WO2025231007A1 - Influenza vaccine compositions and methods of making and using same - Google Patents

Abstract

Disclosed are vaccine compositions, in particular, polyvalent icosahedral compositions for presentation of an HA1 influenza antigen. The disclosed compositions may contain an S particle comprising a norovirus (NoV) S domain and an HA1 influenza antigen, which may be linked via a linker protein domain operatively connected to the norovirus S domain and an influenza antigen. Fusion proteins for producing the vaccine compositions, and methods of using the disclosed vaccine composition are also provided.

Description

INFLUENZA VACCINE COMPOSITIONS AND METHODS

OF MAKING AND USING SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international application which claims priority to and benefit of U.S. provisional application Serial No. 63/640,385, filed April 30, 2024, the contents of which are incorporated in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under TR001425 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

[0003] A Sequence Listing submitted as an ST.26 XML file via the USPTO patent electronic filing system is hereby incorporated by reference. The name of the XML file for the Sequence Listing is CHMC2024-0301b-SEQLIST.xml, the date of the creation of the XML file is April 21, 2025, and the size of the XML file is 16.7 KB (17,185 bytes).

BACKGROUND

[0004] Influenza is an infectious respiratory disease caused by influenza viruses, members of the family Orthomyxoviridae. These viruses are classified into four types or species, named influenza A virus (IAV), influenza B virus (IBV), influenza C virus (ICV), and influenza D virus (IDV), according to genetic and antigenic differences in their nucleoprotein (NP) and matrix protein (Ml), which are two internal proteins of the virions. Despite the implementation of current influenza vaccines, lAVs and IBVs continue to cause seasonal and occasionally pandemic influenza, claiming 250,000-500,000 lives globally each year. In the United States, the Centers for Disease Control and Prevention (CDC) estimate that influenza results in 4.3 to 21 million medical visits, 140,000 to 810,000 hospitalizations, and 12,000 to 61,000 deaths annually. This underscores the persistent threat posed by influenza to public health, highlighting the urgent need for improved countermeasures against this deadly infectious disease. Disclosed herein are compositions and methods directed to the prevention and/or mitigation of influenza infection.

BRIEF SUMMARY

[0005] Disclosed herein are vaccine compositions comprising a nanoparticle platform for presentation of an HA1 influenza antigen, and methods of making and using the same. In certain aspects, the compositions include a modified norovirus (NoV) S domain linked to at least one influenza antigen. In some embodiments, the modified S domain and the HA1 influenza antigen are operatively connected via a linker protein domain. Also provided are methods for making and administering the vaccine compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0007] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0008] FIG. 1 depicts production of the four S-HA1 fusion proteins. (A) depicts a schematic illustration of the S-HA1 fusion protein construct. “S,” denotes the modified norovirus shell (S) domain; “HA1,” denotes the HA1 domain of an influenza virus; “hinge,” refers to the hinge region of norovirus VP1; “His,” denotes the Hisx6 tag. Panels B and C depict SDS-PAGE analysis of the four S-HA1 fusion proteins containing the HA1 antigens from the H1N1 pandemic strain (Hl/pdm), the H1N1 PR8 strain (H1/PR8), an H3N2 influenza virus (H3), and an influenza B virus (IBV). Lanes M show pre-stained protein markers with indicated molecular weights. [0009] FIG. 2 depicts self-assembly of the S-HA1 fusion proteins into pseudovirus nanoparticles (PVNPs) shown by gel filtration chromatography and transmission electron microscopy (TEM). Panels A, B, E, and F depict representative elution curves from gel filtrations chromatography of the S-HA1 fusion proteins containing HA1 antigens from the Hl pdm strain (A), the Hl PR8 strain (B), an H3 strain (E), and an IBV virus (F), respectively. The elution peaks corresponding to the S-HA1 PVNPs and the S-HA1 protein monomers were determined based on the elution peak of the S-HA1 H7 PVNP in the void volume and the GST dimer of ~52 kDa, respectively, as indicated by the red star symbols. The Y-axes indicate A280 absorbances (mAU), while the X-axes indicate the elution volume (mL). The relative protein concentration of each elution peak is shown by its A280 absorbance. Panels C, D, G, and H depict representative TEM micrographs showing PVNPs in the major elution peaks of the gel filtration analyses of the S-HA1 fusion protein of Hl pdm (C), Hl PR8 (D), H3 (G), and IBV (H).

[0010] FIG. 3 depicts a representative TEM micrograph at high magnification and shows the size variations of the S-HA1 PVNPs assembled by the S-HA1 Hl pdm protein. Three typical PVNPs, measuring 28 nm, 35 nm, and 42 nm in diameter, are indicated, respectively.

[0011] FIG. 4 depicts structural models of the S-HA1 PVNPs in three icosahedral symmetries. Panels A to C show three images of the T = 1 S60-HA1 PVNP. Panels D to F show three images of the T = 3 S180-HA1 PVNP. Panels G to I show three images of the T = 4 S240-HA1 PVNP. The images in the left panels (A, D, and G) are viewed from the 5-fold axis; the images in the middle panels (B, E, and H) are viewed from the 3-fold axis; while the images in the right panels (C, F, and I) are viewed from the 2-fold axis. The inner shells of the S-HA1 PVNPs made by norovirus S domains are shown in orange, whereas the trimeric HA1 protrusions made by the influenza virus HA1 domains are shown in green. All structures are shown in cylinder/stud representations.

[0012] FIG. 5 depicts binding of the S-HA1 PVNPs to antibodies against influenza virus or its hemagglutinins (HAs). The X-axis shows various S-HA1 PVNPs, while the Y axis indicates binding signal intensity of the S-HA1 PVNPs to antibodies against the HA proteins of the H1N1 pandemic strain (pdm, left panel); antibodies against the HA protein of a H3N2 strain (middle panel); or mouse serum obtained after challenge with the mouse-adapted H1N1 influenza virus PR8 strain (right panel). LOD, limit of detection.

[0013] FIG. 6 depicts HA 1 -specific IgG responses in mice after immunizations with each of the four S-HA1 PVNPs. HAl-specific serum IgG titers after two (A) and three (B) immunizations with the four S-HA1 PVNPs individually (each represented by a blue, green, red, or brown column) were determined by EIAs, using the corresponding homologous HA1 proteins as capture antigens. The mouse sera after immunization with the S60 nanoparticle (S) were used as negative controls. The Y-axes show the HAl-specific IgG titers, while the X-axes indicate the corresponding immunogens. The coated HA1 proteins as capture antigens are indicated at the tops of the figures. The dashed lines indicate the limit of detection (LOD). Statistical differences between data groups are denoted as “**” for highly significance with P-values <0.01; or “****” for extremely significance with P-values <0.001.

[0014] FIG. 7 depicts the protective efficacies of the S-HA1 PVNPs against mortality and body weight loss in mice caused by challenges with the mouse-adapted H1N1 influenza virus PR8 strain. Shown are survival curves (A) and body weight change curves (B) of mice after immunization with the S-HA1 Hl PR8 PVNP (green lines), the S-HA1 Hl PDM PVNP (blue lines), or S60 nanoparticle control (S, red lines), followed by challenges with mouse-adapted H1N1 influenza virus PR8 strain. Also shown are survival curves (C) and body weight change curves (D) of mice after immunization with the S-HA1 Hl PR8 PVNP (green lines), the S-HA1 H3 PVNP (purple lines), the S-HA1 IBV PVNP (black lines), or S60 nanoparticle control (S, red lines), followed by challenges with the mouse-adapted H1N1 influenza virus PR8 strain. The Y-axes in (A and C) indicate survival rates in percentages, while the Y-axes in panels B and D indicate body weight changes in percentages. All X-axes indicate days post challenge (DPCs). Statistical differences between the survival curves in panels A and C are shown on the right of the figures. DETAILED DESCRIPTION

DEFINITIONS

[0015] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The methods may comprise, consist of, or consist essentially of the elements of the vaccine compositions and/or methods of making or using the vaccine compositions as described herein, as well as any additional or optional element described herein or otherwise useful in an influenza vaccine composition and/or methods of using same.

