored at -20°C until needed) were tested by ELISA to assess NAI activity. In addition, hemagglutinin inhibition assays (HAI) and antibody forensics were performed to assess antibody responses to hemagglutinin antigens following polyvalent vaccination.

[0275] It was demonstrated that rTET-NA retained its immunogenicity after octavalent HA and NA vaccination in ferrets. This is the first demonstration of the feasibility of a fully recombinant octavalent vaccine containing HA and NA. Ferrets immunized with the recombinant octavalent composition developed similar NAI antibody responses as animals immunized with the tetravalent rNA composition alone in a dose- and adjuvant-dependent manner (Figure 7C). As shown in Figure 7C, no interference was observed between HA and NA antigens, and some synergy was detected at the 45 μg dose without adjuvant after the first vaccination. A significant increase in NAI titers was observed after the second vaccination. Induction of NAI titers (and NA ELISA) was confirmed across all four NA subtypes.

[0276] HA-specific antibody responses in octavalently vaccinated ferrets were comparable in both quantity and quality to ferrets immunized with the tetravalent rHA vaccine alone, as demonstrated by HAI and multiple antibody binding ELISA (antibody forensics, AF). As shown in Figure 8, supplementation with tetravalent rHA and tetravalent rTET-NA does not interfere with the magnitude and breadth of the tetravalent rHA immune response elicited against either H3 (Figure 8A-C) or H1 (Figure 8D-F). Results correspond to the second immunization (post-boost) only. Similar results to NA above were demonstrated, with no interference between HA and NA antigens, clear synergy at the 5 μg + AF03 dose, and a dose-sparing effect of AF03. AF data are shown showing a similar breadth of responses elicited by HA antigens of the tetravalent rHA and octavalent formulations (4 rHA + 4 rNA) as determined against a panel of H1 HA and H3 HA (Figure 8B, C, E, F).

[0277] Thus, the addition of a tetravalent recombinant influenza NA vaccine to a tetravalent recombinant influenza HA vaccine induced strong NA-specific humoral immunity in ferrets without interfering with their ability to induce strong HA-specific immunity.

[0278] The effect of the adjuvant (AF03) in this experiment on the antibody responses elicited by recombinant monovalent NA, tetravalent rNA, and octavalent rHA and rNA proteins was also evaluated, and the results are shown in Table 4 below.

[0279] [Table 4]

[0280] AF03 adjuvant increases NAI responses to monovalent and multivalent NA vaccines and shows a dose-sparing effect. For both the quadrivalent and octavalent rHA+rNA vaccines, a dose effect was observed after the first dose and a large NAI boost (e.g., >8-fold) was observed after the second dose.

[0281] Furthermore, the addition of tetravalent rHA to tetravalent rNA did not reduce the immunogenicity of recombinant NA regardless of dose and / or adjuvant, and we observed a clear synergistic effect after the first dose of octavalent rHA+rNA without adjuvant (45 μg) compared to tetravalent rNA without adjuvant (45 μg).

[0282] Example 7 - Evaluation of immunogenicity of multivalent HA and NA in ferrets Part 2 As shown in Table 5 below, a tetravalent vaccine composition including rTET-NA having 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) was combined with a tetravalent vaccine composition including rHA having H1, H3, HBv, and HBy (specifically, from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013). A tetravalent vaccine composition including rHA, H1, H3, HBv, and HBy, but without adjuvant, was used as a control. For each group, n=6 ferrets. mNT (HINT) titers were measured for the following influenza virus strains: A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Iowa / 06 / 2017; and B / Phuket / 3037 / 2013. Results are reported in Table 5 below. Ferrets were injected with the prime vaccine on day 0 and the same dose of boost vaccine on day 21. Bleeds were taken on days -7, 1, 20, 22, and 42. When adjuvants were used, they were mixed with the antigen in a 1:1 ratio.

[0283] [Table 5]

[0284] As shown above, all three adjuvants (AF03, SPA14LD (low dose), and SPA14HD (high dose)) showed equivalent or improved titers compared to the adjuvanted composition. No significant differences were observed between adjuvant responses, even at low doses. Administration of the octavalent recombinant vaccine with adjuvant induced equivalent (within 4-fold) or higher (more than 4-fold) responses compared to administration of the tetravalent recombinant HA vaccine at the highest dose of 35 μg without adjuvant.

