Multivalent influenza vaccine

Abstract

A multivalent vaccine or immunogenic composition is disclosed that includes an influenza virus hemagglutinin (HA) from a standard of care influenza virus strain, or a ribonucleic acid molecule encoding same; and one or more influenza virus HAs identified or designed by machine learning, or one or more ribonucleic acid molecules encoding influenza virus HAs identified or designed by machine learning. 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 dates of, U.S. Provisional Patent Application No. 63 / 253,986, filed October 8, 2021, and U.S. Provisional Patent Application No. 63 / 277,848, filed November 10, 2021, the disclosures of which are incorporated by reference in their entireties herein.

[0002] Disclosed herein is a multivalent influenza vaccine or immunogenic composition comprising multiple influenza virus hemagglutinin (HA) proteins or ribonucleic acid molecules encoding influenza virus HA, the multivalent vaccine or immunogenic composition comprising at least one influenza virus HA (or at least one ribonucleic acid molecule encoding influenza virus HA) having a molecular sequence identified or designed from a machine learning model. Further disclosed herein are methods of using the multivalent influenza vaccine or immunogenic composition. [Background technology]

[0003] Influenza is caused by a virus that primarily attacks the upper respiratory tract, including the nose, throat, and bronchi, and rarely the lungs. Infection usually lasts about a week. It is characterized by the sudden onset of high fever, muscle pain, headache and severe fatigue, dry cough, sore throat, and rhinitis. Most people recover within 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, infection can lead to severe complications of 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.

[0004] Influenza viruses are members of the family Orthomyxoviridae. 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.

[0005] Both HA and NA proteins are the source of the major immunodominant epitopes for virus neutralization and protective immunity, making them important components of influenza preventive vaccines. The genetic makeup of influenza viruses allows for frequent minor genetic changes, known as antigenic drift. Thus, the amino acid sequences of major influenza antigens, including HA and NA, are highly variable across a particular group, subtype, and / or strain. For this reason, current seasonal influenza vaccines are recommended for annual administration and require annual surveillance to account for mutations in HA (antigenic drift) and to adapt to rapidly changing virus strains.

[0006] Certain known licensed influenza vaccine compositions are inactivated vaccines containing whole virions or virions treated with agents that dissolve lipids ("split" vaccines), purified glycoproteins expressed in cell culture ("subunit vaccines"), or live attenuated vaccines. Other types of vaccines have been developed, such as RNA / DNA-based, viral vector-based, etc. These vaccines provide protection by inducing the subject's production of antibodies against antigens, e.g., HA. The antigenic evolution of influenza viruses by mutation alters the HA and, to a lesser extent, the NA. Thus, available vaccines can only protect against strains that have surface glycoproteins that contain identical or cross-reactive epitopes. To provide a sufficient antigenic spectrum, conventional vaccines contain components from several different virus strains, e.g., from both influenza A and B strains. The selection of strains for use in vaccines is reviewed annually for a given year and is based on the recommendations of the World Health Organization (WHO). These recommendations reflect international epidemiological observations.

[0007] The recommended WHO strains are known as standard therapeutic strains and typically include H1N1 subtypes, H3N2 subtypes, B / Yamagata lineage, and B / Victoria lineage. As mentioned above, due to antigenic drift, the selection of standard therapeutic strains must be updated annually to match the expected circulating strains of that year. Thus, commercially available conventional influenza vaccines are typically quadrivalent vaccines that contain four HAs from influenza virus strains, one from each of the subtypes / lineages: H1 (HIN1), H3 (H3N2), B / Yamagata, and B / Victoria. Vaccine compositions may include recombinant HA proteins, inactivated virions, such as split-inactivated virions, or attenuated virions. The WHO must select standard therapeutic strains well before the start of the influenza season to allow manufacturers sufficient time to produce a global vaccine supply, which means that the standard therapeutic strains selected by the WHO do not necessarily match the circulating influenza strains of a particular year. Influenza vaccine efficacy ranges from approximately 40-60% depending on the year and subtype, and is highly variable, especially for A / H3N2. Rapid antigenic drift of A / H3N2 has led to vaccine mismatch in the past, for example in the 2018-2019 season in the Northern Hemisphere. If the recommended standard of care strain selected by the WHO included in a seasonal vaccine formulation differs from the circulating influenza strain of a given season, the antigenic coverage provided by commercially available conventional influenza vaccines is narrowed and therefore the protective efficacy against influenza disease is reduced.

[0008] It would therefore be desirable to be able to supplement the standard of care strains of influenza in a vaccine with additional antigens and / or antigens that may confer additional protection and / or protection against a wider range of influenza strains and drifted HA strains. Summary of the Invention [Means for solving the problem]

[0009] The present disclosure provides a multivalent vaccine or immunogenic composition comprising an influenza virus HA from a standard of care influenza virus strain, or a ribonucleic acid molecule encoding an influenza virus HA from a standard of care influenza strain, and one or more machine learning influenza virus HAs, or ribonucleic acid molecules encoding machine learning influenza virus HAs. The one or more machine learning influenza virus HAs (or ribonucleic acids encoding same) may be selected to provide enhanced and / or broader protection against circulating influenza strains than the standard of care strains, enhancing the efficacy of the vaccine.

[0010] Disclosed herein is a vaccine or immunogenic composition comprising: (a) at least three or at least four influenza virus HAs, or at least three or at least four ribonucleic acid molecules encoding said influenza virus HAs, from a standard of care influenza virus strain; and (b) one or more machine-learning influenza virus HAs, or one or more multiple ribonucleic acid molecules encoding said one or more machine-learning influenza virus HAs, having molecular sequences identified or designed from a machine-learning model. In certain embodiments, the one or more machine-learning influenza virus HAs are selected from H1 HA, H3 HA, HA from B / Victoria lineage, HA from B / Yamagata lineage, or a combination thereof.

[0011] In one aspect, the present invention provides a method for the preparation of a ribonucleic acid molecule comprising: (a) a first influenza virus hemagglutinin (HA), where the first influenza virus HA is an H1 HA from a first standard of care influenza virus strain, or a first ribonucleic acid molecule encoding a first influenza virus H1 HA; and (b) a second influenza virus HA, where the second influenza virus HA is an H3 HA from a second standard of care influenza virus strain, or a second influenza virus H3 HA. Disclosed herein is a vaccine or immunogenic composition comprising: (a) a second ribonucleic acid molecule encoding an HA; (b) a third ribonucleic acid molecule encoding an influenza virus HA, wherein the third influenza virus HA is an HA from a third standard of care influenza virus strain of the B / Victoria lineage, or a third influenza virus HA derived from the B / Victoria lineage; (c) a third ribonucleic acid molecule encoding a third influenza virus HA, wherein the third influenza virus HA is an HA from a third standard of care influenza virus strain of the B / Victoria lineage, or a third influenza virus HA derived from the B / Victoria lineage; (d) a fourth ribonucleic acid molecule encoding a fourth influenza virus HA, wherein the fourth influenza virus HA is an HA from a fourth standard of care influenza virus strain of the B / Yamagata lineage, or a fourth influenza virus HA derived from the B / Yamagata lineage; and (e) one or more machine-learning influenza virus HAs having a molecular sequence identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding the one or more machine-learning influenza virus HAs, wherein the one or more machine-learning influenza virus HAs are selected from an H1 HA, an H3 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, or a combination thereof.Each of the one or more HAs (and independently of the others, if present) having a molecular sequence identified or designed from a machine learning model, or each of the one or more ribonucleic acid molecules (and independently of the others, if present) encoding one or more machine learning influenza virus HAs, may, in certain embodiments, be antigenically distinct, antigenically similar, from a different clade, from the same clade, and / or enhance the protective immune response induced thereby and / or expand the protective immune response induced thereby, as a respective standard of care influenza virus strain HA in the immunogenic composition.

[0012] In certain embodiments, the ribonucleic acid is an mRNA molecule, and in certain embodiments, the ribonucleic acid molecule is encapsulated in a lipid-nanoparticle (LNP). In certain embodiments, the ribonucleic acid molecule is encapsulated in a LNP comprising a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid.

[0013] In various embodiments disclosed herein, the one or more machine learning influenza virus HAs comprise a wild type influenza virus HA molecular sequence, and in certain embodiments, the machine learning influenza virus HAs comprise a non-wild type influenza virus HA molecular sequence. In certain embodiments, the one or more machine learning influenza virus HAs are recombinant influenza virus HAs, and in certain embodiments, the one or more machine learning influenza virus HAs are present in an inactivated influenza virus, such as a split-inactivated virus. In certain embodiments, the multivalent influenza vaccine comprises one or more ribonucleic acid molecules encoding at least one of the one or more machine learning influenza virus HAs.

[0014] In various embodiments, the one or more machine learning influenza virus HAs are a fifth influenza virus HA or a ribonucleic acid molecule encoding a fifth influenza virus HA, and the fifth influenza virus HA is an H3 HA. The fifth influenza virus H3 HA may, in certain aspects, be antigenically distinct from the second influenza H3 HA, antigenically similar to the second influenza H3 HA, enhance the protective immune response induced by the second influenza H3 HA, and / or expand the protective immune response induced by the second influenza H3 HA. The fifth influenza virus H3 HA may, in certain aspects, be from a different clade than the second influenza H3 HA, or from the same clade as the second influenza H3 HA. In certain embodiments, the fifth influenza virus H3 HA is from the 3C.2A clade, and in certain embodiments, the fifth influenza virus H3 HA is from the 3C.3A clade.

[0015] In various embodiments, the one or more machine learning influenza virus HAs are a fifth influenza virus HA or a ribonucleic acid molecule encoding a fifth influenza virus HA, and the fifth influenza virus HA is an H1 HA. The fifth influenza virus H1 HA may, in certain aspects, be antigenically distinct from the first influenza H1 HA, antigenically similar to the first influenza H1 HA, enhance the protective immune response induced by the first influenza H1 HA, and / or expand the protective immune response induced by the first influenza H1 HA. The fifth influenza virus H1 HA may, in certain aspects, be from a different clade than the first influenza H1 HA, or from the same clade as the first influenza H1 HA.

[0016] In certain embodiments of the vaccine or immunogenic composition disclosed herein, the vaccine or immunogenic composition further comprises a sixth influenza virus HA. In certain embodiments, the sixth influenza virus HA is an H3 HA, and in certain embodiments, the sixth influenza virus is an H3 HA with a molecular sequence identified or designed from a machine learning model, or a ribonucleic acid molecule encoding the sixth influenza virus HA. In certain embodiments, the sixth influenza virus HA is an H1 HA, such as an H1 HA with a molecular sequence identified or designed from a machine learning model, or a ribonucleic acid molecule encoding the sixth influenza virus HA. In certain embodiments, the sixth influenza H1 HA is antigenically distinct from the first influenza H1 HA, enhances the protective immune response induced by the first influenza H1 HA, expands the protective immune response induced by the first influenza H1 HA, is from a different clade than the first influenza H1 HA, is from the same clade as the first influenza H1 HA, or is antigenically similar to the first influenza H1 HA. In certain embodiments, the sixth influenza H3 HA is antigenically distinct from the second influenza H3 HA, enhances the protective immune response induced by the second influenza H3 HA, expands the protective immune response induced by the second influenza H3 HA, is from a different clade than the second influenza H3 HA, is from the same clade as the second influenza H3 HA, or is antigenically similar to the second influenza H3 HA.

[0017] In certain embodiments, the vaccine or immunogenic composition disclosed herein further comprises a seventh influenza virus HA derived from the B / Victoria lineage or the B / Yamagata lineage and having a molecular sequence identified or designed from a machine learning model, or a ribonucleic acid molecule encoding the seventh influenza virus HA.

[0018] In certain embodiments, the vaccine or immunogenic composition further comprises a ribonucleic acid molecule encoding a seventh influenza virus HA from the B / Victoria lineage and an eighth influenza virus from the B / Yamagata lineage, or the seventh and eighth influenza virus HAs, having molecular sequences identified or designed from a machine learning model.

[0019] In certain aspects, the machine learning model is trained to predict biological responses, such as human, ferret, or mouse biological responses, and in certain aspects, the biological responses include hemagglutinin inhibition assays (HAI), antibody forensics (AF), or neutralization assays. In certain embodiments, the molecular sequence is an amino acid sequence or a nucleic acid sequence. In certain embodiments, the molecular sequence is an amino acid sequence.

[0020] In various aspects of the vaccine or immunogenic composition disclosed herein, each of the first, second, third, and fourth influenza virus HA is a recombinant influenza virus HA, such as a recombinant influenza virus HA produced by a baculovirus expression system in cultured insect cells. In certain aspects, each of the first, second, third, and fourth influenza virus HA is present in an inactivated influenza virus, such as a split-inactivated virus. In still further aspects, the vaccine or immunogenic composition comprises the first, second, third, and fourth ribonucleic acid molecules described herein.

[0021] In certain embodiments disclosed herein, the first influenza virus HA is an H1 HA from an H1N1 influenza virus strain and the second influenza virus HA is an H3 HA from an H3N2 influenza virus strain.

[0022] In certain embodiments, the vaccine or immunogenic composition further comprises an adjuvant, such as a squalene in water adjuvant, such as AF03, or a liposome-based adjuvant, such as SPA14.

[0023] Another aspect of the present disclosure is directed to a method of immunizing a subject against influenza virus, comprising administering to the subject an immunologically effective amount of a vaccine or immunogenic composition disclosed herein. Similarly, the present disclosure provides an immunologically effective amount of a vaccine or immunogenic composition described herein for use in immunizing a subject against influenza virus. Similarly, the present disclosure also provides a use of an immunologically effective amount of a vaccine or immunogenic composition described herein for the manufacture of a medicament for 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, such as a HA antibody response, in the subject. In certain embodiments of the methods or uses disclosed herein, the subject is a human, and in certain embodiments, the human is 6 months or older, 6-35 months old, at least 2 years old, at least 3 years old, under 18 years old, at least 18 years old, at least 60 years old, at least 65 years old, at least 6 months old and under 18 years old, at least 3 years old and under 18 years old, or at least 18 years old and under 65 years old. In certain embodiments, the vaccine or immunogenic composition is administered or prepared to be administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, inhaled, or intraperitoneally. In certain embodiments, the methods or uses disclosed herein treat or prevent disease caused by either or both seasonal and pandemic influenza strains.

[0024] Also disclosed herein 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 or immunogenic composition disclosed herein. Similarly, the disclosure provides a prophylactically effective amount of a vaccine or immunogenic composition described herein for use in reducing one or more symptoms of influenza virus infection in a subject. Similarly, the disclosure also provides a use of a prophylactically effective amount of a vaccine or immunogenic composition described herein for the manufacture of a medicament for reducing one or more symptoms of influenza virus infection in a subject. In certain aspects, the method or use disclosed herein comprises administering to a subject two doses of a vaccine or immunogenic composition, spaced 2-6 weeks apart, optionally 4 weeks apart.