[0016] As used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0017] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0018] As used herein, the term “effective amount” means the amount of a vaccine composition sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0019] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In some embodiments, the terms refer to an adult human. In further embodiments, the terms refer to a child, i.e., an individual who is under the age of 18. In further embodiments, the terms may refer to a neonate.

[0020] As used herein, the term “antigen” may be used interchangeably with the terms “immunogen” and “immunogenic antigen”, as defined below. An antigen is a substance that is able to combine with the products of an immune response once they are made, but is not necessarily able to induce an immune response (i.e. while all immunogens are antigens, the reverse is not true); however, the antigens that are discussed herein as the subject of the present invention are assumed to be immunogenic antigens, even when referred to as antigens.

[0021] The term “fusion protein” means a protein created through translation of a fusion gene, resulting in a single polypeptide with functional properties derived from each of the original proteins.

[0022] The term “immunity” means the state of having sufficient biological defenses to avoid infection, disease, or other biological invasion by a disease-causing organism. [0023] The term “immunogenicity” means the ability of an immunogen to elicit a humoral and/or cell-mediated immune response.

[0024] The terms “immunogen” and “immunogenic antigen” mean a specific type of antigen that is able to induce or provoke an adaptive immune response in the form of the production of one or more antibodies.

[0025] The terms “immunogenic response” and “immune response” mean an alteration in the reactivity of an organisms' immune system in response to an immunogen. This can involve antibody production, induction of cell-mediated immunity, complement activation or development of acquired immunity or immunological tolerance to a certain disease or pathogen.

[0026] The terms “immunization” and “vaccination” mean the deliberate induction of an immune response and involve effective manipulation of the immune system's natural specificity, as well as its inducibility. The principle behind immunization is to introduce an antigen, derived from a disease-causing organism, which stimulates the immune system to develop protective immunity against that organism, but wherein the antigen itself does not cause the pathogenic effects of that organism. The desired outcome of a prophylactic or therapeutic immune response resulting from an immunization may vary according to the disease. For example, an immune response against a pathogen may inhibit or prevent colonization and replication of the pathogen, effecting protective immunity and the absence or reduction of any disease symptoms. A vaccine against pathogens may also be considered effective if it reduces the number, severity, or duration of symptoms; if it reduces the number of individuals in a population with symptoms; or even if it merely reduces the transmission of an infectious pathogen.

[0027] The term “infection” means the invasion of an animal or plant host's body tissues by a pathogen, as well as the multiplication of the pathogen within the body and the body's reaction to the pathogen and any toxins that it may produce. [0028] The term “vaccine” means a biological preparation or composition that improves immunity to a particular disease. Vaccines are examples of immunogenic antigens intentionally administered to induce an immune response in the recipient.

Influenza Vaccine Compositions

[0029] Disclosed are compositions for immunization of an individual against an influenza infection. In aspects, the compositions may be used for immunization of an individual against an H1N1 influenza infection. In aspects, the compositions may be used for immunization of an individual against an H3N2 influenza infection. In aspects, the compositions may be used for immunization of an individual against an influenza B infection.

[0030] In aspects, the compositions may be used for immunization of an individual against an one or more influenza infection types. For example, in aspects, the vaccine is a monovalent vaccine, displaying a single HA1 influenza antigen type. In aspects, the vaccine is a polyvalent vaccine, displaying at least two, or at least three, or at least four, or more than four HA1 influenza antigen types. For example, the disclosed vaccine compositions may comprise a bivalent vaccine composition, in which two different HA1 influenza antigens are presented via the vaccine and which provide immunity to the two presented antigens. In further aspects, the disclosed vaccine compositions may comprise a trivalent vaccine composition, in which three different HA1 influenza antigens are presented via the vaccine and which provide immunity to the three presented antigens. In further aspects, the disclosed vaccine compositions may comprise a quadrivalent vaccine composition, in which four different HA1 influenza antigens are presented via the vaccine and which provide immunity to the four presented antigens.

PVNP/S60 PVNP

[0031] The disclosed influenza vaccine compositions, in general, employ a non-replicating pseudovirus nanoparticle (PVNP) platform (alternatively referred to as an “S60 PVNP” or “S60 nanoparticle” or “S60 particle” platform, or “platform”) comprising a modified norovirus shell (S) domain has been described, for example, in WO 2024/102697 Al, which is incorporated herein by reference in its entirety. For example, the disclosed vaccine compositions comprise an S60 PVNP that displays an HA1 influenza antigen. In aspects, the influenza vaccine composition comprises an S60 PVNP comprising a modified S domain that displays a domain of an influenza virus which serves as an antigen. As described above, the vaccine composition can comprises one or more influenza virus antigens. In particular, the vaccine compositions comprise the HA1 domain, a surface glycoprotein of the influenza virus that enables the virus to bind to and enter host cells. HA1 is the globular head domain of the hemagglutinin protein, which contains most of the antigenic sites targeted by neutralizing antibodies. The S60 PVNPs disclosed herein can be used to present one or more antigens that, when administered, elicit an immune response characterized by the production of antigen- specific antibodies and/or activation of cellular immune mechanisms, thereby providing protective immunity against influenza infection. For example, in aspects, the disclosed S60-HAl-PVNPs induce robust HAl-specific IgG responses with significantly elevated titers after multiple doses. In aspects, the disclosed S60-HA1 -PVNPs can be administered to improve survival and reduce body weight loss following challenge with a virulent, H1N1 influenza virus,

[0032] In aspects, the influenza vaccine comprises an S60 PVNP that comprises a modified S domain displaying an HA1 domain from the Hl/pdm from the H1N1 virus strain. The Hl/pdm protein refers to the hemagglutinin (HA1) subunit of the H1N1 "pandemic" influenza strain; Hl refers to the subtype of hemagglutinin (HA). In aspects, the influenza vaccine comprises an S60 PVNP that comprises a modified S domain displaying an HA1 domain from the H1/PR8 from the H1N1 vims strain. In aspects, the influenza vaccine comprises an S60 PVNP that comprises a modified S domain displaying an HA1 domain from the H3 from the H3N2 virus strain. In aspects, the influenza vaccine comprises an S60 PVNP that comprises a modified S domain displaying HA1 domain from the influenza B virus strain.

S60 PVNP

[0033] The disclosed vaccine compositions employ a non-replicating pseudovirus nanoparticle

(PVNP) platform referred to as “S60 PVNP,” comprising 60 modified S domains derived from the capsid protein of norovirus. These domains self-assemble into a stable icosahedral structure, displaying antigens such as the influenza HA1 antigen on the nanoparticle surface for immunization. The platform enhances the immunogenicity of otherwise weakly immunogenic antigens by multivalent display on the PVNP.