[0285] Similarly, NAI titers were similarly assessed in ferrets with 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 in Table 6 below.

[0286] [Table 6]

[0287] As shown above, we observed that all three adjuvants (AF03, SPA14HD, and SPA14LD) significantly improved the NAI response over no adjuvant against three of the four strains tested (A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013).

[0288] Furthermore, as shown in Table 7 below, we observed that SPA14HD induced the most significant increase in NAI responses over no adjuvant and significantly improved B strain NAI even at a low dose after a single immunization, which resulted in an NAI response on day 20 before a booster dose was administered.

[0289] [Table 7]

[0290] Example 8 - Multivalent HA and NA vaccine images induce broad NAI immunogenicity in ferrets As shown in Table 8 below, a tetravalent vaccine composition including rTET-NA having N1, N2, BvNA, and ByNA, respectively (specifically, from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013) was combined with a tetravalent vaccine composition including rHA having H1, H3, HBv, and HBy, respectively (specifically, from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013). NAI titers were measured for the following influenza virus strains: A / Singapore / Infimh160019 / 2016; A / Hatay / 4990 / 2016; A / Sweden / 3 / 2017; A / Louisiana / 13 / 2017; A / Townsville51 / 2016; A / Aksaray / 4048 / 2016; A / Perth / 16 / 2009; and A / Ohio13 / 2017. Results are reported in Table 8 below. Ferrets were injected with the prime vaccine on day 0 and the same dose of boost vaccine on day 21. Blood was collected on days 1, 20, 22, and 42.

[0291] [Table 8]

[0292] As shown above, broad-strain NAI responses were demonstrated following administration of the octavalent recombinant vaccine combination. A trend towards higher titers was observed with the SPA14HD adjuvant against all heterologous N2 strains tested.

[0293] Example 9 - Multivalent HA and NA vaccine imaging in a pre-immune ferret model After confirmation of influenza-negative HAI status, pre-immunized ferrets were inoculated with the following four live virus imprinted strains [1 × 10 5 ffu / strain; 0.5 mL per nostril (1 mL total)]. The ferrets were pre-immunized intranasally with a mixture of: A / NewCaledonia / 20 / 1999; A / Perth / 16 / 2009; B / HongKong330 / 2001; and B / Florida / 4 / 2006. On day 21, the ferrets were immunized with rTET-NA with each of N1, N2, BvNA, and ByNA (specifically, A / Michigan / 45 / 2015; A / Si The animals were immunized with an octavalent recombinant protein vaccine composition comprising rHA with H1, H3, HBv, and HBy, respectively, from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013 (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013). Blood was drawn on day 20 to establish baseline titers and again on day 42 (21 days after immunization with recombinant protein) to measure ELLA antibody responses. The results are shown in Table 9 below, and the mean IC50 ratios for each group are shown in Table 10 below.

[0294] [Table 9]

[0295] [Table 10]

[0296] As shown in Tables 9 and 10 above, the recombinant octavalent vaccine composition induced strong ELLA responses to A / Michigan / 45 / 2015, B / Colorado / 06 / 2017, and A / Singapore / Infimh160019 / 2016, regardless of the adjuvant used. We observed weak (inconclusive) responses to A / Perth / 16 / 2009, and responses to B / Phuket / 3037 / 2013 were more difficult to detect due to the high initial baseline. Thus, we observed that infection of pre-immunized ferrets induced some level of cross-reactive ELLA responses to most 2018 / 2019 SOC strains that were boosted by a single dose of the octavalent 2018 / 2019 SOC strain protein vaccine.

[0297] In addition, HA neutralization titers were measured using the HINT mNT protocol and the results are shown in Table 11 below.

[0298] [Table 11]

[0299] As shown in Table 11, similar HINT titers were observed across all groups following immunization with the octavalent vaccine composition.