[0025] In another aspect, disclosed herein is a vaccine composition comprising the immunogenic composition disclosed herein. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 is a model diagram showing hypothetical examples of virus samples, virus 1 and virus 2, scored in an HAI assay, where the HAI titers of virus 1 and virus 2 can be compared to the vaccine virus from the previous season to assess antigenic similarity or dissimilarity of different virus strains. [Diagram 2] 2 is a bar graph showing the mean microneutralization titers for each group of ferrets infected with A / HONGKONG / 45 / 2019 alone (gray), A / ALASKA / 43 / 2019 alone (green), and a combination of A / HONGKONG / 45 / 2019 and A / ALASKA / 43 / 2019 (orange) as described in Example 1. Titers observed for seven strains of the 3C.2 clade are shown on the left, and titers observed for five 3C.3 clade strains are shown on the right. [Diagram 3]3 is a bar graph showing the mean microneutralization titers for each group of ferrets infected with A / HONGKONG / 45 / 2019 alone (green), A / KANSAS / 14 / 2017 alone (blue), and the combination of A / HONGKONG / 45 / 2019 and A / KANSAS / 14 / 2017 (orange) as described in Example 1. Titers observed with seven 3C.2 clade strains are shown on the left, and titers observed with five 3C.3 clade strains are shown on the right. [Figure 4A] FIG. 4A is a graph showing microneutralization titers of influenza virus 3C.2 clade strains (top) and 3C.3 strains (bottom) following co-infection with A / HONGKONG / 45 / 2019 alone (light grey), A / ALASKA / 43 / 2019 alone (dark grey), and a combination of A / HONGKONG / 45 / 2019 and A / ALASKA / 43 / 2019 (orange), as described in Example 1. [Figure 4B] Figure 4B is a graph showing microneutralization titers of 3C.2 clade strains (top) and 3C.3 strains (bottom) of influenza virus following co-infection with A / HONGKONG / 45 / 2019 alone (light grey), A / KANSAS / 14 / 2017 alone (dark grey), and a combination of A / HONGKONG / 45 / 2019 and A / KANSAS / 14 / 2017 (orange), as described in Example 1. [Diagram 5] FIG. 5 is a plot showing the mean neutralization titers following coinfection with the combination of A / HONGKONG / 45 / 2019 and A / ALASKA / 43 / 2019 (blue) and following challenge with the combination of A / HONGKONG / 45 / 2019 and A / KANSAS / 14 / 2017 (orange) relative to the maximum mean single titers for each of the 12 strains evaluated, as described in Example 1. [Figure 6] FIG. 6 is a graph showing the geometric mean titer (GMT) microneutralization assay titers of groups 1-7 and 10 against A / Tasmania / 503 / 2020, A / Victoria / 2570 / 2019, B / Phuket / 3073 / 2013, and B / Washington02 / 2019, respectively, as described in Example 2. [Figure 7] FIG. 7 is a bar graph showing GMT microneutralization assay titers of groups 1-7 and 10 (left bars of each group) against viruses of the 3C.2A clade and groups 1-7 and 10 (right bars of each group) against viruses of the 3C.3A clade, as described in Example 2. [Figure 8] FIG. 8 is a graph showing the geometric mean titer (GMT) microneutralization assay titers of groups 1-7 and 10 against A / Bangladesh / 3190613015 / 2019, A / Hong Kong / 45 / 2019, A / Singapore / INFIMH160019 / 2016, A / Valladolid / 182 / 2017, A / Kansas / 14 / 2017, and A / Mexico / 2356 / 2019, respectively, as described in Example 2. [Figure 9] FIG. 9 is a bar graph showing the coverage rate (GMT values ​​>1:160) of groups 1-7 (left bars of each group) against viruses of the 3C.2A clade and groups 1-7 (right bars of each group) against viruses of the 3C.3A clade, as described in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] 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 (i.e., 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.

[0028] The rate of silent nucleotide substitutions has been shown to be greater than the rate of coding nucleotide substitutions in all influenza virus genes, including the gene for HA (reviewed by Webster et al.; Webster, RG, et al., 1992). However, HA has a much greater rate of coding change than the internal proteins. The greater 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., 1982). Using reassortment antigens to eliminate nonspecific steric hindrance, Kilbourne et al. studied the evolutionary rates of epidemiologically important HA and NA antigens isolated from humans over a 10-year period and found that HA evolves more rapidly than neuraminidase (NA) (Kilbourne, ED, et al., 1990). This was shown for both H1N1 and H3N2 viruses of type A 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.

[0029] Therefore, the addition of HA antigens from supplementary strains may increase vaccine efficacy. This increase in efficacy may be due to two main mechanisms. First, the inclusion of one or more additional HA antigens may allow protection against a wider range of circulating influenza strains, for example, when the circulating strains are matched or antigenically similar to the additional strains but not to the standard therapeutic strain. Second, for circulating strains that are antigenically similar to both the standard therapeutic strain and the additional strains, the effect of a dose of matching or similar antigen in the vaccine can be doubled, which in turn can increase antibody titers and seroconversion rates. Either or both mechanisms may increase vaccine efficacy.

[0030] Thus, disclosed herein are multivalent influenza vaccines that comprise, in addition to an influenza virus HA derived from a standard of care influenza virus strain (and / or a ribonucleic acid molecule encoding such a standard of care influenza virus HA), one or more supplemental HA proteins or ribonucleic acid molecules encoding same, which may be identified or designed using machine learning models.

[0031] definition In order to more readily understand this disclosure, 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.

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that will be apparent to those skilled in the art upon reading this disclosure, etc.

[0033] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in and of itself, imply any importance, priority or order of one claim element relative to another claim element, or the temporal order in which operations of a method are performed, but is merely used as a label (but for the purposes of the use of ordinal terms) to distinguish one claim element having a certain name from another claim element having the same name, in order to distinguish the claim elements.

[0034] 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.

[0035] 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 binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell). In some embodiments, the antigen elicits a humoral response in the organism (e.g., including production of antigen-specific antibodies); alternatively or additionally, in some embodiments, the antigen elicits a cellular response in the organism (e.g., involving T cells whose receptors specifically interact with the antigen). One of skill in the art will appreciate that a particular antigen may elicit an immune response in one or more members of a target organism (e.g., mouse, ferret, rabbit, primate, human), but not 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 induce a specific physiological response in the organism. In some embodiments, for example, the antigen binds 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 the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The antigen includes HA forms described herein.

[0036] Antigenically distinct: As used herein, the term "antigenically distinct" indicates that two antigens (e.g., HA antigens) generate a greater than four-fold antibody response to each other as measured by binding titers or neutralization titers, as described below. HA antigens from different clades can be antigenically distinct.

[0037] Antigenically Similar: As used herein, the term "antigenically similar" indicates that two antigens elicit an antibody response within 4-fold of each other as measured by binding titers or neutralization titers, as described below.

[0038] To assess whether two antigens are antigenically different or similar, a naive ferret model can be used as described in the Examples. In this model, naive ferrets are intranasally infected with live influenza virus and serum is collected to assess antibody responses to the virus. Antibody responses can be measured by hemagglutinin inhibition (HAI) assays to measure virus-antibody binding titers, or by neutralization assays (e.g., microneutralization assays) to measure virus neutralization titers. The efficiency of binding or neutralization of heterologous virus strains can indicate whether the strains are antigenically different or antigenically similar. Figure 1 shows virus samples scored in HAI assays. When comparing circulating virus 1 with vaccine virus, circulating virus 1 differs by one dilution (2-fold difference) and is therefore considered antigenically similar to the vaccine virus of the previous season. When comparing circulating virus 2 with vaccine virus, circulating virus 2 differs by 5 dilutions (32-fold difference) and is therefore considered antigenically similar to the vaccine virus of the previous season.

[0039] 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).

[0040] 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.

[0041] Epitope: As used herein, the term "epitope" includes a portion 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 a selective structure (e.g., linearized).

[0042] 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.

[0043] 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 can include any cell of the body involved in a host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate and / or adaptive immune responses. Methods for measuring immune responses are well known in the art and include, for example, measuring proliferation and / or activity of lymphocytes (such as B cells or T cells), measuring secretion of cytokines or chemokines, measuring inflammation, measuring antibody production, and the like. An antibody response or humoral response is an immune response in which antibodies are produced. A "cellular immune response" is one that is mediated by T cells and / or other white blood cells.

[0044] 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, "immunize" means to induce a protective immune response in a subject against an infectious disease (e.g., influenza).

[0045] Immunologically effective amount: As used herein, the term "immunologically effective amount" means an amount sufficient to immunize a subject.

[0046] 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.

[0047] 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 can be constructed. 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 by reference in their entireties. Machine learning, as used herein, can also include the application of computational tools to analyze and interpret data, for example, bioinformatics analysis, such as phylogenetic analysis. Similarly, "machine learning influenza virus HA" refers to an influenza virus HA identified or designed by machine learning. "Machine learning model" refers to a model that uses algorithms that improve automatically through experience and / or use of data to predict data, such as candidate antigens.

[0048] 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.

[0049] Prevention: The term "prevention," as used herein, refers to preventing, avoiding disease onset, delaying the onset, and / or reducing the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., infection with an influenza virus). In some embodiments, prevention is assessed on a population basis, and an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the occurrence, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition.

[0050] Recombinant: As used herein, the term "recombinant" is intended to refer to a polypeptide that is designed, engineered, prepared, expressed, produced or isolated by recombinant means (e.g., an HA polypeptide described herein), e.g., a polypeptide that is expressed using a recombinant expression vector transfected into a host cell, a polypeptide that is isolated from a recombination, combinatorial polypeptide library, or a polypeptide that is prepared, expressed, produced or isolated by any other means, including splicing selected sequence elements together. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are 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) that do not naturally occur in the same polypeptide.

[0051] 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.

[0052] 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.

[0053] 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 can be performed manually or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs that use 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.).

[0054] 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.

[0055] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, in consecutive nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0056] 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.

[0057] Standard Therapeutic 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 therapeutic strain" or "SOC strain" refers to an influenza strain selected by the World Health Organization (WHO) for inclusion in seasonal vaccine formulations, for example, for the Northern and Southern Hemispheres. Standard therapeutic strains can include past standard therapeutic strains, current standard therapeutic strains, or future standard therapeutic strains.

[0058] 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."

[0059] Vaccine composition: As used herein, the term "vaccine composition" or "vaccine" refers to a composition that generates a protective immune response in a subject. As used herein, a "protective immune response" refers to an immune response that protects a subject from infection (prevents infection or prevents the development of a disease associated with infection) or reduces the symptoms of infection (e.g., infection with influenza virus). Vaccines can induce both prophylactic (preventative) and therapeutic responses. Methods of administration vary depending on the vaccine, but may include inoculation, ingestion, inhalation, or other forms of administration. Inoculation can be delivered by any of a number of routes, including parenterally, such as intravenously, subcutaneously, intraperitoneally, intradermally, or intramuscularly. Vaccines can be administered with adjuvants to enhance the immune response.

[0060] Immunogenic composition: As used herein, the term "immunogenic composition" refers to a composition that generates an immune response, which may or may not be a protective immune response.

[0061] Vaccinate: As used herein, terms such as "vaccinate" refer to administration of a vaccine composition to generate a protective immune response in a subject, e.g., a response against a disease-causing pathogen, such as an influenza virus. Vaccination can occur before, during, and / or after exposure to the disease-causing pathogen and / or before, during, and / or after the onset of one or more symptoms, in some embodiments before, during, and / or immediately after exposure to the pathogen. In some embodiments, vaccination includes multiple administrations of the vaccine composition at appropriate time intervals.

[0062] 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, a wild-type HA polypeptide is found in natural isolates of influenza viruses. A variety of different wild-type HA sequences can be found in the NCBI influenza virus sequence database.

[0063] 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 three major types of influenza: influenza A, influenza B, or influenza C, which infect humans. Influenza A and B cause significant morbidity and mortality annually. It is understood by those skilled in the art that the designation of a virus as a particular type is related to sequence differences in the respective M1 (matrix) protein or P (nucleoprotein). Influenza A viruses are further divided into 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 on the surface of the virus, HA and NA. Currently, there are 18 recognized HA subtypes (H1-H18) and 11 recognized NA subtypes (N1-N11). Group 1 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., 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).

[0064] For convenience, certain abbreviations can be used to refer to the protein constructs and parts thereof described herein. For example, HA can refer to influenza hemagglutinin protein. H1 refers to HA from influenza subtype 1 strains. H3 refers to HA from influenza subtype 3 strains.

[0065] Influenza A subtypes can be further divided into different genetic "clades" and "subclades." For example, A subtype A(H1N1) contains clade 6B.1 and subclade 6B.1A. A subtype A(H3N2) contains clades 3C.2A and 3C.3A, and subclades 3C.2A1, 3C.2A2, 3C2A3, and 3C.2A4. Similarly, B subtype Victoria contains clade V1A and subclades V1A.1, V1A.2, and V1A.3, while B subtype Yamagata contains clades Y1, Y2, and Y3. Finally, the term strain refers to viruses within a subtype that differ from each other in that they have small genetic variations in their genomes.

[0066] Hemagglutinin (HA) Hemagglutinin (HA) is one of the two major influenza surface proteins, along with neuraminidase (NA). The function of HA involves interaction with sialic acid, a terminal molecule that binds to sugar moieties on glycoproteins or glycolipids expressed on the surface of cells. Binding of HA to sialic acid on the cell surface induces endocytosis of the virus by the cell, allowing the virus to enter and infect the cell. Sialic acid is also added to HA as part of the glycosylation process that occurs within the infected cell.

[0067] 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.

[0068] 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. HA0 and mature HA1 and HA2 differ slightly in conformation and antigenic properties, in part because these molecules are tightly folded. Furthermore, HA0 is more stable and resistant to denaturation and proteolysis. Baculovirus / insect cell cultures derived from recombinant HA0 are known to confer protective immunity against influenza.

[0069] The influenza virus HA present in the vaccine or immunogenic composition disclosed herein can be any form of influenza virus HA, including any combination of HA from a standard of care influenza virus strain and machine learning influenza virus HA. For example, in certain embodiments, influenza virus HA from a standard of care influenza strain can be present in the vaccine or immunogenic composition as HA present in an inactivated influenza virus, recombinant influenza virus HA, or ribonucleic acid molecule encoding the aforementioned influenza virus HA, or any combination thereof. In certain further embodiments, one or more machine learning influenza virus HAs can be present in the vaccine or immunogenic composition as HA present in an inactivated influenza virus, recombinant influenza virus HA, or ribonucleic acid molecule encoding the aforementioned machine learning influenza virus HA, or any combination thereof.

[0070] Similarly, in embodiments disclosed herein, the influenza virus HA from a standard of care influenza strain and the HA identified or designed from machine learning can be a wild-type HA, a non-wild-type HA, an HA from a seasonal or pandemic influenza virus strain, and / or any other form of HA known in the art. In certain embodiments disclosed herein, the influenza virus HA is from a pandemic strain or a strain with pandemic potential, including, for example, H1, H2, H3, H5, H7, and / or H10.

[0071] In certain embodiments disclosed herein, the HA from a standard of care influenza virus strain and / or the machine learning influenza virus HA is present in the inactivated influenza virus.

[0072] Certain licensed influenza vaccines may contain formalin-inactivated whole or chemically resolved subunit preparations from multiple influenza subtypes, including, for example, influenza A subtypes H1N1, influenza A H3N2, influenza B / Victoria, and / or influenza B / Yamagata. The seed viruses for such influenza A and B vaccines may be naturally occurring strains (i.e., wild-type strains) that replicate to high titers in the allantoic cavity of chicken eggs or in cultured cells.