[0034] In aspects, the S60 PVNP comprises a modified S domain as described in, for example, U.S. Patent No. 11,833,198 B2, WO 2024/102697 Al, U.S. Patent Application Publication No. 2024/0131145, and which is further described herein. In brief, many defined neutralizing antigens face a common problem of low immunogenicity for non-replicating vaccine development, due to their small sizes with low valences. This problem can be solved via fusion or conjugation of the antigens to a large, polyvalent protein platform for enhanced immunogenicity. Significantly enhanced immunogenicity of antigens can be achieved after displaying via the S60 PVNP as a vaccine platform. The S60 particles can be easily produced, are stable, and are highly immunogenic toward the displayed antigen.

[0035] The S60 PVNP may be referred to interchangeably as a “pseudovirus-like nanoparticle” or “S60-PVNP.” The nanoparticle may be tag-free or may include tags (e.g., HIS tag) for purification purposes. In some embodiments, the PVNP has a diameter of about 20-40 nm. The polyvalent S60 particle with 60 exposed S domain C-termini offers an ideal platform for antigen presentation, leading to enhanced immunogenicity to the displayed antigen for vaccine development. A chimeric S60 particle displaying 60 antigenic proteins can be produced and used to elicit an immunogenic response.

[0036] The S60 PVNP typically comprises an aggregate of 60 modified S domain proteins that self-assemble into a shell with icosahedral symmetry. In some aspects, the S domain comprises or consists of a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1:

MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFV QAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTA GKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIK LIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVE (SEQ ID NO: 1). In aspects, the modified S domain used in the S60 PVNP platform may comprise a sequence having at least 90% sequence identity to SEQ ID NO: 1. In particular, the S60 PVNP may comprise a modified S domain with increasing sequence identities to SEQ ID NO: 1, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In aspects, the S domain is modified, but the hinge region FLVPPTVE (SEQ ID NO: 11) is conserved.

[0037] The S domain is modified to enhance stability and eliminate protease cleavage sites (e.g., via introduction of the R69A mutation). Unlike prior particles containing disulfide- stabilizing mutations (e.g., V57C, Q58C, S136C), the presently disclosed PVNP may be wildtype at positions 57, 58, and 136. In aspects, the S particle comprises a modified S domain that comprises a R69A mutation but is wildtype at one or more of positions V57, Q58, or S136, and M140. In aspects, the modified S domain comprises a R69A mutation but is wildtype at all positions V57, Q58, or S136, and M140.

[0038] The modified S domain sequence of SEQ ID NO: 1 includes a start codon “M.” In certain aspects, the stall codon “M” can be omitted, such as where a signal peptide is appended to the modified S domain sequence of SEQ ID NO: 1 .

[0039] In some embodiments, the PVNP comprises a chimeric fusion protein comprising a modified S domain, a peptide linker, and an HA1 influenza antigen. The linker joins the C-terminus of the S domain to the N-terminus of the HA1 antigen. Upon self-assembly, the nanoparticle displays 60 exposed C-termini arranged in a triangular pattern, each displaying an HA1 antigen on the nanoparticle surface.

[0040] The PVNP may comprise (display) one or more HA1 influenza antigen types as described herein. In one aspect, the PVNP comprises an HA1 influenza antigen. In aspects, the PVNP comprises an HA1 influenza antigen and is capable of eliciting HA1 influenza antigenspecific IgA and/or IgG antibodies in an individual administered the influenza vaccine. In aspects, the PVNP comprises 60 sites for antigen presentation. Linker

[0041] In aspects, the S60 PVNP may include a linker that operatively connects the antigen to the S domain. In aspect, the linker comprises an amino acid sequence of a length sufficient to provide space and certain flexibility between the S domain protein particle and the displayed antigens. The linker is typically a short peptide of one to ten amino acid units, or three to six amino acids, that connect the C-terminus of the S domain to the displayed antigens. The linker provides space and certain flexibility between the S60 particle and the displayed antigens, which helps the independent folding of the S domain and the displayed antigens. A longer linker may be used as necessary. The amino acid length of the linker should be sufficient to allow flexibility of the protein domains to form the claimed compositions. In one aspect, the non-replicating S60-PVNP may comprise a linker having a sequence selected from HHHH (SEQ ID NO: 2), GGGG (SEQ ID NO: 3), GSGS (SEQ ID NO: 4), HHHHHH (SEQ ID NO: 5) and SSRVDGGGGG (SEQ ID NO: 6). In one aspect, the linker may comprise the sequence GGGG (SEQ ID NO: 3). In one aspect, the linker may comprise the sequence SSRVDGGGGG (SEQ ID NO: 6. In aspects, the linker comprises or consists of GGGG (SEQ ID NO: 3). In aspects, the linker comprises or consists of SSRVDGGGGG (SEQ ID NO: 6).

HA1 Antigens

[0042] In one aspect, a non-replicating influenza pseudovirus nanoparticle (PVNP) comprises a modified S domain and an HA1 influenza antigen, with the HA1 antigen displayed on the surface of the nanoparticle (referred to herein as “S60-HA1-PVNP”). The S60-HA1-PVNP can be formed by the self-assembly of fusion proteins comprising a modified S domain and an influenza HA1 antigen, which may further comprise a linker. In certain aspects, the S60-HA1-PVNP induces an immune response to influenza infection. In some embodiments, administration of the S60-HA1- PVNP reduces the severity or likelihood of one or more symptoms of influenza infection in a subject. [0043] In aspects, the influenza HA1 antigen is fused to (operatively linked, or, in other aspects, contiguous with) the S domain. In aspects, the influenza HA1 antigen is operatively connected to the S domain via a linker, as described herein. In aspects, the HA1 influenza antigen is operatively connected to the S domain via any amino acid sequence which allows for presentation of the antigen.

[0044] In certain aspects, the HA1 antigen of the S60-HA1-PVNP is derived from the H1N1 pandemic strain (Hl/pdm) and comprises the following sequence:

CKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEEL REQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPK LSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKV RDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGS GIIISDTPVHD (SEQ ID NO: 7).

[0045] In aspects, the influenza HA1 antigen of the S60-HA1-PVNP is that of HA1 antigens from the H1N1 PR8 strain (H1/PR8) having the following sequence:

CRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELR EQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKN SYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVR DQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHE (SEQ ID NO: 8).

[0046] In aspects, the influenza HA1 antigen of the S60-HA1-PVNP is that of HA1 antigens from the H3N2 influenza virus (H3) having the following sequence:

CDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRS LVASSGTLEFNNESFNWTGVTQNGTSSACKRGSNNSFFSRLNWLTHLKFKYPALNVTM PNNEKFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSR ISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGK (SEQ ID NO: 9). [0047] In aspects, the influenza HA1 antigen of the S60-HA1-PVNP is that of HA1 antigens from the influenza B virus (IBV) having the following sequence:

CPNCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRG YENIRLSTQNVINAEKAPGGPYRLGTSGSCPNATSRSGFFATMAWAVPGDNNKTATGPL TVEVPYICTKGEDQITVWGFHSDSKTRMRSLYGDSNPQKFTSSANGVTTHYVSQIGGFP DQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIG (SEQ ID NO: 10).

[0048] It will be understood that antigen sequences used to generate the antigen peptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference sequence, provided that the resulting antigen elicits at least a partial immune response in an individual administered the composition having the antigen.