[0300] Example 10 - Protection of multivalent recombinant vaccines in mice against homologous H1N1 and drifted H1N1 Mice were immunized on day 0 (priming) and day 21 (booster) with a recombinant octavalent vaccine composition comprising rTET-NA with N1, N2, BvNA, and ByNA, respectively (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Colorado / 06 / 2017; and B / Phuket / 3037 / 2013) and rHA with H1, H3, HBv, and HBy, respectively (specifically from strains A / Michigan / 45 / 2015; A / Singapore / Infimh160019 / 2016; B / Maryland / 15 / 2016; and B / Phuket / 3037 / 2013) at a dose of 0.2 μg / strain. Control mice were administered PBS. N=5 for both groups. Bleeding was performed 3 weeks after the prime dose and 3 weeks after the booster dose. Mice were challenged with 5LD of A / Belgium / 145 / 2009 on day 42. 50 (5 times the lethal dose to induce 50% mortality), or 5LD of Wisconsin / 588 / 2019. 50 The participants were challenged with this diet and their weight changes were monitored.

[0301] Compared to control mice, 5LD of A / Belgium / 145 / 2009 50 It was demonstrated that mice were well protected from weight loss during a 2-week monitoring period following infection with V. vaccinatum. Vaccinated mice had a 100% survival rate compared to control mice, which had a 100% mortality rate by day 8 post-infection.

[0302] Furthermore, compared with control mice, 5LD of Wisconsin / 588 / 2019 50 Mice were better protected from weight loss for up to two weeks after infection with vaccinated mice, but demonstrated 100% survival in both vaccinated and control mice.

[0303] Also, it should 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. Optional or optionally means that the subsequently described event or circumstance may or may not occur, and the description includes the event or circumstance that 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 can be expressed herein as from about one particular value and / or to about another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent, it will be understood that the particular value forms another embodiment. It will be further understood that each of the endpoints of the ranges has meaning both in relation to the other endpoint, and independently of the other endpoint. All references cited in this disclosure are incorporated herein by reference in their entirety.

Claims

1. An immunogenic composition comprising a plurality of recombinant influenza virus proteins, wherein the plurality of recombinant influenza virus proteins comprises: a first recombinant influenza virus hemagglutinin (HA), wherein said first recombinant influenza virus HA is an H1 HA; a second recombinant influenza virus HA, wherein said second recombinant influenza virus HA is an H3 HA; a third recombinant influenza virus HA, wherein said third recombinant influenza virus HA is derived from the B / Victoria lineage; a fourth recombinant influenza virus HA, wherein the fourth recombinant influenza virus HA is derived from the B / Yamagata lineage; a first recombinant influenza virus neuraminidase (NA), wherein the first recombinant influenza virus NA is an N1 NA; a second recombinant influenza virus NA, wherein said second recombinant influenza virus NA is N2 NA; a third recombinant influenza virus NA, wherein the third recombinant influenza virus NA is derived from the B / Victoria lineage; and A fourth recombinant influenza virus NA, wherein the fourth recombinant influenza virus NA is derived from the B / Yamagata lineage. An immunogenic composition comprising:

2. 2. The immunogenic composition of claim 1, wherein each of the first, second, third, and fourth recombinant influenza virus NAs is a modified recombinant influenza virus NA.

3. The modified recombinant influenza virus NA comprises four modified monomeric NA molecules, each of which comprises the head region of the NA of the influenza virus, but The immunogenic composition of claim 2, comprising a modified recombinant tetrameric influenza virus NA comprising a modified monomeric NA molecule that lacks the cytoplasmic tail, the transmembrane region, and all or substantially all of the stalk region of the NA of the virus, wherein the modified monomeric NA molecule forms a modified recombinant tetrameric NA when expressed in a host cell.

4. The immunogenic composition of claim 3 , wherein each modified recombinant monomeric influenza virus NA comprises a heterologous tetramerization domain.

5. The immunogenic composition of claim 3, wherein each modified recombinant monomeric influenza virus NA does not contain a heterologous oligomerization domain.

6. 5. The immunogenic composition of claim 4, wherein the heterologous tetramerization domain is a Staphylothermus marinus tetrabrachion tetramerization domain, a GCN4 leucine zipper tetramerization domain, a tetramerization domain derived from a paramyxovirus phosphoprotein, or a human vasodilator-stimulated phosphoprotein (VASP) tetramerization domain.

7. 2. The immunogenic composition of claim 1, wherein each of the recombinant influenza virus HAs is produced in a baculovirus expression system in cultured insect cells.

8. 2. The immunogenic composition of claim 1, wherein each of the recombinant influenza virus NAs is produced in Chinese hamster ovary (CHO) cells.

9. 2. The immunogenic composition of claim 1, wherein the immunogenic composition does not comprise inactivated influenza virions or live attenuated influenza virions.