[0073] Alternatively, the strains may be reassortant viruses that have the correct surface antigen genes. Reassortant viruses are those that have the characteristics of each parental strain due to segmentation of the viral genome. When two or more influenza virus strains infect a cell, these viral segments mix to create progeny virions that contain a variety of genes from both parents.Reverse genetics methods used to generate infectious reassortant viruses are well known to those of skill in the art and include, but are not limited to, methods using plasmids described in Neuman et al, 1999, Proc Natl Acad Sci USA, 96(16):9345-9350; Neumann et al, 2005, Proc Natl Acad Sci USA, 102(46):16825-16829; Zhang et al, 2009, J Virol, 83(18):9296-9303; Massin et al, 2005, J Virol, 79(21):13811-13816; Murakami et al, 2008, 82(3):1605-1609; and / or methods using plasmids described in Neuman et al, 1999, Proc Natl Acad Sci USA, 96(16):9345-9350; Neumann et al. al,2005,Proc Natl Acad Sci USA,102(46):16825-16829;Zhang et al,2009,J Virol,83(18):9296-9303;Massin et al,2005,J Virol,79(21):13811-13816;Murakami et al. al,2008,82(3):1605-1609;Koudstaal et al,2009,Vaccine,27(19):2588-2593;Schickli et al,2001,Philos Trans R Soc Lond Biol Sci,356(1416):1965-1973;Nicolson et al. al,2005,Vaccine,23(22):2943-2952;Legastelois et al. et al., 2007, Influenza Other Respi Viruses, 1(3):95-104; Whiteley et al., 2007, Influenza Other Respi Viruses, 1(4):157-166.

[0074] Thus, the HA protein disclosed herein includes the HA present in the inactivated virion. In certain embodiments, the inactivated virus is a split inactivated virus. In certain embodiments, disclosed herein is an influenza virus HA present in the inactivated virus, wherein the HA is selected from an H1 HA from a standard care influenza virus, an H3 HA from a standard care influenza virus, an HA from a standard care influenza virus strain of B / Victoria lineage, or an HA from a standard care influenza virus of B / Yamagata lineage.

[0075] In certain embodiments, disclosed herein is a machine-learned influenza virus HA, wherein the HA is present in an inactivated virus, and the machine-learned HA is selected from one or more of an H1 HA, an H3 HA, an HA from a B / Victoria lineage, an HA from a B / Yamagata lineage, or a combination thereof.

[0076] Also disclosed herein are vaccines or immunogenic compositions comprising recombinant HA, including recombinant HA derived from standard of care influenza virus strains and / or machine learning recombinant HA.

[0077] Isolation, propagation, and purification of influenza virus strains for cloning the desired HA gene can be performed by any method known in the art, such as the method disclosed in U.S. Pat. No. 5,762,939, incorporated herein by reference.

[0078] Recombinant HA antigens are expressed in cells, such as insect cells, infected with a viral-hemagglutinin vector. The primary gene product is unprocessed full-length HA (rHA0), which is not secreted but remains associated with the peripheral membrane of infected cells. In insect cells, this rHA0 is glycosylated with N-linked high mannose glycans, and there is evidence that rHA0 forms trimers post-translationally and subsequently accumulates in the cytoplasmic cell membrane.

[0079] The rHA0 can 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, such as affinity or gel chromatography, antigen binding, DEAE ion exchange, or lentil lectin affinity chromatography. The purified rHA0 can then be resuspended in an isotonic buffer solution. In certain embodiments, the rHA0 is purified to at least about 80%, e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0080] In certain embodiments, full-length uncleaved (HA0) hemagglutinin antigens from influenza viruses can be produced using baculovirus expression vectors in cultured insect cells and further purified, for example, under non-denaturing conditions. Two or more (e.g., three, four or more) purified hemagglutinin antigens from influenza A and / or influenza B strains can be mixed to generate a multivalent influenza vaccine.

[0081] Baculoviruses are DNA viruses of the family Baculoviridae. These viruses are known to have a narrow host range that is restricted mainly 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.

[0082] 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 it is under the transcriptional control of the polyhedrin promoter. The recombinant baculovirus, including the recombinant baculovirus encoding the recombinant HA protein, can then be replicated in a variety of insect cell lines. The recombinant HA protein can also be expressed in other expression vectors, including, for example, entomopoxviruses (insect poxviruses), cytoplasmic polyhedrosis viruses (CPVs), and transformation of insect cells with the recombinant HA gene.

[0083] HA-encoding ribonucleic acid molecule Also disclosed herein are ribonucleic acid molecules, such as mRNA molecules, that encode one or more of the influenza virus HAs disclosed herein. The ribonucleic acid molecules, such as mRNA, can encode standard of care influenza virus strains, such as any one of the combinations of H1 HA, H3 HA, HA from B / Victoria lineage, or HA from B / Yamagata lineage. In certain embodiments, the ribonucleic acid molecules, such as mRNA, can encode machine learning influenza virus HAs, such as any one of the combinations of H1 HA, H3 HA, HA from B / Victoria lineage, or HA from B / Yamagata lineage. In certain embodiments, the ribonucleic acid molecules are encapsulated in lipid-nanoparticles (LNPs).

[0084] Exemplary mRNAs and LNPs are disclosed, for example, in International Application No. PCT / US2021 / 058250, filed November 5, 2021, the entire contents of which are incorporated by reference.

[0085] Any known LNP formulation may be used in the embodiments disclosed herein. In certain embodiments, LNPs include a mixture of four lipids: ionized (e.g., cationic) lipids, polyethylene glycol (PEG)-conjugated lipids, cholesterol-based lipids, and helper lipids such as phospholipids. LNPs are used to encapsulate ribonucleic acid molecules (e.g., mRNA). The encapsulated mRNA molecules may be composed of naturally occurring ribonucleotides, chemically modified nucleotides, or a combination thereof, and may individually or collectively code for one or more proteins.

[0086] Ionizable lipids facilitate mRNA encapsulation and may be cationic lipids, which provide a positively charged environment at low pH and facilitate efficient encapsulation of the negatively charged mRNA drug substance.

[0087] Contemplated PEGylated lipids include C6-C PEG-Ceramides, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). 20 (For example, 8, C 10 , C 12 , C 14 , C 16 , or C 18Examples of PEGylated lipids include, but are not limited to, polyethylene glycols (PEGs) of up to 5 kDa length covalently attached to lipids having alkyl chains of up to 1 kDa length. In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG); N,N-ditetradecylacetamide-polyethylene glycol (e.g., ALC-0159); or 1-monomethoxypolyethylene glycol-2,3-dimyristylylglycerol (e.g., PEG2000-DMG).

[0088] PEG preferably has a high molecular weight, for example, 2000-2400 g / mol. In some embodiments, PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, or C8 PEG2000. The PEGylated lipid component provides control of nanoparticle size and stability. The addition of such components can prevent complex aggregation, increase circulation life, and provide a means to increase delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al., FEBS Letters (1990) 268(1):235-7). These components can be selected to be rapidly exchanged from the pharmaceutical composition in vivo (see, for example, U.S. Pat. No. 5,885,613).

[0089] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Pat. No. 5,744,335), imidazole cholesterol ester ("ICE"; WO 2011 / 068810), β-sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0090] The helper lipid enhances the structural stability of the LNP and aids the LNP in endosomal escape. It improves the uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is a zwitterionic lipid with fusogenic properties to enhance the uptake and release of the drug payload. In certain embodiments, the helper lipid is a phospholipid. Examples of helper lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-oleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serinecholine (DOPS), 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (DEPE), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC); 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0091] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof.

[0092] In certain embodiments disclosed herein, the LNPs comprise: (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, ALC-0315, or SM-102; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0093] In certain embodiments disclosed herein, the LNPs comprise (i) ALC-0315 as the cationic lipid, (ii) N,N ditetradecylacetamido-polyethylene glycol (e.g., ALC-0159) as the PEGylated lipid, (iii) DSPC as the helper lipid, and (iv) cholesterol. In certain embodiments, the LNPs comprise (i) ALC-0315 as the cationic lipid in a molar ratio of about 25% to about 65%, e.g., about 46.3%; (ii) N,N ditetradecylacetamido-polyethylene glycol (e.g., ALC-0159) as the PEGylated lipid in a molar ratio of about 0.5% to about 2.6%, e.g., 1.6%; (iii) DSPC as the helper lipid in a molar ratio of about 5% to about 15%, e.g., 9.4%; and (iv) cholesterol in a molar ratio of about 20% to about 60%, e.g., 42.7%.

[0094] The molar ratios of the LNP components above may enhance the effectiveness of the LNP in delivering mRNA. The molar ratios of cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid are A:B:C:D (where A+B+C+D=100%). In some embodiments, the molar ratio of cationic lipid in the LNP to total lipid (i.e., A) is 35-50%. In some embodiments, the molar ratio of PEGylated lipid component to total lipid (i.e., B) is 0.25-2.75%. In some embodiments, the molar ratio of cholesterol-based lipid to total lipid (i.e., C) is 20-50%. In some embodiments, the molar ratio of helper lipid to total lipid (i.e., D) is 5-35%. In some embodiments, the (PEGylated lipid+cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNP has a molar ratio of cationic lipid to helper lipid greater than 1.

[0095] To calculate the actual amount of each lipid contained in the LNP formulation, first, the molar amount of the cationic lipid is determined based on the desired N / P ratio (where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP). Next, the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid.

[0096] In certain embodiments, the LNPs contain cationic lipids, PEGylated lipids, cholesterol-based lipids, and helper lipids in a molar ratio of 40:1.5:28.5:30. In further particular embodiments, the LNPs contain (i) OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14, (ii) DMG-PEG2000, (iii) cholesterol; and (iv) DOPE in a molar ratio of 40:1.5:28.5:30.

[0097] If desired, the LNP or LNP formulation may be multivalent. In some embodiments, the LNP may carry ribonucleic acid molecules (e.g., mRNAs) encoding two or more antigens, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens, from the same or different pathogens. For example, the LNP may carry multiple ribonucleic acid molecules (e.g., mRNAs), each encoding a different antigen; or may carry a polycistronic mRNA that can be translated into two or more antigens (e.g., each antigen coding sequence is separated by a nucleotide linker that encodes a self-cleaving peptide, such as a 2A peptide). LNPs carrying different ribonucleic acid molecules (e.g., mRNAs) typically contain (encapsulate) multiple copies of each mRNA molecule. For example, LNPs carrying or encapsulating two different ribonucleic acid molecules (e.g., mRNAs) typically carry multiple copies of each of the two different ribonucleic acid molecules (e.g., mRNAs).

[0098] In some embodiments, a single LNP formulation can include multiple species (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of LNPs, each of which carries a different ribonucleic acid molecule (eg, mRNA).

[0099] In some embodiments, a vaccine or immunogenic composition disclosed herein comprises a ribonucleic acid molecule encoding a polypeptide derived from one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) influenza virus proteins selected from H1 HA, H3 HA, HA from B / Victoria lineage, and / or HA from B / Yamagata lineage. In further embodiments, a vaccine or immunogenic composition disclosed herein contains four ribonucleic acid molecules (e.g., mRNAs), where a first ribonucleic acid molecule encodes an H1 HA derived from a first standard of care influenza virus strain, a second ribonucleic acid molecule encodes an H3 HA derived from a second standard of care influenza virus strain, a third ribonucleic acid molecule encodes an HA derived from a third standard of care influenza virus strain derived from the B / Victoria lineage, and a fourth ribonucleic acid molecule encodes an HA derived from a fourth standard of care influenza virus strain derived from the B / Yamagata lineage. In certain embodiments, the vaccine or immunogenic composition further comprises one or more ribonucleic acid molecules (e.g., mRNA) encoding one or more machine-learning influenza virus HAs disclosed herein, wherein the one or more machine-learning influenza virus HAs are selected from an H1 HA, an H3 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, or a combination thereof.

[0100] In certain embodiments, the vaccine or immunogenic composition disclosed herein may comprise one or more self-amplifying ribonucleic acids, such as one or more self-amplifying mRNAs encoding influenza virus HA. Antigen expression from conventional mRNA is proportional to the number of mRNA molecules successfully delivered from the vaccine or immunogenic composition to a subject. However, self-amplifying mRNA comprises a genetically engineered replicon derived from a self-replicating virus, and therefore can be added to the vaccine or immunogenic composition at a lower dose than conventional mRNA while achieving comparable results.

[0101] The self-amplifying mRNA may encode any of the influenza virus HAs disclosed herein, including, for example, an H3 HA from a standard of care influenza virus, an H1 HA from a standard of care influenza virus, an HA from a standard of care influenza virus of the B / Victoria lineage, an HA from a standard of care influenza virus of the B / Yamagata lineage, and / or one or more machine learning influenza virus HAs.

[0102] Ribonucleic acid molecules (e.g., mRNA) can be unmodified (i.e., containing only natural ribonucleotides A, U, C, and / or G linked by phosphodiester bonds) or chemically modified (e.g., containing nucleotide analogs such as pseudouridines (e.g., N-1-methylpseudouridine), 2'-fluororibonucleotides, and 2'-methoxyribonucleotides, and / or phosphorothioate bonds). Ribonucleic acid molecules (e.g., mRNA) can include a 5' cap and a poly-A tail. In certain embodiments, the one or more ribonucleic acid molecules comprise one or more modified nucleotides, and in certain embodiments, the one or more modified nucleotides are selected from pseudouridine, methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine. In one embodiment, the modified nucleotide is methylpseudouridine, in particular 1N-methylpseudouridine. In certain embodiments, all uridines in the ribonucleic acid molecule are replaced by pseudouridines, such as methylpseudouridines, such as 1N-methylpseudouridine.

[0103] Each ribonucleic acid molecule may be included in the compositions disclosed herein in an amount effective to induce an immune response in a subject to which the composition is administered. In certain embodiments, each ribonucleic acid molecule may be included in the vaccine or immunogenic composition disclosed herein in an amount ranging from, for example, about 0.1 mg to about 150 mg, e.g., about 5 mg to about 120 mg, about 10 mg to about 60 mg, or about 15 mg to about 45 mg. In certain embodiments, each ribonucleic acid molecule is included in the vaccine or immunogenic composition in an amount sufficient to encode, for example, about 5 mg to about 120 mg, e.g., about 10 mg to about 60 mg, or about 15 mg to about 45 mg of influenza virus HA.

[0104] The nucleic acids and / or LNPs can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharma- ceutical acceptable excipients to stabilize the nucleic acids and / or LNPs (e.g., to extend the shelf life of a vaccine product), to facilitate administration of the LNP pharmaceutical composition, and / or to enhance in vivo expression of the nucleic acids. Examples of such excipients are parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0105] The LNP compositions of the present disclosure can be provided in a frozen liquid form or in a lyophilized form. A variety of cryoprotectants can be used, including but not limited to sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. Once formulated with a cryoprotectant, the LNP compositions can be frozen (or lyophilized and stored frozen) at -20°C to -80°C. The LNP compositions can be provided to the patient in a buffered aqueous solution (thawed if previously frozen, or reconstituted in a buffered aqueous solution at the bedside if previously lyophilized). The buffer is preferably isotonic, e.g., suitable for intramuscular or intradermal injection. In some embodiments, the buffer is phosphate buffered saline (PBS).