Fusion Proteins

[0049] As described above, the S60 PVNPs can be formed by the self-assembly of fusion proteins. Disclosed are various fusion proteins that may be used to form the S60 PVNPs for use in vaccine compositions. The fusion proteins can be used for the manufacture of a vaccine composition comprising an S60 PVNP that displays the influenza antigen, such as a HA1 antigen as described herein. In aspects, the fusion proteins aggregate to form the antigen-presenting PVNP, which serves as the basis of a vaccine composition to elicit an immune response against the selected antigen in an individual administered the composition.

[0050] In general, the fusion proteins used to form the S60 PVNPs comprises a S domain and an HA1 influenza antigen. The fusion proteins may further include a linker connecting the S domain to the HA1 influenza antigen. In aspects, the S domain is a modified S domain.

[0051] In aspects, the disclosed fusion proteins comprise a modified S domain, an HA antigen, and a linker. In general, the fusion protein may comprise, from the N-terminus to the C-terminus direction, a norovirus (NoV) S domain, a linker, and an HA1 influenza antigen sequence. The fusion protein may comprise a modified S domain having a mutation to the trypsin site as described above; a linker protein domain operatively connected to the modified S domain; and an antigen domain operatively connected to the linker. Expression of the fusion proteins may be carried out using methods known in the art and as disclosed herein.

[0052] For example, the fusion proteins may comprise the modified S-domain comprising the hinge region of norovirus, a linker, and an HA1 influenza antigen sequence as described herein. In aspects, the hinge region comprises or consists of the sequence FLVPPTVE (SEQ ID NO: 11).

H1N1 2009 Pandemic Strain Fusion Protein

[0053] As previously described, the S domain is conjugated to an HA1 influenza antigen and assembled into an S60 PVNP platform, which presents the antigen for immunization following administration to an individual. The S60 PVNP platform is formed through the self-aggregation of the fusion protein. Thus, in aspects, the disclosed S60 PVNPs are formed from one or more of the disclosed fusion proteins.

[0054] In aspects, the vaccine composition comprises an S60 PVNP displaying an HA1 from the H1N1 2009 Pandemic Strain. In this aspect, the fusion protein forming the platform comprises a S domain, such as a modified S domain, and an HA1 influenza antigen, wherein the HA1 influenza antigen is the HA1 antigen from that of the Hl/pdm H1N1 virus strain. For example, the Hl/pdm strain can be that of the A/H1N1 2009 pandemic strain (GenBank AC#: ACP41105.1, A/Califomia/04/2009/H1N1, amino acid C59 to D291, 233 aa), referred as pandemic or PDM. In aspects, the HA1 antigen portion of the fusion protein is derived from the above-referenced GenBank AC#: ACP41105.1 sequence that has been codon-optimized for Escherichia coli 33T expression systems. For example, in aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 95% homology to MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFV QAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTA GKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIK LIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPrVEHHHHCKLRGVAPLHL GKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFER FEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKE VLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYY WTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDHHHHHH (SEQ ID NO: 12), in which the hinge region is FLVPPTVE (SEQ ID NO: 11) and the linker HHHH (SEQ ID NO: 2) is in double underline. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 96% homology to SEQ ID NO: 12. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 97% homology to SEQ ID NO: 12. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 98% homology to SEQ ID NO: 12. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 99% homology to SEQ ID NO: 12. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having 100% homology to SEQ ID NO: 12.

H1N1 A/H1N] PR8 Strain Fusion Protein

[0055] In aspects, the vaccine composition comprises an S60 PVNP displaying an HA1 from the H1/PR8 H1N1 virus strain. In this aspect, the fusion protein forming the platform comprises a S domain, such as a modified S domain, and an HA1 influenza antigen, wherein the HA1 influenza antigen is the HA1 antigen from that of the H1/PR8 H1N1 strain. In aspects, the H1/PR8 H1N1 strain is A/H1N1 PR8 strain (GenBank AC#: EF467821.1, A/Puerto Rico/8/1934/HlNl, C59 to E290, 232 aa), referred as PR8. In aspects, the HA1 antigen portion of the fusion protein is derived from the above-referenced GenBank AC#: EF467821.1 sequence that has been codon-optimized for Escherichia coli 33T expression systems. For example, in aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 95% homology to MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFV QAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTA GKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIK LIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPrVEHHHHCRLKGIAPLQLG KCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERF EIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEV LVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYY WTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHEHHHHHH (SEQ ID NO: 13), in which the hinge region is FLVPPTVE (SEQ ID NO: 11) and the linker HHHH (SEQ ID NO: 2) is in double underline. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 96% homology to SEQ ID NO: 13. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 97% homology to SEQ ID NO: 13. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 98% homology to SEQ ID NO: 13. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 99% homology to SEQ ID NO: 13. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having 100% homology to SEQ ID NO: 13.

A/H3N2 Strain Fusion Protein

[0056] In aspects, the vaccine composition comprises an S60 PVNP displaying an HA1 from the H3N2 virus strain. In this aspect, the fusion protein forming the platform comprises a S domain, such as a modified S domain, and an HA1 influenza antigen, wherein the HA1 influenza antigen is the HA1 antigen from that of “H3” from the H3N2 virus strain. In aspects, the H3 strain is A/H3N2 strain (GenBank AC#: ABJ53460.1, A/Westem Australia/69/2005/H3N2, amino acid C68 to K292, 225 aa), referred as H3. In aspects, the HA1 antigen portion of the fusion protein is derived from the above-referenced GenBank AC#: ABJ53460.1 sequence that has been codon- optimized for Escherichia coli 33T expression systems. For example, in aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 95% homology to MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFV QAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTA GKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIK LIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPrVEHHHHCDSPHQILDGEN CTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNN ESFNWTGVTQNGTSSACKRGSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIW GVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDI LLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKHHHHHH (SEQ ID NO: 14), in which the hinge region is FLVPPTVE (SEQ ID NO: 11) and the linker HHHH (SEQ ID NO: 2) is in double underline. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 96% homology to SEQ ID NO: 14. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 97% homology to SEQ ID NO: 14. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 98% homology to SEQ ID NO: 14. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 99% homology to SEQ ID NO: 14. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having 100% homology to SEQ ID NO: 14.

B/Yamagata Strain Fusion Protein

[0057] In aspects, the vaccine composition comprises an S60 PVNP displaying an HA1 from the Influenza B strain. In this aspect, the fusion protein forming the platform comprises a S domain, such as a modified S domain, and an HA1 influenza antigen, wherein the HA1 influenza antigen is the HA1 antigen from that of the influenza B virus strain. In aspects, the influenza B strain is a B/Yamagata stain (GenBank AC#: AAD02807.1, B/Yamagata/ 16/88, amino acid C54 to G290, 237 aa), referred as IBV. In aspects, the HA1 antigen portion of the fusion protein is derived from the above-referenced GenBank AC#: AAD02807.1 sequence that has been codon-optimized for Escherichia coli 33T. For example, in aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 95% homology to MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFV QAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTA GKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIK LIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPrVEHHHHCPNCLNCTDLD VALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYENIRLSTQNVI NAEKAPGGPYRLGTSGSCPNATSRSGFFATMAWAVPGDNNKTATGPLTVEVPYICTKG EDQITVWGFHSDSKTRMRSLYGDSNPQKFTSSANGVTTHYVSQIGGFPDQTEDGGLPQS GRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGHHHHHH (SEQ ID NO: 15), in which the hinge region is FLVPPTVE (SEQ ID NO: 11) and the linker HHHH (SEQ ID NO: 2) is in double underline. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 96% homology to SEQ ID NO: 15. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 97% homology to SEQ ID NO: 15. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 98% homology to SEQ ID NO: 15. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having at least 99% homology to SEQ ID NO: 15. In further aspects, the fusion protein comprises, consists of, or consists essentially of a sequence having 100% homology to SEQ ID NO: 15.