10. 2. The immunogenic composition of claim 1, wherein each of the recombinant influenza virus HAs and / or each of the recombinant influenza virus NAs is derived from a standard of care influenza strain.

11. 2. The immunogenic composition of claim 1, wherein the H1 HA is derived from an H1N1 influenza virus strain and / or the H3 HA is derived from an H3N2 influenza virus strain.

12. the N1 NA is derived from an H1N1 influenza virus strain, and / or the N2 NA is derived from an H3N2 influenza virus strain; or 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 the N2 NA is derived from an H3N2 influenza virus strain; or 2. The immunogenic composition of claim 1, wherein the H1 HA and the N1 NA are derived from the same H1N1 influenza virus strain, and the H3 HA and N2 NA are derived from the same H3N2 influenza virus strain.

13. The multiple types of recombinant influenza virus proteins include: a first recombinant influenza virus HA, wherein said first recombinant influenza virus HA is an H1 HA; a second recombinant influenza virus HA, wherein said second recombinant influenza virus HA is an H3 HA; A third recombinant influenza virus HA, The viral HA is a third recombinant influenza virus HA derived from the B / Victoria lineage; a fourth recombinant influenza virus HA, wherein the fourth recombinant influenza virus HA is derived from the B / Yamagata lineage; a first recombinant influenza virus NA, wherein said first recombinant influenza virus NA is an N1 NA; a second recombinant influenza virus NA, wherein said second recombinant influenza virus NA is an N2 NA; a third recombinant influenza virus NA, wherein the third recombinant influenza virus NA is derived from the B / Victoria lineage; and A fourth recombinant influenza virus NA, wherein the fourth recombinant influenza virus NA is derived from the B / Yamagata lineage. The immunogenic composition of claim 1, comprising:

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

15. each of the recombinant influenza virus HAs is present in the 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 5 μg to about 45 μg; 2. The immunogenic composition of claim 1, wherein each of the recombinant influenza virus NAs is present in the 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.

16. A vaccine comprising the immunogenic composition of any one of claims 1 to 15 and a pharmaceutical carrier.

17. 17. The vaccine of claim 16 for use in immunizing a subject against influenza virus.

18. By immunizing, (i) influenza virus infection in said subject is prevented, and / or (ii) generating a protective immune response in said subject; and / or (iii) Disease caused by either or both seasonal and pandemic influenza strains is treated or prevented; 18. The vaccine of claim 17.

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

20. 17. The vaccine of claim 16 for use in reducing one or more symptoms of influenza virus infection, the method comprising administering a prophylactically effective amount of the vaccine to a subject.

21. 17. The method of claim 16 for use in enhancing or expanding a protective immune response in a subject.

17. The vaccine of claim 16, wherein the method comprises administering to the subject an immunologically effective amount of a vaccine, wherein the vaccine increases 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%, and optionally the method comprises administering to the subject two doses of the vaccine, 2 to 6 weeks apart, optionally 4 weeks apart.

22. An immunogenic composition comprising a plurality of recombinant influenza virus proteins, wherein the plurality of recombinant influenza virus proteins comprise three recombinant influenza virus HAs and three recombinant influenza virus NAs, wherein the three recombinant influenza virus HAs are selected from H1 HA, H3 HA, HA derived from the B / Victoria lineage, HA derived from the B / Yamagata lineage, or a combination thereof, and the three recombinant influenza virus HAs are selected from H1 HA, H3 HA, HA derived from the B / Victoria lineage, HA derived from the B / Yamagata lineage, or a combination thereof.

23. a first recombinant influenza virus HA, which is a first recombinant influenza virus hemagglutinin HA, wherein said first recombinant influenza virus HA is an H1 HA; a second recombinant influenza virus HA, wherein said second recombinant influenza virus HA is an H3 HA; a third recombinant influenza virus HA, wherein said third recombinant influenza virus HA is derived from the B / Victoria lineage; a first recombinant influenza virus NA, wherein said first recombinant influenza virus NA is an N1 NA; a second recombinant influenza virus NA, wherein the second recombinant influenza virus NA is an N2 NA; and a third recombinant influenza virus NA, the third recombinant influenza virus NA being derived from the B / Victoria lineage; The immunogenic composition of claim 22, comprising:

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