[0106] Machine Learning To complement the protection provided by currently available quadrivalent vaccines, which contain HA molecules from the four standard of care influenza virus strains selected by the WHO each year, the vaccine or immunogenic compositions and methods disclosed herein further comprise one or more machine-learning influenza virus HAs having molecular sequences identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding the one or more machine-learning influenza virus HAs, wherein the one or more machine-learning influenza virus HAs are selected from an H1 HA, an H3 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, or a combination thereof.

[0107] In embodiments disclosed herein, the vaccine or immunogenic composition may include, in addition to an HA from a standard of care influenza virus strain, one or more machine-learned influenza virus HAs having molecular sequences identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding one or more machine-learned influenza virus HAs disclosed above. In certain embodiments, the one or more machine-learned HAs are selected from an H1 HA, an H3 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, or a combination thereof.

[0108] The machine learning HA disclosed herein can be any form of HA, including HA present in an inactivated virus or recombinant HA, or a ribonucleic acid molecule disclosed herein, including an HA nucleic acid molecule (e.g., mRNA) encoding any of the foregoing HA.

[0109] Any machine learning algorithm may be used in selecting one or more machine learning influenza virus HAs. For example, the machine learning algorithms and methods disclosed in PCT Application WO 2021 / 080990A1 (titled Systems and Methods for Designing Vaccines), WO 2021 / 080999A1 (titled Systems and Methods for Predicting Biological Responses), U.S. Provisional Application No. 63 / 319,692 (titled Machine-Learning Techniques in Protein Design for Vaccine Generation), and U.S. Provisional Application No. 63 / 319,700 (titled Machine-Learning Techniques in Protein Design for Vaccine Generation), which are incorporated herein by reference in their entireties, are contemplated herein.

[0110] In certain embodiments, a predictive machine learning model of influenza antigenicity may be constructed to enable prediction of antibody titers in animal models and / or humans. In certain embodiments, the machine learning model may extract feature values ​​from input data of a training set, where features are variables that are considered potentially relevant regardless of whether an input data item has an associated characteristic. An 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.

[0111] A set of influenza sequences (e.g., target strains) to protect against can then be identified and a selection algorithm constructed. 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 representative of one or more molecular sequences, the first time series data set representing the one or more molecular sequences and, for each of the one or more molecular sequences, one or more circulations of pathogenic strains that include 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 from the driver model output data representing one or more predicted molecular sequences based on the received first time series data set, ii) applying a translational model configured to predict a biological response to the molecular sequences for a plurality of translational axes to the output data representing the predicted one or more molecular sequences to generate first translational response data representing one or more first translational responses corresponding to a particular translational axis of the plurality of translational axes based on the one or more predicted molecular sequences in the output data, iii) adjusting one or more parameters of the driver model based on the first translational response data, and iv) repeating steps i-iii a number of times to generate trained translational response data representing one or more trained translational responses corresponding to the particular translational axis. The one or more operations include selecting a set of trained driver models from the plurality of driver models based on the one or more trained translational responses.The one or more operations include: applying the trained driver models to a second time series dataset to generate, for each trained driver model in the set of trained driver models, trained output data representing one or more predicted molecular sequences for a particular season; applying the translation model to the final output data to generate second translation response data representing one or more second translation responses, for each translation axis of the plurality of translation axes; and selecting a subset of the trained driver models of the set of trained driver models based on the second translation response data.

[0112] 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.

[0113] The output data representing one or more predicted molecular sequences based on the received first time series data set can 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 can 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 can 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.

[0114] The plurality of translational axes may include at least one of a ferret neutralization axis, a ferret antibody forensics (AF) axis, a ferret hemagglutination inhibition assay (HAI) axis, a mouse neutralization axis, a mouse AF axis, a mouse HAI axis, a human neutralization 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.

[0115] Selecting a set of trained driver models from the plurality of driver models can 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 can 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.

[0116] 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.

[0117] In one aspect, a system is provided. The system includes a computer-readable memory including computer-executable instructions. The system includes at least one processor configured to execute executable logic including at least one machine learning model trained to predict one or more molecular sequences, and when the at least one processor executes the computer-executable instructions, the at least one processor is configured to perform one or more operations. The one or more operations include receiving time series data indicating one or more molecular sequences, and for each of the one or more molecular sequences, receiving one or more circulations of a pathogenic strain that includes the molecular sequence as a natural antigen. The one or more operations include processing the time series data through one or more data structures that store one or more portions of the executable logic included in the machine learning model, and predicting one or more molecular sequences based on the time series data.

[0118] Predicting the one or more molecular sequences based on the time series data can include predicting one or more immunological properties that the predicted one or more molecular sequences will confer for future use. Predicting the one or more molecular sequences based on the time series data can include predicting one or more molecular sequences that generate a maximized agglutinative biological response across all pathogenic strains in the time series data. Predicting the one or more molecular sequences based on the time series data can 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 can be used to design a vaccine for a pathogenic strain circulating at a time after one or more circulations of the time series data.

[0119] The machine learning model can include a recurrent neural network.

[0120] In certain embodiments, a data processing system for predicting a biological response is provided. The system includes a computer readable memory including computer executable instructions. The system includes at least one processor configured to execute executable logic including at least one machine learning model trained to predict a biological response, and when the at least one processor executes the computer executable instructions, the at least one processor performs one or more operations. The one or more operations include receiving first sequence data of a first molecular sequence. The one or more operations include receiving second sequence data of a second molecular sequence. The one or more operations include predicting a biological response to the second molecular sequence based at least in part on the received first and second sequence data.

[0121] The one or more operations can include receiving non-human biological response data corresponding to the first molecular sequence and the second molecular sequence. The one or more operations can further include predicting a biological response based at least in part on the non-human biological response data. The one or more operations can include encoding the first sequence data and the second sequence data as amino acid mismatches.

[0122] The first molecular sequence may comprise a candidate antigen, and the second molecular sequence may comprise a known viral strain.

[0123] Predicting the biological response can include predicting a human biological response. Predicting the biological response can include predicting at least one human biological response and at least one non-human biological response. The biological response can include an antibody titer. The machine learning model can include a deep neural network.

[0124] Machine learning methods can 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 can be used to efficiently process inherently sparse data, for example by reducing the dimensionality of the data, when compared to traditional approaches. At least some of the described systems and methods can exploit non-linear relationships in the received data to increase prediction accuracy compared to traditional approaches. At least some of the described systems and methods can be used to simultaneously predict human and non-human biological responses. At least some of the described systems and methods can be used to predict outcomes that are not experimentally observed.

[0125] In certain embodiments, a system of one or more computers can 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 can 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 outcome object, ii) a nucleic acid capable of producing the protein defined by the discrete outcome object, and iii) a delivery vehicle capable of producing the protein defined by the discrete outcome 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.

[0126] 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 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 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.

[0127] 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 that includes at least one of i) a protein defined by the discrete result object, ii) a nucleic acid capable of generating the protein defined by the discrete result object, and iii) a delivery vehicle capable of generating the protein defined by the discrete result object. Other embodiments of this aspect include corresponding computer systems, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the operations of the method.

[0128] 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 amino acid sequence and the second amino acid sequence including a respective character or a respective character string. The conversion of the discrete data object to the continuous data object includes generating, for each first discrete value, a weight vector of weight values, each weight value representing a likelihood that the first discrete value represents a particular amino acid, generating, for each weight value of each weight vector, a property vector of property values, each property value representing a physicochemical property of a particular amino acid, and combining the weight vector and the property vector to generate a first continuous value of the continuous data object. Each weight vector has 20 weight values, each weight value corresponding to one of the 20 possible amino acids. The conversion of the continuous result object to the discrete result object may include determining, for each second continuous value, a respective single amino acid, the determined single amino acids forming a plurality of second discrete values. 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.

[0129] One general aspect includes a non-transitory computer-readable medium storing instructions that, when executed by one or more processors, cause the one or more processors to perform operations that may include: receiving a discrete data object including a plurality of first discrete values, the discrete data object including one or more amino acid sequences; converting the discrete data object into a continuous data object including a plurality of first continuous values; applying a continuous data algorithm to the continuous data object to generate a continuous result object including a plurality of second continuous values; converting the continuous result object into a discrete result object including a plurality of second discrete values, and manufacturing a vaccine comprising at least one of i) a protein defined by the discrete result object, ii) a nucleic acid capable of producing the protein defined by the discrete result object, and iii) a delivery vehicle capable of producing the protein defined by the discrete result object. Other embodiments of this aspect include corresponding computer systems, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the operations of the method.

[0130] Implementations may include one or more of the following features: The medium may include a first amino acid sequence and a second amino acid sequence, each of the first amino acid sequence and the second amino acid sequence including a respective character or a respective character string. The conversion of the discrete data object to the continuous data object includes generating, for each first discrete value, a weight vector of weight values, each weight value representing a likelihood that the first discrete value represents a particular amino acid, generating, for each weight value of each weight vector, a property vector of property values, each property value representing a physicochemical property of a particular amino acid, and combining the weight vector and the property vector to generate a first continuous value of the continuous data object. Each weight vector has 20 weight values, each weight value corresponding to one of the 20 possible amino acids. The conversion of the continuous result object to the discrete result object may include determining, for each second continuous value, a respective single amino acid, the determined single amino acids forming a plurality of second discrete values. Implementations of the techniques described may include hardware, a method or process, or computer software on a computer accessible medium.

[0131] In certain embodiments, disclosed herein is an algorithm 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 the reduced space (e.g., the model predicts the mean and variance from the input sequence using embedded coordinates chosen from a normal distribution with predicted mean and variance); b) 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.

[0132] 2) Train an immune response prediction model based on the position of antigens (vaccine candidates) and readout strains (target sequences) in reduced dimensional space [input: antigens and readouts embedded by the model from step 1, output: measures of immune response such as antibody titers].

[0133] 3) Sample candidate vaccine component expressions from the reduced space and rank the candidate vaccine component expressions by their predictive performance against the target sequence using the model described in step 2 to identify the top candidates.

[0134] 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.

[0135] A system of one or more computers can 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 can 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 an operation in which each of the generated amino acid sequences is 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.

[0136] 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.

[0137] 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 a viral amino acid sequence; and generating at least one reduced dimensional viral sequence in the reduced dimensional space. and; providing each of the candidate sequences and at least one of the reduced dimensionality 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.

[0138] 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.

[0139] 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 viral amino acid sequences; generating at least one reduced dimensional viral sequence in the reduced dimensional space. generating a sequence; providing each of the candidate sequences and at least one of the reduced dimensionality 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.

[0140] Implementations may include one or more of the following features: A medium in which generating a plurality of reduced dimensional arrays may include creating a representation of a wild-type amino acid sequence using a variational autoencoder that predicts mean and variance values ​​of input data. Each of the reduced dimensional arrays may include a 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 a candidate score, the potency score defining a measure of a biological response between the first sequence and the second sequence. Implementations of the described approaches may include hardware, methods or processes, or computer software on a computer-accessible medium.

[0141] These and other aspects, features, and implementations may be expressed as methods, apparatus, systems, components, program products, methods of conducting a business, means or steps for performing a function, and in other manners, and will become apparent from the following description, including the claims.

[0142] Implementations of the present disclosure may provide the following advantages: Compared to conventional approaches, vaccines can be designed for future pathogenic seasons to confer more protection in terms of biological response volume against at least one pathogenic strain of the future pathogenic season. Compared to conventional approaches, vaccines can be designed for future pathogenic seasons to 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 that may confer "more protection" because they cross-react with more strains than frequently observed strains can be evaluated and the effectiveness of vaccination against them can be predicted.

[0143] Methods for measuring biological responses The vaccines or immunogenic compositions disclosed herein induce a biological response (e.g., an immunological response) when administered to a subject. These biological responses can be used to compare vaccines or immunogenic compositions to determine, for example, whether a vaccine or immunogenic composition enhances or broadens an immune response compared to a vaccine or immunogenic composition that does not include one or more machine learning HAs.

[0144] An exemplary assay that can be used to measure biological responses is 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 present 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 compared 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).

[0145] Another approach to measure biological responses is to measure a potentially larger set of antibodies that are elicited by the human or animal immune response and that may not necessarily affect hemagglutination in an HAI assay. A common approach for this is to utilize enzyme-linked immunosorbent assay (ELISA) technology, in which 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 results in a readily detectable product. There are many variations of this type of in vitro assay. One such variation is called antibody forensics (AF), a multiplex bead array 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 a proportionally higher or lower measurement than the corresponding HAI titer for the hemagglutinin molecule of one virus compared to the red hemagglutinin molecule of another virus; in other words, these two measurements, AF and HAI, generally do not correlate linearly.

[0146] Another method of measuring humoral immune responses includes virus neutralization assays (e.g., microneutralization assays), in which antibody titers are measured in permissive cell cultures after incubating virus with serial dilutions of antibody / serum samples by reduction in plaques, foci, and / or fluorescent signal, depending on the specific neutralization assay technique.

[0147] Vaccine or Immunogenic Composition The present disclosure provides a multivalent vaccine or immunogenic composition comprising an influenza virus HA from a standard of care influenza virus strain (e.g., HA from at least three or at least four standard of care influenza strains) or a ribonucleic acid molecule encoding an influenza virus HA from a standard of care influenza strain, and one or more ribonucleic acid molecules encoding one or more influenza virus HAs or one or more machine-learned influenza virus HAs having molecular sequences identified or designed from a machine learning model.

[0148] In certain embodiments, (a) a first influenza virus hemagglutinin (HA), where the first influenza virus HA is an H1 HA from a first standard of care influenza virus strain, or a first ribonucleic acid molecule encoding the first influenza virus H1 HA; (b) a second influenza virus HA, where the second influenza virus HA is an H3 HA from a second standard of care influenza virus strain, or a second ribonucleic acid molecule encoding a second influenza virus H3 HA; (c) a third influenza virus HA, where the third influenza virus HA is an HA from a third standard of care influenza virus strain of the B / Victoria lineage, or a third ribonucleic acid molecule encoding a third influenza virus HA from the B / Victoria lineage; (d) a fourth influenza virus HA, where the fourth influenza virus HA is an HA from a fourth standard of care influenza virus strain of the B / Yamagata lineage, or a fourth ribonucleic acid molecule encoding a fourth influenza virus HA from the B / Yamagata lineage; (e) one or more machine-learning influenza virus HAs having molecular sequences identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding one or more machine-learning influenza virus HAs, wherein the one or more machine-learning influenza virus HAs are selected from an H1 HA, an H3 HA, an HA from B / Victoria lineage, an HA from B / Yamagata lineage, or a combination thereof; Disclosed herein is a vaccine or immunogenic composition comprising:

[0149] In certain embodiments of the vaccine or immunogenic composition disclosed herein, the one or more machine learning influenza virus HAs include a fifth influenza virus HA, the fifth influenza virus HA is an H3 HA, and the fifth influenza H3 HA is antigenically distinct from the second influenza H3 HA. In certain embodiments, the fifth influenza H3 HA is antigenically similar to the second influenza H3 HA. In certain embodiments, the fifth influenza H3 HA enhances or expands the protective immune response induced by the second influenza H3 HA. In certain embodiments, the fifth influenza H3 HA is from a different clade than the second influenza H3 HA, and in certain embodiments, the fifth influenza H3 HA is from the same clade as the second influenza H3 HA. In certain embodiments, the fifth H3 HA is from the 3C.2A clade, and in certain embodiments, the fifth H3 HA is from the 3C.3A clade. In certain embodiments, the one or more machine-learned influenza virus HAs comprise two or more H3 HAs, such as two, three, or four H3 HAs.