Vaccine Compositions

[0058] In aspects, the vaccine compositions may further comprise one or more pharmaceutically-acceptable carriers, for example, a solvent, dispersion media, coating, stabilizing agent, diluent, preservative, antibacterial and/or antifungal agent, isotonic agent, adsorption delaying agent, adjuvant, or combinations thereof. In aspects, the administered composition comprises a pharmaceutically acceptable carrier. In aspects, the administered composition comprises one or more of an adjuvant and a preservative.

[0059] In one aspect, the vaccine composition comprises physiological saline. Optionally, a protectant may be included, for example, an anti-microbiological active agent, such as for example Gentamycin, Merthiolate, and the like. Stabilizing agents which may be used include saccharides, trehalose, mannitol, saccharose and the like, which may be added in an amount sufficient to increase and/or maintain product shelf-life. The disclosed herein may include known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as, for example, saline or a corresponding plasma protein solution may be used. Exemplary diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Exemplary isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Exemplary stabilizers may include albumin and alkali salts of ethylenediaminetetraacetic acid, among others.

[0060] In one aspect, disclosed is a container, for example a container suited for delivery to an individual in need thereof, for example a capsule, a vial, or a syringe, comprising at least one dose of the immunogenic composition as disclosed herein. The container may comprise, for example, 1 to 250 doses of the immunogenic composition, or in other aspects, 1, 10, 25, 50, 100, 150, 200, or 250 doses of the immunogenic composition. In one aspect, each of the containers may comprise a single dose of the vaccine composition, or more than one dose of the vaccine composition and may further comprises an anti-microbiological active agent. Those agents may include, for example, antibiotics such as Gentamicin and Merthiolate and the like.

Kits

[0061] A further aspect relates to a kit. The kit may comprise any of the containers described above and an instruction manual, including the information for the delivery of the immunogenic composition disclosed above. For example, instructions related to intramuscular application of at least one dose may be provided for lessening the severity of clinical symptoms associated with an infection of an antigen as disclosed here. The kits and/or compositions may further include an immune stimulant such as keyhole limpet hemocyanin (KLH), or incomplete Freund's adjuvant (KLH/ICFA). Any other immune stimulant known to a person skilled in the art may also be used. In one aspect, the adjuvant is aluminum hydroxide.

Methods of Making

[0062] In one aspect, a method of making the disclosed polyvalent icosahedral structures is disclosed. The method may comprise the steps of a) making a first region comprising a modified NoV S domain protein, wherein the modification comprises a mutation sufficient to destruct an exposed protease cleavage site (wherein the mutation prevents protein degradation), preferably an R69A mutation, and b) recombinantly expressing the first region having a modified NoV S domain protein with a linker and an antigen. In certain aspects, the PVNP composition may be effectively produced in E.coli. In aspects, the PVNP composition is produced via a prokaryotic system. In aspects, the PVNP composition is produced via a eukaryotic system. In aspects, the PVNP composition is produced via both a prokaryotic system and a eukaryotic system.

Method of Immunizing an Individual

[0063] Further provided are methods for treating an individual using the disclosed vaccine compositions for improving an immune response to an influenza virus. For example, the disclosed vaccine compositions may be administered to an individual for immunization against one or more of an H1N1 influenza infection, an H3N2 influenza infection, an influenza B infection and combinations thereof. In aspects, the HA1 antigen comprises a sequence derived from a specific influenza strain selected from H1N1, H3N2, or influenza B lineages. In aspects, the HA1 antigen is a consensus or mosaic sequence derived from multiple influenza strains.

[0064] In aspects, the method of reducing the severity of an influenza infection and/or eliciting an immune response in a subject against influenza in an individual in need thereof is disclosed, the method comprising administering an S60 PVNP comprising an HA1 influenza antigen, or vaccine composition containing an S60 PVNP comprising an HA1 influenza antigen as disclosed herein to the individual.

[0065] In one aspect, a method of immunizing an individual in need thereof against an influenza infection is disclosed. In aspects, the individual may be a human subject at increased risk of influenza infection, such as an elderly individual, an immunocompromised individual, a healthcare worker, or a pediatric subject. The method may further comprise identifying the subject as having an increased risk of severe influenza-related complications prior to administration. [0066] In one aspect, a method of inducing an antigen specific immune response in an individual is disclosed, the method comprising administering any of the vaccine compositions described herein to an individual in an amount effective to produce an antigen specific immune response. In some aspects, the antigen specific immune response may comprise a T cell response. In some aspects, the antigen specific immune response may comprise a B cell response.

[0067] In aspects, the method comprises boosting a pre-existing immune response in a previously vaccinated or previously infected individual.

[0068] In some aspects, the vaccine is administered to the individual via a route selected from intramuscular administration, intradermal administration and subcutaneous administration. In some aspects, the administering step comprises contacting a muscle tissue of the individual with a device suitable for injection of the composition. In some aspects, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition in combination with electroporation.

[0069] In some aspects, the vaccine composition may be administered to the subject by intradermal or intramuscular injection. The method may comprise administering an S60 PVNP comprising an HA1 influenza antigen, or vaccine composition containing an S60 PVNP comprising an HA1 influenza antigen as disclosed here. In one aspect, the administration may be sufficient to induce a neutralizing antibody titer against the antigen. The disclosed an S60 PVNP comprising an HA1 influenza antigen, or vaccine composition containing an S60 PVNP comprising an HA1 influenza antigen may be used to elicit an immune response in an individual in need thereof, comprising administering an S60 PVNP comprising an HA1 influenza antigen, or vaccine composition containing an S60 PVNP comprising an HA1 influenza antigen disclosed herein, to the individual.

[0070] In some aspects, the method may comprise a single administration of the vaccine composition. In some aspects, the method may further comprise administering a booster dose of the vaccine. In aspects, the administration comprises one dose, or two doses, or three doses of the influenza vaccine.

[0071] In aspects, the method comprises co-administering the S60 PVNP vaccine composition with one or more other vaccines, such as a SARS-CoV-2 vaccine, a pneumococcal vaccine, or a Tdap vaccine.

[0072] In aspects, the composition is administered to an individual via a route selected from one or more of intradermal injection, intramuscular injection, or by intranasal administration. In aspects, the composition is administered using a microneedle patch, a transdermal patch, or an oral formulation.

[0073] In some aspects, the method further comprises evaluating protection against influenza vims infection, reduction in clinical symptoms, viral load, or hospitalization following administration.

Dosing

[0074] In one aspect, provided are methods comprising administering the vaccine compositions to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.

[0075] In certain aspects, the compositions may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic or prophylactic effect. The dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain aspects, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens may be used. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.

[0076] In some aspects, the method comprises administering the vaccine composition in a prime-boost regimen at intervals of 2 to 12 weeks. In certain aspects, the vaccine composition is formulated for sustained release or depot delivery.

Dosage Forms

[0077] The vaccine composition described herein may be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).

[0078] Liquid Dosage Forms

[0079] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain aspects for parenteral administration, compositions may be mixed with solubilizing agents, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.

[0080] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated and may include suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0081] Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0082] Pulmonary Dosage Forms

[0083] Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation may be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

[0084] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.