[0150] In certain further embodiments of the vaccine or immunogenic composition disclosed herein, the one or more machine learning influenza virus HAs are a fifth influenza virus HA, the fifth influenza virus HA is an H1 HA, and the fifth influenza H1 HA is antigenically distinct from the first influenza H1 HA. In certain embodiments, the fifth influenza H1 HA is antigenically similar to the first influenza H1 HA. In certain embodiments, the fifth influenza H1 HA enhances or expands the protective immune response induced by the first influenza H1 HA. In certain embodiments, the fifth influenza H1 HA is from a different clade than the first influenza H1 HA, and in certain embodiments, the fifth H1 HA is from the same clade as the first influenza H1 HA. In certain embodiments, the H1 HA is from the 6B.1 clade, and in certain embodiments, the H1 HA is from the 6B.1A subclade. In certain embodiments, the one or more machine-learned influenza virus HAs include two or more H1 HAs, for example two, three, or four H1 HAs.

[0151] In certain further embodiments of the vaccine or immunogenic composition disclosed herein, the one or more machine learning influenza virus HAs are a fifth influenza virus HA from the B / Victoria lineage, the fifth influenza being antigenically distinct from the third influenza virus HA. In certain embodiments, the fifth influenza virus HA is antigenically similar to the third influenza virus HA. In certain embodiments, the fifth influenza virus HA enhances or expands the protective immune response induced by the third influenza HA. ​​In certain embodiments, the fifth influenza virus HA is from a different clade than the third influenza virus HA, and in certain embodiments, the fifth influenza virus HA is from the same clade as the third influenza virus HA. In certain embodiments, the fifth influenza virus HA is from the V1A clade of B / Victoria, and in certain embodiments, the fifth influenza virus HA is from the V1A.1, V1A.2, or V1A.3 subclade of B / Victoria. In certain embodiments, the one or more machine-learned influenza virus HAs include two or more HAs from the B / Victoria lineage, such as two, three, or four HAs from the B / Victoria lineage.

[0152] In certain further embodiments of the vaccine or immunogenic composition disclosed herein, the one or more machine learning influenza virus HAs are a fifth influenza virus HA from the B / Yamagata lineage, the fifth influenza being antigenically distinct from the fourth influenza virus HA. In certain embodiments, the fifth influenza virus HA is antigenically similar to the fourth influenza virus HA. In certain embodiments, the fifth influenza virus HA enhances or expands the protective immune response induced by the fourth influenza virus HA. In certain embodiments, the fifth influenza virus HA is from a different clade than the fourth influenza virus HA, and in certain embodiments, the fifth influenza virus HA is from the same clade as the fourth influenza virus HA. In certain embodiments, the fifth influenza virus HA is from the Y1 clade of B / Yamagata, and in certain embodiments, the fifth influenza virus HA is from the Y2 clade of B / Yamagata. In certain embodiments, the fifth influenza virus HA is from the Y3 clade of B / Yamagata. In certain embodiments, the one or more machine-learned influenza virus HAs include two or more HAs from the B / Yamagata lineage, such as two, three, or four HAs from the B / Yamagata lineage.

[0153] In certain aspects of the disclosure, the vaccine or immunogenic composition comprises a sixth influenza virus HA (wherein the sixth influenza virus HA is an H1 HA or an H3 HA having a molecular sequence identified or designed from a machine learning model), or a nucleic acid molecule encoding the sixth influenza virus HA.

[0154] In certain embodiments, the sixth influenza is an H1 HA that is antigenically distinct from the first influenza H1 HA, enhances or expands the protective immune response induced by the first influenza H1 HA, is from a different clade than the first influenza H1 HA, is from the same clade as the first influenza H1 HA, or is antigenically similar to the first influenza H1 HA. In certain embodiments, the sixth influenza is an H3 HA that is antigenically distinct from the second influenza H3 HA, enhances or expands the protective immune response induced by the second influenza H3 HA, is from a different clade than the second influenza H3 HA, is from the same clade as the second influenza H3 HA, or is antigenically similar to the second influenza H3 HA.

[0155] In certain embodiments of the vaccine or immunogenic compositions disclosed herein, the first influenza virus HA is an H1 HA from an H1N1 influenza virus strain and the second influenza virus HA is an H3 HA from an H3N2 influenza virus strain.

[0156] One or more of the HAs in the multivalent vaccine or immunogenic composition may be recombinant HA and may be formulated and packaged alone or in combination with other recombinant HA antigens, such as HA from a standard of care influenza virus strain and / or machine learning HA. In certain embodiments, the recombinant HA is formulated with one, two, or three additional recombinant HA antigens, such as one, two, or three additional recombinant antigens from a standard of care influenza virus strain. In certain embodiments, the recombinant HA is formulated with three additional recombinant HA antigens to produce a quadrivalent vaccine or immunogenic composition. In certain embodiments, the vaccine or immunogenic composition may contain four recombinant antigens from a standard of care influenza virus strain and one or more, such as one, two, three, or four machine learning influenza virus HAs.

[0157] In certain embodiments, the vaccine or immunogenic composition may comprise recombinant H3 HA, recombinant H1 HA, recombinant HA derived from the B / Victoria lineage, recombinant HA derived from the B / Yamagata lineage, and recombinant machine learning H3 HA.

[0158] In certain embodiments, the vaccine or immunogenic composition may comprise recombinant H3 HA, recombinant H1 HA, recombinant HA derived from the B / Victoria lineage, recombinant HA derived from the B / Yamagata lineage, and recombinant machine learning H1 HA.

[0159] In certain embodiments, the vaccine or immunogenic composition may comprise recombinant H3 HA, recombinant H1 HA, recombinant HA derived from the B / Victoria lineage, recombinant HA derived from the B / Yamagata lineage, recombinant machine learning H3 HA, and recombinant machine learning H1 HA.

[0160] In certain embodiments, the vaccine or immunogenic composition may comprise recombinant H3 HA, recombinant H1 HA, recombinant HA from the B / Victoria lineage, recombinant HA from the B / Yamagata lineage, recombinant machine learning H3 HA, recombinant machine learning H1 HA, and recombinant machine learning HA from the B / Victoria lineage.

[0161] In certain embodiments, the vaccine or immunogenic composition may comprise recombinant H3 HA, recombinant H1 HA, recombinant HA from the B / Victoria lineage, recombinant HA from the B / Yamagata lineage, recombinant machine learning H3 HA, recombinant machine learning H1 HA, and recombinant machine learning HA from the B / Yamagata lineage.

[0162] In certain embodiments, the vaccine or immunogenic composition may comprise recombinant H3 HA, recombinant H1 HA, recombinant HA from the B / Victoria lineage, recombinant HA from the B / Yamagata lineage, recombinant machine learning H3 HA, recombinant machine learning H1 HA, recombinant machine learning HA from the B / Victoria lineage, and recombinant machine learning HA from the B / Yamagata lineage.

[0163] In any of the embodiments in which the vaccine or immunogenic composition comprises a recombinant HA, one or more of the recombinant HAs in the vaccine or immunogenic composition can be replaced by one or more HAs present in an inactivated influenza virus or by one or more ribonucleic acid molecules encoding an influenza virus HA. For example, in certain embodiments, the vaccine or immunogenic composition can comprise an inactivated influenza virus H3 HA, an inactivated influenza virus H1 HA, an inactivated influenza virus HA from the B / Victoria lineage, an inactivated influenza virus HA from the B / Yamagata lineage, and a recombinant machine learning H3 HA or a ribonucleic acid encoding a machine learning influenza virus H3 HA. In certain embodiments, the vaccine or immunogenic composition can comprise a ribonucleic acid encoding an influenza virus H3 HA, a ribonucleic acid encoding an influenza virus H1 HA, a ribonucleic acid encoding an influenza virus HA from the B / Victoria lineage, a ribonucleic acid encoding an influenza virus HA from the B / Yamagata lineage, and a recombinant machine learning H3 HA or a ribonucleic acid encoding a machine learning influenza virus H3 HA.

[0164] One or more of the HAs in a multivalent vaccine or immunogenic composition may be present in an inactivated influenza virus as disclosed herein and may be formulated and packaged alone or in combination with other HAs, such as HAs from standard of care influenza virus strains and / or machine learning HAs, such as recombinant HAs, other HAs present in the inactivated influenza virus, or ribonucleic acid encoding HAs.

[0165] In certain embodiments, the HA present in the inactivated influenza virus is formulated with one, two, or three additional HAs present in the inactivated influenza virus, such as one, two, or three additional HAs from a standard of care influenza virus strain. In certain embodiments, the HA present in the inactivated influenza virus is formulated with three additional HAs present in the inactivated influenza virus to generate a quadrivalent vaccine or immunogenic composition. In certain embodiments, the vaccine or immunogenic composition may contain four HAs present in the inactivated influenza virus from a standard of care influenza virus strain and one or more, such as one, two, three, or four, machine learning influenza virus HAs.

[0166] In certain embodiments, a vaccine or immunogenic composition may comprise an H3 HA, an H1 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, and a machine learning H3 HA, where each HA in the composition is present in an inactivated influenza virus.

[0167] In certain embodiments, a vaccine or immunogenic composition may comprise an H3 HA, an H1 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, and a machine learning H1 HA, where each HA in the composition is present in an inactivated influenza virus.

[0168] In certain embodiments, a vaccine or immunogenic composition may comprise an H3 HA, an H1 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, a machine learning H3 HA, and a machine learning H1 HA, where each HA in the composition is present in an inactivated influenza virus.

[0169] In certain embodiments, a vaccine or immunogenic composition may comprise an H3 HA, an H1 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, a machine learning H3 HA, a machine learning H1 HA, and a machine learning HA from the B / Victoria lineage, wherein each HA in the composition is present in an inactivated influenza virus.

[0170] In certain embodiments, a vaccine or immunogenic composition may comprise an H3 HA, an H1 HA, an HA antigen from the B / Victoria lineage, an HA from the B / Yamagata lineage, a machine learning H3 HA, a machine learning H1 HA, and a machine learning HA from the B / Yamagata lineage, wherein each HA in the composition is present in an inactivated influenza virus.

[0171] In certain embodiments, a vaccine or immunogenic composition may comprise an H3 HA, an H1 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, a machine learning H3 HA, a machine learning H1 HA, a machine learning HA from the B / Victoria lineage, and a machine learning HA from the B / Yamagata lineage, wherein each HA in the composition is present in an inactivated influenza virus.

[0172] In any of the embodiments in which the vaccine or immunogenic composition comprises an HA present in an inactivated influenza virus, one or more HAs present in the inactivated influenza virus can be replaced by one or more recombinant HAs or by one or more ribonucleic acid molecules encoding influenza virus HAs.

[0173] Each recombinant HA may be included in a composition 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 HA may be present in a vaccine or immunogenic composition disclosed herein in an amount ranging from about 5 μg to about 120 μg, e.g., from about 10 μg to about 60 μg or from about 15 μg to about 45 μg. In certain embodiments, each recombinant HA is present in a vaccine or immunogenic composition disclosed herein in an amount of about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, or about 60 μg.

[0174] The ribonucleic acid molecule encoding HA may be present in a vaccine or immunogenic composition disclosed herein, respectively, in an amount ranging from about 5 μg to about 120 μg, such as from about 10 μg to about 60 μg or from about 15 μg to about 45 μg. In certain embodiments, the ribonucleic acid molecule encoding HA is present in a vaccine or immunogenic composition disclosed herein in an amount of about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, or about 100 μg.

[0175] Each HA present in the inactivated virus may be included in the compositions disclosed herein in an amount effective to induce an immune response in a subject to which the composition is administered. In certain embodiments, each HA present in the inactivated virus may be present in the vaccine or immunogenic composition disclosed herein in an amount ranging from, for example, about 5 μg to about 120 μg, e.g., from about 10 μg to about 100 μg, from about 10 μg to about 60 μg, or from about 15 μg to about 45 μg. In certain embodiments, each HA present in the inactivated virus is present in a vaccine or immunogenic composition disclosed herein in an amount of about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, or about 100 μg.

[0176] Further disclosed herein is a vaccine or immunogenic composition comprising: (a) a first influenza HA, where the first influenza virus HA is an H1 HA from a first standard of care influenza virus strain, or a first ribonucleic acid molecule encoding a first influenza virus H1 HA; (b) a second influenza virus HA, where the second influenza virus HA is an HA from a second standard of care influenza virus strain of the B / Victoria lineage, or a 23rd ribonucleic acid molecule encoding a second influenza virus HA from the B / Victoria lineage; (c) a third influenza virus HA, where the third influenza virus HA is an HA from a third standard of care influenza virus strain of the B / Yamagata lineage, or a third ribonucleic acid molecule encoding a third influenza virus HA from the B / Yamagata lineage; and (d) a fourth influenza virus HA, wherein the fourth influenza virus HA is a machine-learned influenza virus H3 HA having a molecular sequence identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding the machine-learned influenza virus H3 HA.

[0177] Further disclosed herein is a vaccine or immunogenic composition comprising: (a) a first influenza virus HA, where the first influenza virus HA is an H3 HA from a first standard of care influenza virus strain, or a first ribonucleic acid molecule encoding a first influenza virus H3 HA; (b) a second influenza virus HA, where the second influenza virus HA is an HA from a second standard of care influenza virus strain of the B / Victoria lineage, or a second ribonucleic acid molecule encoding a second influenza virus HA from the B / Victoria lineage; (c) a third influenza virus HA, where the third influenza virus HA is an HA from a third standard of care influenza virus strain of the B / Yamagata lineage, or a third ribonucleic acid molecule encoding a third influenza virus HA from the B / Yamagata lineage; and (d) a fourth influenza virus HA, wherein the fourth influenza virus HA is a machine-learned influenza virus H1 HA having a molecular sequence identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding the machine-learned influenza virus H1 HA.

[0178] In certain embodiments, the vaccine or immunogenic composition disclosed herein further comprises a ribonucleic acid molecule encoding an additional influenza virus H1 HA, and an additional influenza virus H3 HA, an influenza virus HA from the B / Victoria lineage, and / or an influenza virus HA from the B / Yamagata lineage, wherein each additional HA has a molecular sequence identified or designed from a machine learning model, or an additional machine learning influenza virus HA.

[0179] In certain embodiments, the vaccine or immunogenic composition is a tetravalent HA vaccine. In certain embodiments, the vaccine or immunogenic composition is a pentavalent HA vaccine. In certain embodiments, the vaccine or immunogenic composition is a hexavalent HA vaccine. In certain embodiments, the vaccine or immunogenic composition is a heptavalent HA vaccine. In certain embodiments, the vaccine or immunogenic composition is an octavalent HA vaccine. In certain embodiments, the vaccine or immunogenic composition is a multivalent vaccine or immunogenic comprising more than eight different HA molecules.

[0180] The vaccine or immunogenic composition may also further comprise an adjuvant, which may include a suspension of minerals to which the antigen is adsorbed, including alum, aluminum salts, such as aluminum hydroxide / oxyhydroxide (AlOOH), aluminum phosphate (AlPO4), aluminum hydroxyphosphate sulfate (AAHS), and / or potassium aluminum sulfate, or a water-in-oil emulsion in which a solution of the antigen is emulsified in mineral oil, such as Freund's incomplete adjuvant, which may also contain killed mycobacteria to further enhance antigenicity (Freund's complete adjuvant). Immunostimulatory oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants (see, for example, U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199).Adjuvants also include biological molecules such as lipids and costimulatory molecules. Exemplary biological adjuvants include AS04 (Dierlaurent, A. M. et al, J. Immunol., 2009, 183:6186-6197), IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, and 41 BBL.

[0181] 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 population of 90% of the oil droplets by volume having a size of less than 200 nm.

[0182] In certain embodiments, the polyoxyethylene alkyl ether has the formula CH3-(CH2) x-(O-CH2-CH2) n -OH, where n is an integer from 10 to 60, and x is an integer from 11 to 17. In a particular embodiment, the polyoxyethylene alkyl ether surfactant is polyoxyethylene (12) cetostearyl ether.

[0183] 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.

[0184] In certain embodiments, the adjuvant further comprises at least one alditol, including, but not limited to, glycerol, erythritol, xylitol, sorbitol, and mannitol.

[0185] In certain embodiments, the hydrophilic nonionic surfactant has a hydrophilic lipophilic balance (HLB) of at least 10. In certain embodiments, the hydrophobic nonionic surfactant has an HLB of less than 9.

[0186] 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. 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.

[0187] 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.

[0188] In certain embodiments, the adjuvant comprises AF03, as described in Klucker et al., J. Pharm. Sci. 2012, 101(12):4490-500, which is incorporated herein by reference in its entirety. In certain embodiments, the adjuvant comprises a liposome-based adjuvant, such as SPA14, as described, for example, in WO 2022 / 090359, which is incorporated herein by reference in its entirety.

[0189] In addition to HA 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 can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the vaccine or immunogenic composition to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pharma- ceutically acceptable salts to adjust osmotic pressure, preservatives, stabilizers, buffers, sugars, amino acids, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0190] 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.

[0191] 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 herein by reference. Intradermal compositions may also be administered by devices that limit the effective penetration length of the needle into the skin, such as those described in WO 1999 / 34850, which is incorporated herein by reference, and functional equivalents thereof. Also suitable are jet injection devices that deliver liquid vaccines to the dermis by liquid jet injectors or by a needle that pierces the stratum corneum and generates 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, 5,312,335, 5,503,627, 5,503,628, 5,503,629, 5,603,710, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335, 5,503,627, 5,503,628, 5,503,629, 5,603,710, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335, 5,503,627, 5,503,629 ... US Patent No. 5,064,413, US Patent No. 5,520,639, US Patent No. 4,596,556, US Patent No. 4,790,824, US Patent No. 4,941,880, US Patent No. 4,940,460, WO 1997 / 37705, and WO 1997 / 13537 (all of which are incorporated herein by reference).Also suitable is ballistic powder / particle delivery device, using compressed gas to accelerate the powder form of vaccine through the outer layer of the skin to the dermis.In addition, conventional syringes can be used in the classical Mantoux method of intradermal administration.

[0192] 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, 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., based on Ringer's dextrose), and the like. Preservatives and other additives may also be included, such as antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0193] kit Further disclosed herein are kits for the vaccine or immunogenic composition, which may include a suitable container containing the vaccine or immunogenic composition, or multiple containers containing the various components of the vaccine or immunogenic composition, optionally together with instructions for use.

[0194] In certain embodiments, the kit may include, for example, multiple containers containing: (a) a first influenza virus HA, where the first influenza virus HA is an H1 HA from a first standard of care influenza virus strain; (b) a second influenza virus HA, where the second influenza virus HA is an H1 HA from a second standard of care influenza virus strain; (c) a third influenza virus HA, where the third influenza virus HA is an HA from a third standard of care influenza virus strain of the B / Victoria lineage; and (d) a fourth influenza virus HA, where the fourth influenza virus HA is an HA from a fourth standard of care influenza virus strain of the B / Yamagata lineage, and a second container containing one or more machine-learning influenza virus HAs having a molecular sequence identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding the one or more machine-learning influenza virus HAs, where the one or more machine-learning influenza virus HAs are selected from an H1 HA, an H3 HA, an HA from the B / Victoria lineage, an HA from the B / Yamagata lineage, or a combination thereof.

[0195] In certain embodiments, each of the first, second, third, and fourth influenza virus HAs in the first container is a recombinant influenza virus HA, and the one or more machine-learning influenza virus HAs in the second container are recombinant influenza virus HAs. Alternatively, the one or more machine-learning influenza virus HAs in the second container are present in an inactivated virus, or the second container comprises one or more ribonucleic acid molecules encoding the one or more machine-learning influenza virus HAs.

[0196] In certain embodiments, each of the first, second, third, and fourth influenza virus HAs in the first container are present in an inactivated influenza virus, and the one or more machine-learned influenza virus HAs in the second container are present in an inactivated virus. Alternatively, the one or more machine-learned influenza virus HAs in the second container are recombinant HAs, or the second container comprises one or more ribonucleic acid molecules encoding the one or more machine-learned influenza virus HAs.

[0197] In certain embodiments, each of the first, second, third, and fourth influenza virus HAs in the first container are present as ribonucleic acid molecules encoding the respective influenza virus HAs, and each of the one or more machine-learned influenza virus HAs in the second container are present as ribonucleic acid molecules encoding the respective influenza virus HAs. Alternatively, the one or more machine-learned influenza virus HAs in the second container are recombinant HAs or are present in inactivated influenza viruses.

[0198] Nucleic acids, cloning and expression systems The present disclosure further provides nucleic acid molecules encoding the disclosed HA. The nucleic acid can be used, for example, to express recombinant HA that can be used in or as a component of a vaccine or immunogenic composition. The nucleic acid can comprise DNA or RNA and can be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence described herein encompasses DNA molecules having the particular sequence and, unless the context requires otherwise, RNA molecules having the particular sequence in which U is replaced by T, or derivatives thereof, such as pseudouridine. Other nucleotide derivatives or modified nucleotides can be incorporated into the disclosed nucleic acid molecules encoding HA.

[0199] 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 disclosed herein. The present disclosure further provides host cells that contain one or more of the above constructs.

[0200] Also provided are methods for making HA encoded by these nucleic acid molecules. HA polypeptides may be produced using recombinant techniques. Recombinant protein production and expression are well known in the art and may be carried out using conventional procedures such as those disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Ed. 2012), Cold Spring Harbor Press: for example, expression of HA polypeptides may be achieved by culturing under appropriate conditions a host cell containing a nucleic acid molecule encoding HA as disclosed herein. After production by expression, HA may be isolated and / or purified using any suitable technique and then used as desired.

[0201] Systems for cloning and polypeptide expression in a variety of different host cells are well known in the art. Any protein expression system (e.g., stable or transient) that is compatible with the constructs disclosed herein can be used to produce the HA described herein.

[0202] Suitable vectors can be chosen or constructed to contain appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.

[0203] To express recombinant HA, a nucleic acid encoding HA can be introduced into a host cell. Introduction can be by 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, such as vaccinia, or baculovirus for insect cells. For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using bacteriophage. These techniques are well known in the art. (See, for example, "Current Protocols in Molecular Biology", Ausubel et al. eds., John Wiley & Sons, 2010). DNA introduction can be followed by a selection method (e.g., antibiotic resistance) to select for cells containing the vector.

[0204] 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 an insect cell.

[0205] How to use The present disclosure provides methods of administering a vaccine or immunogenic composition described herein to a subject. The methods may 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 or immunogenic composition comprising an HA and / or ribonucleic acid molecule described herein and an optional adjuvant, in an amount effective to vaccinate the subject against influenza virus. Similarly, the present disclosure provides a vaccine or immunogenic composition comprising an HA and / or ribonucleic acid molecule described herein for use in vaccinating a subject against influenza virus.

[0206] The present disclosure also provides the use of a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein for the manufacture of a medicament for vaccinating a subject against influenza virus. The present disclosure also provides a method of vaccinating a subject against influenza virus comprising administering to the subject an immunologically effective amount of a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein and an optional adjuvant. Similarly, the present disclosure provides a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein for use in immunizing a subject against influenza virus. The present disclosure also provides the use of a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein for the manufacture of a medicament for immunizing a subject against influenza virus.

[0207] 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.

[0208] The immunization methods (or related uses) provided herein can elicit a broad neutralizing immune response against one or more influenza viruses. Thus, in various embodiments, the compositions described herein can 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) can elicit an improved immune response against one or more seasonal influenza strains (e.g., standard of care strains). For example, the improved immune response can be an improved humoral immune response. In some embodiments, the immunization methods (or related uses) can elicit an improved immune response against one or more pandemic influenza strains. In some embodiments, the immunization methods (or related uses) can elicit an improved immune response against one or more swine influenza strains. In some embodiments, the immunization methods (or related uses) can elicit an improved immune response against one or more avian influenza strains.

[0209] Also provided is a method of preventing influenza virus disease in a subject, comprising administering to the subject a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule 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 or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein and an optional adjuvant for use in preventing influenza virus disease in a subject. The present disclosure also provides the use of a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein and an optional adjuvant for the manufacture of a medicament for the prevention of influenza virus disease in a subject.

[0210] Also provided is a method of inducing an immune response against influenza virus HA in a subject, comprising administering to the subject a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein and an optional adjuvant. Similarly, the present disclosure provides a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein and an optional adjuvant for use in inducing an immune response against influenza virus HA in a subject. The present disclosure also provides the use of a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein and an optional adjuvant for the manufacture of a medicament for inducing an immune response against influenza virus in a subject.

[0211] A vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein and an optional adjuvant may be administered prior to or after the onset of one or more symptoms of influenza infection. That is, in some embodiments, a vaccine or immunogenic composition 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, such as when the subject comes into contact with other individuals or livestock (e.g., pigs) known or suspected to be infected with 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 or immunogenic composition 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 or believed to have been exposed to influenza virus. In some embodiments, the subject is at risk or susceptible to influenza infection, such as when the subject is known or believed to have been exposed to influenza virus. In some embodiments, a subject is known or considered to be exposed to influenza virus if the subject has been in contact with other individuals or livestock (e.g., pigs) known or suspected to be infected with pandemic influenza virus, and / or if the subject is present or present in an area where influenza infection is known or considered to be epidemic or endemic. The vaccine or immunogenic compositions disclosed herein may be used to treat or prevent disease caused by seasonal or pandemic influenza strains, or both.

[0212] Vaccine or immunogenic compositions 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 method of administration.

[0213] 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 rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig).

[0214] In some embodiments, the vaccine or immunogenic compositions described herein are administered to a human subject. In certain embodiments, the human subject is 6 months or older, 6 months to 35 months, at least 2 years old, at least 3 years old, 36 months to 8 years old, 9 years old or older, at least 6 months to under 18 years old, or at least 3 years to under 18 years old. In some embodiments, the human subject is an infant (under 36 years old). In some embodiments, the human subject is a child or adolescent (under 18 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 to under 65 years old). In some embodiments, the methods and uses of the vaccine or immunogenic compositions described herein include a prime boost vaccination method. Prime boost vaccination involves administering a priming vaccine followed, after a period of time, by administering a boosting vaccine to the subject. The immune response is "primed" upon administration of the priming vaccine and "boosted" upon administration of the boosting vaccine. The priming vaccine can comprise a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein and an optional adjuvant. Similarly, the boosting vaccine can comprise a vaccine or immunogenic composition comprising the HA and / or ribonucleic acid molecule described herein and an optional adjuvant. The priming vaccine or immunogenic composition can be, but need not be, the same as the boosting vaccine. The boosting vaccine is generally administered 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.

[0215] The vaccine or immunogenic composition may be administered using any suitable route of administration, including, for example, parenteral delivery, as described above.

[0216] Typically, the HA and / or ribonucleic acid molecules described herein and optional adjuvants are administered together as components of the same vaccine or immunogenic composition. However, the HA and / or ribonucleic acid molecules described herein need not be administered as part of the same vaccine or immunogenic composition. That is, the HA and / or ribonucleic acid molecules described herein and optional adjuvants can be administered sequentially to a subject, if desired.

[0217] Representative embodiments of the present disclosure 1. (a) a first influenza virus hemagglutinin (HA), where the first influenza virus HA is an H1 HA from a first standard of care influenza virus strain, or a first ribonucleic acid molecule encoding a first influenza virus H1 HA; (b) a second influenza virus HA, where the second influenza virus HA is an H3 HA from a second standard of care influenza virus strain, or a second ribonucleic acid molecule encoding a second influenza virus H3 HA; (c) a third influenza virus HA, where the third influenza virus HA is an HA from a third standard of care influenza virus strain of the B / Victoria lineage, or a third ribonucleic acid molecule encoding a third influenza virus HA from the B / Victoria lineage; (d) a fourth influenza virus HA, where the fourth influenza virus HA is an HA from a fourth standard of care influenza virus strain of the B / Yamagata lineage, or a fourth ribonucleic acid molecule encoding a fourth influenza virus HA from the B / Yamagata lineage; (e) one or more machine-learning influenza virus HAs having molecular sequences identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding one or more machine-learning influenza virus HAs, wherein the one or more machine-learning influenza virus HAs are selected from an H1 HA, an H3 HA, an HA from B / Victoria lineage, an HA from B / Yamagata lineage, or a combination thereof; 20. An immunogenic composition comprising:

[0218] 2. The immunogenic composition of embodiment 1, wherein the ribonucleic acid molecule is an mRNA molecule.

[0219] 3. The immunogenic composition of embodiment 1 or 2, wherein the ribonucleic acid molecule is encapsulated in a lipid-nanoparticle (LNP).

[0220] 4. The immunogenic composition according to any one of embodiments 1 to 3, wherein the molecular sequence is an amino acid sequence or a nucleic acid sequence.

[0221] 5. The immunogenic composition of any one of embodiments 1-4, wherein the one or more machine-learned influenza virus HAs comprise a wild-type influenza virus HA molecular sequence.

[0222] 6. The immunogenic composition of any of embodiments 1-5, wherein the one or more machine-learned influenza virus HAs comprise a non-wild-type influenza virus HA molecular sequence.

[0223] 7. The immunogenic composition of any one of embodiments 1-6, wherein the one or more machine-learned influenza virus HAs comprise a recombinant influenza virus HA.

[0224] 8. The immunogenic composition of any of embodiments 1-6, wherein the one or more machine learning influenza virus HAs are present in an inactivated influenza virus, optionally a split-inactivated virus.

[0225] 9. An immunogenic composition according to any one of embodiments 1 to 6, comprising a ribonucleic acid molecule encoding at least one of one or more machine learning influenza virus HAs.

[0226] 10. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H3 HA, and the fifth influenza H3 HA is antigenically distinct from the second influenza H3 HA.

[0227] 11. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H3 HA, and the fifth influenza H3 HA enhances a protective immune response induced by the second influenza H3 HA.

[0228] 12. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H3 HA, and the fifth influenza H3 HA expands the protective immune response induced by the second influenza H3 HA.

[0229] 13. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H3 HA, and the fifth influenza H3 HA is from a different clade than the second influenza H3 HA.

[0230] 14. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H3 HA, and the fifth influenza H3 HA is antigenically similar to the second influenza H3 HA.