[0085] Multi-Dose and Repeat-Dose Administration

[0086] In some aspects, the vaccine compositions may be administered in two or more doses (referred to herein as “multi-dose administration”). Such doses may comprise the same components or may comprise components not included in a previous dose. Such doses may comprise the same mass and/or volume of components or an altered mass and/or volume of components in comparison to a previous dose. In some aspects, multi-dose administration may comprise repeat-dose administration. As used herein, the term “repeat-dose administration” refers to two or more doses administered consecutively or within a regimen of repeat doses comprising substantially the same components provided at substantially the same mass and/or volume. In some aspects, subjects may display a repeat-dose response. As used herein, the term “repeat-dose response” refers to a response in a subject to a repeat-dose that differs from that of another dose administered within a repeat-dose administration regimen. In some aspects, such a response may be the expression of a protein in response to a repeat-dose comprising the vaccine composition.

[0087] In one aspect, a method of eliciting an immune response, in particular, an immune response to an HA1 influenza antigen, sufficient to deter, prevent, decrease the likelihood of obtaining, reduce the severity of, and/or mitigate an influenza infection, in an individual in need thereof is disclosed. In this aspect, the method may include the step of administering a vaccine composition as disclosed above to an individual in need thereof. The disclosed compositions may be administered to an individual according to any method known in the art. The vaccine compositions may be administered prophylactically to an individual suspected of having a future exposure to the antigen incorporated into the vaccine composition. In certain aspects, provided is a method of providing an immune response that protects an individual receiving the composition from infection, or reduces or lessens the severity of the clinical symptoms associated from an infection. Dosage regimen may be a single dose schedule or a multiple dose schedule (e.g., including booster doses) with a unit dosage form of the composition administered at different times. The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the antigenic compositions disclosed herein in an amount sufficient to produce the desired effect, which compositions are provided in association with a pharmaceutically acceptable excipient (e.g., pharmaceutically acceptable diluent, carrier or vehicle). The vaccine may be administered in conjunction with other immunoregulatory agents.

EXAMPLES

[0088] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

[0089] The following example demonstrates that bioengineered pseudovirus nanoparticles (PVNPs) displaying the receptor-binding HA1 domains of influenza viruses elicit strong HA1- specific antibody responses and protect against influenza virus challenges.

[0090] The continually high disease burden of influenza and the relatively low effectiveness of current influenza vaccines call for enhanced vaccine strategies. Applicant previously generated unique S-HA1 pseudovirus nanoparticles (PVNPs) displaying the receptor binding HA1 antigens of the H7N9 subtype as an influenza vaccine candidate and characterized their features in biochemistry, biophysics, structure, and immune response. In this follow up study, new S-HA1 PVNPs displaying the HA1 antigens of other common influenza viruses, including two H1N1 strains, one H3N2 strain, and an influenza B virus, respectively, were created. It was found that the recombinant PVNPs react well with antibodies against hemagglutinins (HAs) or mouse sera obtained after influenza virus challenge. 3D structural models were constructed to comprehend the structural features and size variations of the S-HA1 PVNPs. The PVNPs are immunogenic, eliciting high titers of HAl-specific serum antibodies that recognize commercial HA1 proteins. Importantly, the S-HA1 PVNP representing the H1N1 PR8 strain provided mice with 100 % protection against mortality caused by challenge with the mouse-adapted influenza virus of the same PR8 strain. The S-HA1 PVNP representing the H1N1 2009 pandemic strain conferred mice with 50 % protection against mortality caused by challenge with the 1934 PR8 strain, despite the two strains circulating 75 years apart. The data demonstrated the feasibility of generating S-HA1 PVNPs to display HA1 antigens of diverse influenza A and B viruses. The readily available S- HA1 PVNPs hold promise as influenza vaccines, presenting a novel approach to combat the deadly influenza disease.

[0091] Influenza viruses typically exhibit a spherical or filamentous shape, with sizes ranging from 100 to over 300 nm. Each influenza virion is encapsulated by a protein capsid and a lipid envelope, containing a single-stranded, negative-sense RNA genome. Two spike proteins, hemagglutinin (HA) and neuraminidase (NA), decorate the viral surface by extending beyond the protein capsid and lipid envelope. Distinct numbers are assigned to HAs and NAs to identify IAV subtypes based on the combinations of HA and NA. The HA spike glycoprotein plays a crucial role in the early stages of virus infection. First, the virion recognizes and binds to host receptors, typically the sialic acid portions of sialoglycans on the epithelial cells in the host respiratory track, facilitating viral attachment. Subsequently, the virion liberates its genome into the cytoplasm through membrane fusion with the host cells. Therefore, the HA spike is an important vaccine target.

[0092] The HA spike protein is composed of two main domains: the HA1 domain, which forms the distal head of the HA spike protein, and the HA2 domain, which constitutes the stalk region of the spike. HA1 is responsible for binding to the host receptor to initiate viral infection and serves as the immunodominant antigen. Compared with HA2 stalk, the HA1 sequences are more variable, playing a major role in the antigenic drifts and epochal evolution of influenza viruses. Nevertheless, conserved antigenic supersites were identified at or near the receptor-binding site of the HA1 domains, and these conserved antigenic supersites have been demonstrated to be targets of broadly neutralizing antibodies [5-7]. In addition, epitopes with sequences of limited variability were found neighboring the receptor-binding site and have been shown to be protective against influenza virus infection [8], These epitopes have been proposed as targets for a universal influenza vaccine. Applicant previously created a unique pscudovirus nanoparticlc (PVNP) named S-HA1 PVNP [9], for displaying the HA1 antigens of an avian H7N9 influenza virus on the norovirus S nanoparticle [10]. Significantly, the PVNP displayed H7 HAls retained their trimer formation, were recognized by H7 specific antibody, bound to sialoglycan receptors with 2,3 linked sialic acids, and agglutinated human red blood cells [9]. Importantly, the S-HA1 PVNP was immunogenic, eliciting strong titers of HAl-specific antibody that showed high cross-subtype hemagglutination inhibition titers. Here, Applicant further demonstrated that the PVNP technology, established based on the H7N9 virus, can be utilized to generate new S-HA1 PVNPs displaying HA1 antigens of other common influenza virus strains, including those from H1N1 and H3N2 subtypes, as well as IBV lineages. Notably, it was demonstrated for the first time that the S-HA1 PVNPs protected mice against mortality and body weight loss caused by challenge with homologous and heterologous influenza viruses.

[0093] Materials and methods

[0094] Plasmids for the expressions of the S-HA1 fusion proteins

[0095] DNA sequences encoding HA1 antigens, each representing the A/H1N1 2009 pandemic strain (GenBank AC#: ACP41105.1, A/California/04/2009/H1N1, amino acid C59 to D291, 233 aa), referred as pandemic or PDM, having the following sequence: CKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEEL REQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPK LSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKV RDQEGRMNYYWTLVEPGDKITFE ATGNLV VPRYAFAMERN AGS GIIISDTPVHD (SEQ ID NO: 7); the A/H1N1 PR8 strain (GenBank AC#: EF467821.1, A/Puerto Rico/8/1934/HlNl, C59 to E290, 232 aa), referred as PR8, having the following sequence: CRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELR EQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKN SYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVR DQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHE (SEQ ID NO: 8); an A/H3N2 strain (GenBank AC#: ABJ53460.1, A/Westem Australia/69/ 2005/H3N2, amino acid C68 to K292, 225 aa), referred as H3, having the following sequence: CDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRS LVASSGTLEFNNESFNWTGVTQNGTSSACKRGSNNSFFSRLNWLTHLKFKYPALNVTM PNNEKFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSR ISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGK (SEQ ID NO: 9); and a B/Yamagata stain (GenBank AC#: AAD02807.1, B/Yamagata/16/88, amino acid C54 to G290, 237 aa), referred as IBV, having the following sequence: CPNCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRG YENIRLSTQNVINAEKAPGGPYRLGTSGSCPNATSRSGFFATMAWAVPGDNNKTATGPL TVEVPYICTKGEDQITVWGFHSDSKTRMRSLYGDSNPQKFTSSANGVTTHYVSQIGGFP DQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIG (SEQ ID NO: 10), were codon-optimized to Escherichia coli 33T and synthesized by GenScript (Piscataway, NJ). Each of these DNA fragments was cloned into the previously constructed pET- 24b (Novagen)-based plasmid for the production of the S-HA1 fusion protein of an H7N9 influenza virus [9]. This process involved replacing the HA1 H7 -encoding sequences with the newly synthesized HAl-encoding DNA fragments, respectively. A linker (HHHH, (SEQ ID NO: 2)) was present between the S and the HA1 domains with a His tag at the C-terminus of the HA1 domain (FIG. 1, panel A).

[0096] Recombinant S-HA1 protein production

[0097] His-tagged S-HA1 proteins were expressed in E. coli (BL21/DE3 strain) and purified under denaturing conditions using a protocol described in the HisPur™ Cobalt resin manual (Thermo Fisher Scientific) as described previously [11], Briefly, following overnight induction with 0.4 mM IPTG (isopropyl P-D-l-thiogalactoyranoside), the bacteria were harvested and subsequently resuspended with 6 M guanidine hydrochloride (GnHCl) in phosphate buffer-NaCl (50 mM NaPCU, 300 mM NaCl, pH 7.2). After sonication, His-tagged S-HA1 proteins in the clarified bacterial lysate supernatant were purified using cobalt resins, which bind to the His-tag. For refolding, the purified proteins were sequentially dialyzed overnight against phosphate buffer (50 mM NaPCE, 300 mM NaCl, pH 7.4) containing 6 M, 4 M, 2 M and 1 M urea, respectively, to remove the GnHCl and reduce the urea in the protein solutions. Finally, the protein in phosphate buffer with 1 M urea was dialyzed against Redox buffer (100 mM Tris, 400 mM L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 1 mM oxidized glutathione, pH 8.0) for 16 hours. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Purified proteins were analyzed by SDS-PAGE using 12 % separating gels, as described elsewhere [12],

[0098] Gel filtration chromatography [0099] This method was conducted to evaluate PVNP formations of the S-HA1 proteins, as described previously [13-15]. The analyses were performed using an A’ KTA Fast Performance Liquid Chromatography system (FPLC, A"KTA pure™ 25 L, GE Healthcare Life Sciences) equipped with a size exclusion column (Superdex 200, 10/300 GL, 25 mL bed volume, GE Healthcare Life Sciences). The column was calibrated using gel filtration calibration kits (GE Healthcare Life Sciences). The elution peaks corresponding to the S-HA1 PVNPs and the S-HA1 monomers were determined using the previously made S-HA1 H7 PVNP (-3.12 MDa) [9] and GST-dimers (54 kDa) [16]. The relative concentrations of proteins in the effluent were measured via A280 absorbance.

[0100] Electron microscopy

[0101] The morphology of the S-HA1 PVNPs was examined by negative stain transmission electronic microscopy (TEM), following a procedure described previously [10]. In brief, purified S-HA1 PVNPs were applied to grids (FCF200-CV-50, Electron Microscopy Sciences). After negative staining with 1 % ammonium molybdate, the air-dried grids were observed using a Hitachi microscope (model H-7650) at 80 kV, with magnifications ranging from 15,000x to 40,000x.

[0102] Structural modeling of the S-HA1 PVNPs

[0103] Three-dimensional (3D) structural models of the S-HA1 Hl PDM PVNPs in T = 1, T = 3, and T = 4 icosahedral symmetries were generated using UCSF ChimeraX software (version 1.7) [17], as previously described [11]. The electron density map obtained from cryogenic electron-microscopy (cryoEM) of the S60-HA1 H7 PVNP [9] served as a template. Known structures of norovirus inner shells, each representing T= l, T = 3, orT = 4 icosahedral symmetries [11,18-20], and the HA1 protein trimers (PDB code: 3LZG) of the H1N1 influenza virus (A/California/04/2009/H1N1) were docked into the respective regions of the template. The structural analysis of the S-HA1 Hl PDM PVNP models and the generation of the model images were also performed using UCSF ChimeraX. [0104] Binding of the S-HA1 PVNPs to HA-specific antibodies

[0105] Antigenic reactivity of the S-HA1 PVNPs to HA-specific antibodies was measured by enzyme immunoassays (EIAs). Briefly, the S-HA1 PVNPs at ~2.5 pg/mL were coated onto 96- well microtiter plates. After blocking with 5 % skim milk, the coated PVNPs were incubated with one of the three antibodies: 1) rabbit anti-HINl pandemic strain (California/ 04/2009) HA (Sino Biological, Inc.) at a 1:2000 dilution; 2) rabbit anti-H3N2 (A/Brisbane/10/2007) HA (Sino Biological, Inc.) at a 1:2000 dilution; or 3) in-house-made mouse serum obtained after challenge with the mouse-adapted H1N1 influenza virus PR8 strain (A/Puerto Rico/8/1934) at a 1:5000 dilution. The bound antibodies were detected by horseradish peroxidase (HRP)-conjugated secondary antibodies: goat anti-rabbit IgG (MP Biomedicals) at a 1:5000 dilution or goat antimouse IgG (MP Biomedicals) at a 1:5000 dilution.

[0106] BALB/c mice from the Jackson Laboratory (Bar harbor, ME) were maintained under pathogen free conditions at the Division of Veterinary Services of Cincinnati Children’s Hospital Medical Center (CCHMC). 40 female mice at -6 weeks of age were randomly divided into five groups with 8 mice in each group (n = 8). Mice in each group were vaccinated with one of following immunogens: (1) the S-HA1 PVNPs of the A/ H1N1/California/04/2009 strain (referred to as S-HA1 Hl PMD); (2) the S-HA1 PVNPs of the A/HINl/Puerto Rico/8/1934 strain (S-HA1 Hl PR8); (3) the S-HA1 PVNPs of the A/H3N2/Westem Australia/69/2005 strain (S-HA1 H3); (4) the S-HA1 PVNPs of the B/Yamagata/16/88 strain (S-HA1 IBV); and (5) the S60 PVNPs as platform control (S). The immunogens at 10 pg/mouse/dose were mixed with Imject Alum Adjuvant (Thermo Fisher Scientific, aluminum hydroxide, 40 mg/mL) at 1:1 volume ratio as described previously [21]. The ready-to-use immunogens were administered intramuscularly in the thigh muscle at volume of -80 pL/mouse/dose for three times at 2- week intervals. Blood was collected two weeks after the second and the third immunization via tail veins [22], respectively, for serum sample preparations using an established protocol [22] .