[0231] 15. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H3 HA, and the fifth influenza H3 HA is from the same clade as the second influenza H3 HA.

[0232] 16. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H1 HA, and the fifth influenza H1 HA is antigenically distinct from the first influenza H1 HA.

[0233] 17. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H1 HA, and the fifth influenza H1 HA enhances the protective immune response induced by the first influenza H1 HA.

[0234] 18. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H1 HA, and the fifth influenza H1 HA expands the protective immune response induced by the first influenza H1 HA.

[0235] 19. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H1 HA, and the fifth influenza H1 HA is from a different clade than the first influenza H1 HA.

[0236] 20. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H1 HA, and the fifth influenza H1 HA is antigenically similar to the first influenza H1 HA.

[0237] 21. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H1 HA, and the fifth influenza H1 HA is from the same clade as the first influenza H1 HA.

[0238] 22. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs are a fifth influenza virus HA, and the fifth influenza virus HA is an H3 HA from the 3C.2A clade.

[0239] 23. The immunogenic composition of any of embodiments 1-9, wherein the one or more machine learning influenza virus HAs is a fifth influenza virus HA, and the fifth influenza virus HA is an H3 HA from the 3C.3A clade.

[0240] 24. The immunogenic composition of any of embodiments 1-15, further comprising a sixth influenza virus HA.

[0241] 25. The immunogenic composition of embodiment 24, wherein the sixth influenza virus HA is an H1 HA having a molecular sequence identified or designed from a machine learning model, or a ribonucleic acid molecule encoding the sixth influenza virus HA.

[0242] 26. The immunogenic composition of embodiment 25, wherein the sixth influenza H1 HA is antigenically different from the first influenza H1 HA, the sixth influenza H1 HA enhances the protective immune response induced by the first influenza H1 HA, the sixth influenza H1 HA expands the protective immune response induced by the first influenza H1 HA, the sixth influenza H1 HA is from a different clade from the first influenza H1 HA, the sixth influenza H1 HA is from the same clade as the first influenza H1 HA, or the sixth influenza H1 HA is antigenically similar to the first influenza H1 HA.

[0243] 27. The immunogenic composition according to any one of embodiments 24 to 26, further comprising a seventh influenza virus HA from the B / Victoria lineage having a molecular sequence identified or designed from a machine learning model, or a ribonucleic acid molecule encoding the seventh influenza virus HA.

[0244] 28. The immunogenic composition according to any one of embodiments 24 to 27, further comprising an eighth influenza virus HA from the B / Yamagata lineage having a molecular sequence identified or designed from a machine learning model, or a ribonucleic acid molecule encoding the eighth influenza virus HA.

[0245] 29. An immunogenic composition according to any of embodiments 1 to 28, wherein the machine learning model is trained to predict a biological response.

[0246] 30. The immunogenic composition of embodiment 29, wherein the biological response is a human, ferret, or mouse biological response.

[0247] 31. The immunogenic composition of embodiment 29 or 30, wherein the biological response comprises a hemagglutinin inhibition assay (HAI), antibody forensics (AF), or neutralization assay.

[0248] 32. The immunogenic composition of any one of embodiments 1-31, wherein each of the first, second, third, and fourth influenza virus HAs is a recombinant influenza virus HA.

[0249] 33. The immunogenic composition of any one of embodiments 1 to 31, wherein each of the first, second, third, and fourth influenza virus HAs is present in an inactivated influenza virus.

[0250] 34. The immunogenic composition according to any one of the preceding embodiments, comprising a first, a second, a third and a fourth influenza virus HA as ribonucleic acid molecules.

[0251] 35. The immunogenic composition according to any one of embodiments 7 to 34, wherein each of the recombinant influenza virus HAs is produced by a baculovirus expression system in cultured insect cells.

[0252] 36. The immunogenic composition of any of embodiments 1-35, wherein the first influenza virus HA is an H1 HA derived from an H1N1 influenza virus strain, and the second influenza virus HA is an H3 HA derived from an H3N2 influenza virus strain.

[0253] 37. The immunogenic composition of any of embodiments 1 to 36, further comprising an adjuvant.

[0254] 38. The immunogenic composition of embodiment 37, wherein the adjuvant comprises a squalene in water adjuvant or a liposome-based adjuvant.

[0255] 39. The immunogenic composition of embodiment 38, wherein the squalene adjuvant in water comprises AF03.

[0256] 40. The immunogenic composition of embodiment 38, wherein the liposome-based adjuvant comprises SPA14.

[0257] 41. An immunogenic composition according to any of the preceding embodiments, wherein each ribonucleic acid molecule comprises one or more modified nucleotides.

[0258] 42. The immunogenic composition of any of embodiments 1-41, formulated for intramuscular injection.

[0259] 43. An immunogenic composition according to any of the preceding embodiments, wherein the ribonucleic acid molecule is encapsulated in an LNP comprising a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid.

[0260] 44. A method of immunizing a subject against influenza virus, comprising administering to the subject an immunologically effective amount of the immunogenic composition of any one of embodiments 1 to 43.

[0261] 45. The method of embodiment 44, for preventing influenza virus infection in a subject.

[0262] 46. ​​The method of embodiment 44 or 45, which generates a protective immune response in a subject.

[0263] 47. The method of embodiment 46, wherein the protective immune response comprises an HA antibody response.

[0264] 48. The method of any one of embodiments 44 to 47, wherein the subject is a human.

[0265] 49. The method according to any one of embodiments 44 to 48, wherein the immunogenic composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

[0266] 50. The method according to any one of embodiments 44 to 49, for treating or preventing a disease caused by either or both seasonal and pandemic influenza strains.

[0267] 51. The method of any one of embodiments 44-50, wherein the subject is a human and the human is at least 6 months old, 6-35 months old, at least 2 years old, at least 3 years old, under 18 years old, at least 18 years old, at least 60 years old, at least 65 years old, at least 6 months old under 18 years old, at least 3 years old under 18 years old, or at least 18 years old under 65 years old.

[0268] 52. A method for alleviating one or more symptoms of influenza virus infection, comprising administering to a subject a prophylactically effective amount of the immunogenic composition of any one of embodiments 1-43.

[0269] 53. The method of any one of embodiments 44-52, comprising administering to the subject two doses of the immunogenic composition, spaced 2-6 weeks apart, optionally 4 weeks apart.

[0270] 54. A vaccine composition comprising an immunogenic composition according to any one of embodiments 1 to 43.

[0271] 55. The method according to any one of embodiments 44 to 53, wherein the immunogenic composition is a vaccine composition.

[0272] The present disclosure will be better understood with reference to the following examples. EXAMPLES

[0273] The following examples should be considered illustrative, and not limiting, of the scope of the above disclosure.

[0274] 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.

[0275] Hemagglutinin inhibition (HAI) assay: Prior to the HAI assay, serum was treated with receptor-destroying enzyme (RDE; Denka Seiken, Co., Japan) to inactivate nonspecific inhibitors. RDE-treated serum was 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). The homologous virus panels used are described in the Examples below and were grown in eggs unless otherwise indicated. Plates were covered and incubated at room temperature for 20 min (or 45-60 min), 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. Plates were mixed by vortexing, covered, and the RBCs were allowed to settle at room temperature for approximately 30 min to 1 h. The HAI titer was determined by the reciprocal dilution of the last well containing non-agglutinated RBCs.

[0276] HINT mNT Influenza Protocol: Neutralizing titers against influenza strains were measured as described in Jorquera, PA et al, Insights into antigenic advancement of influenza A(H3N2)viruses, 2011-2018, Sci. Reports 9, 2676 (2019). Briefly, serial two-fold dilutions of RDE-treated serum, 1:20 to 1:2,560, were mixed with an equal volume of virus, approximately 1000 focus-forming units (FFU), and incubated at 37°C for 60 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 ALEXA FLUOR® 488-conjugated secondary antibody (Thermo Fisher Scientific; Waltham, MA). Cells were washed and plates were scanned with CTL IMMUNOSPOT® Cell Imaging v2 (CTL, Cleveland, OH). Counts from plates were transferred to Graphpad Prism software and neutralization titers 50 (NT50) were calculated using a sigmoidal dose response, variable slope, non-linear progression. NT50 titers of serum samples that inhibited virus infection to 50% of virus input only control wells were the titers calculated from the sigmoidal curve. This assay measures inhibition of virus 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, B / Victoria, and B / Yamagata.

[0277] Example 1 - Immune response to administration of multiple H3 HA strains To determine the effect on HA immune responses, multiple H3 HAs were administered in a naive ferret model. Ferrets were infected with a single H3N2 inactivated virus or a cocktail of two H3N2 inactivated viruses to determine whether the antibody responses elicited by the cocktail showed broader breadth than responses elicited by a single virus against an antigenically diverse panel of viral readouts.

[0278] Viruses selected for co-infection were from the same clade 3C.2A (similar viruses) or different clades 3C.2A and 3C.3A (dissimilar viruses) and are shown in Table 1 below.

[0279] [Table 1] The readout panel included viruses from the period 2016–2019 and was representative of circulating strains from two antigenically distinct clades, 3C.2A and 3C.3A, to assess cross-clade coverage. The following seven 3C.2A viruses were used in the readout panel: A / Valladolid / 182 / 2017, A / Alaska / 43 / 2019, A / Bangladesh / 3190613015 / 2019, A / Hongkong / 45 / 2019, A / Victoria / 617 / 2017, A / Peru / 9519 / 2019, and A / Singapore / INFIMH-16-0019 / 2016. Five 3C.3A viruses were used in the readout panel: A / Kansas / 14 / 2017, A / Brisbane / 34 / 2018, A / Mexico / 2356 / 2019, A / Suriname / 0902 / 2019, and A / Indiana / 08 / 2018.

[0280] Naive ferrets (2 per group) were inoculated intranasally with (1) A / Hongkong / 45 / 2019 alone; (2) A / Alaska / 43 / 2019 alone; (3) A / Kansas / 14 / 2017 alone; (4) a 1:1 combination of A / Hongkong / 45 / 2019 and A / Alaska / 43 / 2019; or (5) a 1:1 combination of A / Hongkong / 45 / 2019 and A / Kansas / 14 / 2017. Each virus was administered at the same dose, 5 log 10 The vaccine was given in focus forming units (FFU). Blood was taken on day -8, ferrets were immunized on day 0, and blood was taken again on day 14.

[0281] High microneutralization titers against lead-out viruses covering the 3C.2A and 3C.3A clades were observed from ferrets infected with the A / HONGKONG / 45 / 2019+A / KANSAS / 14 / 2017 virus combination. See Figure 3 and Figure 4B. In contrast, ferrets infected with an antigenically similar virus cocktail containing the A / HONGKONG / 45 / 2019+A / ALASKA / 43 / 2019 combination showed a more clade-restricted response, with high titers observed against lead-out strains from the 3C.2A clade. See Figure 2 and Figure 4B. The combination of virus cocktails does not appear to interfere with the responses elicited against each individual strain within the cocktail, similar to those observed with single infections. See Figure 5.

[0282] A mixture of H3 HAs from opposing clades (3C.2A and 3C.3A) demonstrated an additive effect of antibody responses indicative of cross-clade spread with the highest neutralization titers. Thus, the overall responses observed with this 3C.2A+3C.3A cocktail were similar to those observed with each single infection with 3C.2A and 3C.3A viruses. Although the same 3C.2A clade virus mixture did not induce titers as large as the 3C.2A+3C.3A virus cocktail with spread to the 3C.3A clade, this mixture demonstrated that the addition of A / ALASKA / 43 / 2019 virus to the standard of care virus A / HONGKONG / 45 / 2019 boosted antibody titers across the entire readout panel. See Figure 2, which compares (1) the titers of A / Hongkong / 45 / 2019 and (2) the combination of A / Hongkong / 45 / 2019 and A / Alaska / 43 / 2019. These data indicate that combining different H3 HAs can increase range and improve coverage of antigenically diverse influenza viruses.

[0283] Example 2 - Immunogenicity in a naive ferret model evaluating tetravalent and pentavalent influenza vaccines The aim of this study was to determine whether mixing two H3 HAs delivered in a modified non-replicating (MNR) mRNA formulation would induce additive, synergistic or antagonistic effects on the HA immune response in a naive ferret model. This study further evaluated the feasibility of a pentavalent influenza vaccine (PIV) containing an additional H3 strain to extend coverage compared to a quadrivalent influenza vaccine (QIV) without the additional strain.

[0284] Naive ferrets used to evaluate polyvalent vaccine immunogenicity were vaccinated twice (on days 0 and 21) with one of the following 10 groups, spaced 21 days apart, as described in Table 2 below: Group (1) a mixture of five mRNAs encoding HA antigens, four of which were selected from the 2021-2022 Northern Hemisphere WHO standard of care (WHO SOC) strains (H1, H3, BVic, and BYam) (specifically, the A / Wisconsin / 588 / 2019 (H1N1) strain, the A / Tasmania / 503 / 2020 (H3N2) strain, the B / Washington / 02 / 2019 (Victoria lineage) strain, and the B / Phuket / 3073 / 2013 (Yamagata lineage) strain), one of which was selected by machine learning to protect against clade H3C.2A (specifically, the wild-type A / Norway / 2629 / 2015), and HA mRNA from each strain was present in an amount of 15 mg; Group (2) a mixture of five mRNAs encoding HA antigens, four of which were WHO SOC strains, one of which was selected by machine learning to protect against clade H3C.2A (specifically, non-wild-type A / Design / H3S25 / 2019), with HA mRNA from each strain present in an amount of 15 μg; Group (3) a mixture of five mRNAs encoding HA antigens, four of which were WHO SOC strains, one of which was selected by machine learning to protect against clade H3C.3A (specifically, wild-type A / Washington / 526 / 2019), with HA mRNA from each strain present in an amount of 15 μg; group (4) a mixture of five mRNAs encoding HA antigens, four of which were the WHO SOC strains mentioned above and one of which was an additional WHO SOC strain, A / Kansas / 14 / 2017, selected to provide clade H3C.3A protection, with HA mRNA from each strain present in an amount of 15 μg; Group (5) a mixture of five mRNAs encoding HA antigens, four of which were the WHO SOC strains mentioned above, one of which was the 2019–2020 Northern Hemisphere WHO SOC strain A / Kansas / 14 / 2017, with HA mRNAs from H1, BYam, and BVicn each present in amounts of 15 μg each, and mRNAs from two H3 strains (A / Tasmania / 503 / 2020 and A / Kansas / 14 / 2017) each present in amounts of 7.5 μg; group (6) a mixture of four mRNAs encoding WHO SOC strains, mRNA from each of the H1, BYam, and BVic strains was present in an amount of 15 μg, and mRNA from the H3 strain was present in an amount of 30 μg; group (7), a mixture of four mRNAs encoding WHO SOC strains, with mRNA from each of the four strains present in an amount of 15 μg; group (8) a mixture of four recombinant HA proteins selected from WHO SOC strains, recombinant HA from each of the four strains was present in an amount of 45 μg; Group (9) a mixture of four inactivated viruses selected from the WHO SOC strains, each of the four strains was present in an amount of 60 μg; and Group (10) phosphate buffered saline (PBS).

[0285] With the exception of groups 8-10, all vaccine formulations contained a single encapsulated (single subtype / LNP) MNR mRNA HA, which were combined into a single formulation prior to immunization to generate the desired vaccine combination.

[0286] Ferrets were immunized by intramuscular inoculation on days 0 and 21, and humoral responses were assessed on day 49, one month after the second immunization, by microneutralization (mNT) assay, which measures functional HA antibody responses.

[0287] [Table 2]

[0288] [Table 3]

[0289] For each group, n = 6 ferrets were tested for mNT antibody titers against the following egg-amplified influenza virus strains: A / Tasmania / 503 / 2020, A / Victoria / 2570 / 2019, B / Phuket / 3073 / 2013, and B / Washington / 02 / 2019, and geometric mean titer (GMT) values ​​were calculated. The results are shown in Figure 6.

[0290] The upper titer limit for H1 (A / Victoria / 2570 / 2019) was 6,000-7,000. As shown above and in Figure 6, ferrets that received the tetravalent mRNA vaccine (groups 6 and 7) generated functional antibody responses to all four homologous influenza subtypes, H1N1, H3N2, B / Yamagata, and B / Victoria, by day 49. H1 responses were robust in all of groups 1-9 (where the upper titer limit was set at 1:6,000).

[0291] In B / Victoria responses in ferrets inoculated with PIV containing 15 mg HA mRNA per strain (groups 1-4), neutralization titers elicited against B / Washington / 02 / 2019 were comparable to those elicited from ferrets inoculated with QIV containing 15 mg HA mRNA per strain (group 7). Similarly, similar B / Yamagata responses were observed in groups 2-4 and 7, except that in group 1, four of the ferrets did not develop homologous neutralization titers against B / Phuket / 3073 / 2013, and overall titers were significantly lower than those in group 7 (p<0.0001 in a mixed model analysis). Without being bound by theory, the results may indicate technical issues with immunization or strain-specific effects, since the addition of H3 strains in the other PIV groups did not result in a significant reduction in mNT titers.

[0292] There was no statistically significant difference in antibody responses measured on day 49 between the 15 μg dose of H3 A / Tasmania / 503 / 2020 or the 30 μg dose of H3A / Tasmania / 503 / 2020 QIV mRNA vaccine groups 6 and 7 (p=0.95, mixed model analysis), suggesting that doubling the H3 antigen dose did not increase neutralizing antibody titers in this dose range.

[0293] Ferrets immunized with the 7.5 μg dose of H3 in the PIV formulation (group 5) showed significantly higher A / Tasmania / 503 / 2020 mNT titers than ferrets immunized with the QIV formulation (groups 6 and 7, which received 15 μg and 30 μg of the H3 component, respectively) ( p < 0.05; mixed model analysis).

[0294] Groups 1-4, each containing two H3 strains (30 μg total H3) in the PIV formulation, demonstrated comparable homologous A / Tasmania / 503 / 2020 mNT titers within 2-fold (groups 6 and 7) of the QIV control GMT titers (not significant by mixed model analysis). Ferrets in group 5 immunized with the lower dose (7.5 μg of each H3 strain) elicited a significantly higher homologous response than those in groups 6 and 7, with responses 2.3-fold higher (p<0.05 by mixed model analysis). Significantly, the data indicate that the addition of H3 strains to make the PIV formulation did not interfere with the homologous mNT responses elicited by the H1, H3, B / Victoria, or B / Yamagata WHO SOC strains.

[0295] This study also explored whether coverage of the H3 antigenic space could be expanded by adding a second H3 strain from machine learning selection or from a previous WHO SOC selection to the QIV to generate a PIV (e.g., groups 1-5). The WHO 2021-2022 SOC H3 strain A / Tasmania / 503 / 2020 (3C.2A circulating clade) was part of the QIV formulation, but the PIV formulation included two additional H3 clade 3C.2A strains as a fifth strain: the machine learning selected wild-type strain A / Norway / 2629 / 2015 (group 1) and the machine learning selected non-wild-type strain A / Design / H3S25 / 2019 (group 2). These PIV formulations containing two 3C.2A H3 strains showed a slight increase in GMT mNT titers compared to the single 3C.2A formulation in QIV (Group 7) and did not show any negative interference with the heterologous response when co-administered with similar HA to naive ferrets. Results are shown in Table 3 below. See also Figure 7 which shows the mNT GMT values ​​for the 3C.2A and 3C.3A strains for both PIV (Groups 1-5) and QIV (Groups 6 and 7) vaccine formulations.

[0296] [Table 4]

[0297] In general, ferrets immunized with two H3s from the 3C.2A clade (groups 1 and 2) did not expand into the 3C.3A space, but the machine learning designed H3 strain A / Design / H3S25 / 2019 efficiently neutralized 50% of the 3C.3A viruses, unlike the QIV controls groups 6 and 7. See Figure 9.

[0298] Alternatively, adding an H3 strain from the 3C.3A clade to the H3 3C.2A strain in a tetravalent vaccine, as was done in groups 3-5, demonstrated at least an additive effect on antibody responses compared to the heterologous multi-clade 3C.2A and 3C.3A viruses, as shown in Figure 8 and reported in Table 4 below.

[0299] For each of groups 1-7, mNT titers were measured for the following cell-amplified influenza virus strains: A / Bangladesh / 3190613015 / 2019; A / Mexico / 2356 / 2019; A / Valladolid / 182 / 2017; A / Brisbane / 75 / 2019; A / Tasmania / 503 / 2020; A / HongKong / 45 / 2019; A / Kansas / 14 / 2017; and A / Singapore / Infimh160019 / 2016. A / Mexico / 2356 / 2019 and A / Kansas / 14 / 2017 are both clade 3C.3A. A / Bangladesh / 3190613015 / 2019; A / Brisbane / 75 / 2019; A / Tasmania / 503 / 2020; and A / HongKong / 45 / 2019 are clade / subclade 3C.2A1b. A / Valladolid / 182 / 2017 is clade / subclade 3C.2A4 and A / Singapore / Infimh160019 / 2016 is clade / subclade 3C.2A1. The results are reported in Table 4 below.

[0300] [Table 5]

[0301] In addition to increased mNT titers, as shown in Table 4 above and Figure 8, strain coverage in the diverse 3C.3A clade was 100% in PIV formulation groups 3-5, while no coverage of the 3C.3A clade was observed in QIV formulation groups 6 and 7, as shown in Figure 9. Maximizing coverage in a multi-clade season by delivery of two distinct H3HAs represents a potential improvement in efficacy of quadrivalent standard of care vaccine formulations.

[0302] Also, as used in this disclosure and the appended claims, it should be noted that 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. 1. An immunogenic composition comprising: (a) at least three or at least four influenza virus hemagglutinins (HAs) from standard of care influenza virus strains, or at least three or at least four ribonucleic acid molecules encoding influenza virus HAs; and (b) one or more machine-learned influenza virus HAs having molecular sequences identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding the one or more machine-learned influenza virus HAs, wherein the one or more machine-learned influenza virus HAs are selected from the group consisting of H1 HA, H3 HA, HA from B / Victoria lineage, HA from B / Yamagata lineage, or a combination thereof.

2. (a) a first influenza virus hemagglutinin (HA), wherein said first influenza virus HA is an H1 HA from a first standard of care influenza virus strain, or a first ribonucleic acid molecule encoding said first influenza virus H1 HA; (b) a second influenza virus HA, wherein said second influenza virus HA is an H3 HA from a second standard of care influenza virus strain, or a second ribonucleic acid molecule encoding a second influenza virus H3 HA; (c) a third influenza virus HA, wherein the third influenza virus HA is from a third standard of care influenza virus strain of the B / Victoria lineage, or a third ribonucleic acid molecule encoding a third influenza virus HA of the B / Victoria lineage; (d) one or more machine-learned influenza virus HAs having molecular sequences identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding the one or more machine-learned influenza virus HAs; The immunogenic composition of claim 1, comprising:

3. (a) a first influenza virus hemagglutinin (HA), wherein said first influenza virus HA is an H1 HA from a first standard of care influenza virus strain, or a first ribonucleotide encoding said first influenza virus H1 HA; a nucleic acid molecule; (b) a second influenza virus HA, wherein said second influenza virus HA is an H3 HA from a second standard of care influenza virus strain, or a second ribonucleic acid molecule encoding said second influenza virus H3 HA; (c) a third influenza virus HA, wherein the third influenza virus HA is an HA from a third standard of care influenza virus strain of the B / Victoria lineage, or a third ribonucleic acid molecule encoding the third influenza virus HA from the B / Victoria lineage; (d) a fourth influenza virus HA, wherein the fourth influenza virus HA is an HA from a fourth standard of care influenza virus strain of B / Yamagata lineage, or a fourth ribonucleic acid molecule encoding the fourth influenza virus HA from B / Yamagata lineage; (e) one or more machine-learned influenza virus HAs having molecular sequences identified or designed from a machine-learning model, or one or more ribonucleic acid molecules encoding said one or more machine-learned influenza virus HAs, wherein said one or more machine-learned influenza virus HAs are selected from H1 HA, H3 HA, HA from B / Victoria lineage, HA from B / Yamagata lineage, or a combination thereof; An immunogenic composition comprising:

4. The immunogenic composition of claim 1 , wherein the ribonucleic acid molecule is an mRNA molecule.

5. The immunogenic composition of claim 1, wherein the mRNA molecule is encapsulated in a lipid-nanoparticle (LNP).

6. The immunogenic composition according to any one of claims 1 to 5, wherein the molecular sequence is an amino acid sequence or a nucleic acid sequence.

7. The immunogenic composition of any one of claims 1 to 5, wherein the one or more machine-learned influenza virus HAs comprise a wild-type influenza virus HA molecular sequence or a non-wild-type influenza virus HA molecular sequence.

8. 6. The immunogenic composition of any one of claims 1 to 5, wherein the one or more machine-learned influenza virus HAs are recombinant influenza virus HAs, the one or more machine-learned influenza virus HAs are present in an inactivated influenza virus, optionally a split-inactivated virus, or at least one of the one or more machine-learned influenza virus HAs is encoded by a ribonucleic acid molecule.

9. the one or more machine-learned influenza virus HAs include a first machine-learned influenza virus HA, wherein the first machine-learned influenza virus HA is an H3 HA; (a) the first machine-learning influenza virus H3 HA is antigenically distinct from the second influenza virus H3 HA, or the first machine-learning influenza virus H3 HA is from a different clade than the second influenza virus H3 HA; (b) the first machine-learned influenza virus H3 HA enhances or expands the protective immune response induced by the second influenza H3 HA; or (c) the first machine-learning influenza virus H3 HA is antigenically similar to the second influenza virus H3 HA or the first machine-learning influenza virus H3 HA is from the same clade as the second influenza virus H3 HA; The immunogenic composition according to any one of claims 2 to 5.

10. the one or more machine-learned influenza virus HAs include a first machine-learned influenza virus HA, wherein the first machine-learned influenza virus HA is an H1 HA; (a) the first machine-learned influenza virus H1 HA is antigenically distinct from the first influenza virus H1 HA or the first machine-learned influenza virus H1 HA is from a different clade than the first influenza virus H1 HA; (b) the first machine-learned influenza virus H1 HA enhances or expands the protective immune response induced by the first influenza virus H1 HA; or (c) the first machine-learned influenza virus H1 HA is antigenically similar to the first influenza virus H1 HA or is from the same clade as the first influenza virus H1 HA. The immunogenic composition according to any one of claims 2 to 5.

11. 6. The immunogenic composition of any one of claims 1-5, wherein the one or more machine-learned influenza virus HAs comprise a first machine-learned influenza virus HA, wherein the first machine-learned influenza virus HA is an H3 HA from the 3C.2A clade or the 3C.3A clade.

12. The immunogenic composition of any one of claims 2 to 5, wherein the one or more machine-learned influenza virus HAs comprise a first machine-learned influenza virus HA and a second machine-learned influenza virus HA.

13. 13. The immunogenic composition of claim 12, wherein the first machine-learned influenza virus HA is an H3 HA and the second machine-learned influenza virus HA is an H1 HA having a molecular sequence identified or designed from a machine-learning model, or a ribonucleic acid molecule encoding the second machine-learned influenza virus HA.

14. the second machine-learned influenza virus H1 HA is antigenically distinct from the first influenza virus H1 HA, or the second machine-learned influenza virus H1 HA is antigenically distinct from the first influenza virus H1 HA. the second machine-learning influenza virus H1 HA enhances the protective immune response induced by the first influenza virus H1 HA; the second machine-learning influenza virus H1 HA expands the protective immune response induced by the first influenza virus H1 HA; the second machine-learning influenza virus H1 HA is from a different clade than the first influenza virus H1 HA; 14. The immunogenic composition of claim 13, wherein the HA is from the same clade as the first influenza virus H1 HA or the second machine-learned influenza virus H1 HA is antigenically similar to the first influenza virus H1 HA.

15. 15. The immunogenic composition of claim 14, further comprising a third machine-learned influenza virus HA from the B / Victoria lineage having a molecular sequence identified or designed from a machine-learning model, or a ribonucleic acid molecule encoding the third machine-learned influenza virus HA.

16. 6. The immunogenic composition of any one of claims 1 to 5, wherein the machine learning model is trained to predict biological responses such as hemagglutinin inhibition assay (HAI), antibody forensics (AF), or neutralization assay.

17. The immunogenic composition of any one of claims 3 to 5, wherein each of the first, second, third, and fourth influenza virus HAs is a recombinant influenza virus HA, or each of the first, second, third, and fourth influenza virus HAs is present in an inactivated influenza virus, or each of the first, second, third, and fourth influenza virus HAs is a ribonucleic acid molecule.

18. 9. The immunogenic composition of claim 8, wherein each of the recombinant influenza virus HAs is produced in cultured insect cells using a baculovirus expression system.

19. The immunogenic composition of any one of claims 2 to 5, wherein the first influenza virus HA is an H1 HA derived from an H1N1 influenza virus strain, and the second influenza virus HA is an H3 HA derived from an H3N2 influenza virus strain.

20. The immunogenic composition of any one of claims 1 to 5, further comprising an adjuvant such as a squalene in water adjuvant or a liposome-based adjuvant.

21. The immunogenic composition of any one of claims 1 to 5, wherein each ribonucleic acid molecule comprises one or more modified nucleotides and is encapsulated in an LNP comprising a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid.

22. An immunogenic composition according to any one of claims 1 to 5 for use in a method for immunising a subject against influenza virus.

23. 23. The immunogenic composition of claim 22, wherein immunizing the subject comprises preventing influenza virus infection in the subject or generating a protective immune response in the subject.

24. 23. The immunogenic composition of claim 22, wherein the influenza virus is one or both of a seasonal influenza strain and a pandemic influenza strain.

25. 23. The immunogenic composition of claim 22, wherein the subject is a human, and the human is at least 6 months old, 6-35 months old, at least 2 years old, at least 3 years old, under 18 years old, at least 18 years old, at least 60 years old, at least 65 years old, at least 6 months and under 18 years old, at least 3 years old and under 18 years old, or at least 18 years old and under 65 years old.

26. 6. The immunogenic composition of any one of claims 1 to 5 for use in reducing one or more symptoms of influenza virus infection in a subject.

27. A vaccine composition comprising the immunogenic composition of any one of claims 1 to 5.

28. The immunogenic composition of claim 22, wherein the immunogenic composition is a vaccine composition.

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