[0107] Determination of HA 1- specific IgG titers [0108] HA 1 -specific IgG titers in mouse sera after immunization with the S-HA1 PVNPs were assessed using EIAs, as described elsewhere [9]. Recombinant HA1 proteins corresponding to the HA1 antigens of Hl, H3, and IBV influenza viruses in our S-HA1 PVNPs were purchased from Sino Biological as capture antigens. The HA1 proteins were coated to 96-well microtiter plates at ~2 ng/pL. After blocking of the coated antigens with 5 % nonfat milk, mouse sera at serial twofold dilutions were added. The bound IgG was detected by goat-anti-mouse IgG-HRP conjugates (1:5000, MP Biomedicals). The HAl-specific IgG titers were defined as the highest dilutions of sera displaying positive signals (OD450 > 0.2).

[0109] Mouse influenza virus challenge model

[0110] This was essentially performed as described previously [15, 22, 34], Briefly, the five mouse groups were immunized with the different S-HA1 PVNPs and the S60 PVNP control, and their serum samples were collected as described above. Three weeks after the third immunization, the immunized mice were challenged with the mouse-adapted influenza virus PR8 (H1N1) strain at the 100% lethal dose (LD100), which was 50 pL of the virus stock at approximately 2.4 x 10⁶ plaque forming units per mL (PFU/mL). The viruses were administered intranasally with 25 pL to each nostril of the mice under general anesthesia. Body weights, survival rates, and morbidity of the mice were monitored daily for 10 days. A weight loss equal to or exceeding 20 % served as the endpoint for euthanizing moribund mice, as required by the animal use protocol.

[0111] Statistical analysis

[0112] Statistical comparisons between two sets of data were conducted using GraphPad Prism 10.0 (GraphPad Software, Inc.) through unpaired t-test. Data groups of survival rates were analyzed for statistical differences using Log-rank (Mantel-Cox) test. Statistical comparisons were considered non- significant (ns) if P-values were > 0.05, significant (*) if P-values were < 0.05, highly significant (**) if P-values were < 0.01, and extremely significant if P-values were < 0.001 (***), or < 0.0001 (****), respectively.

[0113] Results [0114] Production of the S-HA1 fusion proteins

[0115] The HA1 domains of four influenza viruses, two (Hl/pdm and Hl/ PR8) representing the H1N1 subtype, one (H3) representing the H3N2 subtype, and one (IBV) representing an IBV lineage, were individually fused to the norovirus shell (S) domain, each with a C-terminal His tag (FIG. 1, A). The four S-HA1 fusion proteins were produced using the E. coli expression system and purified through the His-tag binding cobalt resin via a denaturing approach. After removal of the denaturing reagents from the protein solutions by dialysis, the proteins were refolded in a refolding buffer (Redox buffer). SDS-PAGE analysis of the refolded proteins revealed a single major band for each S-HA1 fusion protein, showing slightly different mobilities around 52 kDa. The final protein yields ranged between 20 and 30 mg/L of bacterial culture (FIG. 1, panels B and C).

[0116] Self-assembly of the S-HA1 PVNPs

[0117] Gel-filtration chromatography was employed to examine whether the refolded S-VP4 fusion proteins self-assemble into PVNPs. The elution curves of the gel-filtration showed that each of the four S-HA1 fusion proteins was eluted as a single major peak with a very large molecule weight (FIG. 2, panels A, B, E, and F), corresponding to the elution peak of the previously generated S-HA1 H7 PVNP [9] with a calculated molecular weight of -3.12 MDa. On the other hand, the elution peaks corresponding to the S-HA1 protein monomers were minor. Therefore, the four S-HA1 fusion proteins most likely self-assemble into PVNPs efficiently. To further verify the self-formation of the S-HA1 PVNPs, samples from the major elution peaks of the gel filtrations for the four S-HA1 proteins were observed under transmission electronic microscopy (TEM). This revealed typical PVNP morphologies in all four protein samples (FIG. 2, panels C, D, G, and H).

[0118] PVNP size variations

[0119] While the PVNP sizes varied, four distinct size groups were recognized. Since all the four S-HA1 PVNPs exhibited similar size variations, the S-HA1 Hl/pdm PVNPs were used as an example to analyze their size variations. The majority of the S-HA1 Hl PVNPs appeared to have diameters of about 28 nm or 35 nm (FIG. 3, A). Additionally, a small number of larger PVNPs, approximately 42 nm in diameter, were also observed, along with some smaller PVNPs measuring <25 nm in diameter. Based on the propensity of recombinant VP1 of norovirus to form T = 1, T = 3, and T = 4 VLPs in vitro [18-20], and the comparison between the PVNPs of this study and the S60-HA1 H7 PVNP with known structures solved by cryogenic electronic microscopy (cryoEM) [9], the ~28 nm PVNPs should exhibit T = 1 icosahedral symmetry, comprising 60 S-HA1 proteins. The other two larger PVNPs are expected to display T = 3 and T = 4 icosahedral symmetry, consisting of 180 and 240 S-HA1 proteins, respectively. However, the symmetry of the smaller PVNPs with diameters <25 nm remains elusive.

[0120] Structural modeling of three S-HA1 Hl PVNPs

[0121] The 3D structure of the S60-HA1 PVNP of an avian H7N9 virus in a T= 1 icosahedral symmetry has previously been solved by cryoEM [9]. Its 60 HA1 antigens were found to form 20 trimeric protrusions on the PVNP surface. Since the PVNPs generated in this study (FIG. 3) exhibited similar morphologies under TEM to those of S-HA1 H7 PVNPs under TEM, it was assumed that the S-HA1 PVNPs made in this study share the same basic structural features with those of the S-HA1 H7 PVNPs [9], Three structural models of the S-HA1 Hl PVNPs were constructed, each representing the S-HA1 Hl pdm PVNP in T = 1, T = 3, or T= 4 icosahedral symmetry (FIG. 4). These models were generated using the UCSF ChimeraX software, employing the cryoEM density map of the S60-HA1 H7 PVNP [9] as a template, along with the known structures of norovirus interior shells in the three icosahedral symmetries [11,18-20], and the H1N1 HA1 trimers (PDB code: 3LZG) of the influenza virus pandemic strain (A/California/04/2009) as structural modules. The structures viewed from 5-fold (FIG. 4, panels A, D, and G), 3-fold (FIG. 4, panels B, E, and H), and 2-fold (FIG. 4, panels C, F, and I) axes are shown in FIG. 4. All three PVNPs share a common organization that consists of an inner shell made by the shell (S) domains of norovirus VP1 and multiple trimeric surface protrusions made by the HA1 domains of influenza virus. The major differences among the three models are their numbers of S-HA1 fusion proteins, their sizes, their icosahedral symmetries, and the numbers of HA1 trimers on the surface (FIG. 4).

[0122] Recognition of the S-HA1 PVNPs by antibodies against influenza viruses

[0123] This was demonstrated through EIA assays. The S-HA1 PVNPs reacted well with antibodies against HA proteins of homologous influenza viruses (FIG. 5). Specifically, the S-HA1 PVNPs with the HA1 antigens of the H1N1 pandemic strain or PR8 strain bound well to the antibody against the H1N1 HA protein of the pandemic strain (FIG. 5, left panel). The S-HA1 H3 PVNP reacted well with the antibody against the H3N2 HA protein (FIG. 5, middle panel). It was noted that the mouse serum obtained after challenge with the mouse-adapted influenza virus PR8 strain bound significantly stronger to the S-HA1 PVNP containing the HA1 antigens of the same PR8 strain compared with the binding to the S-HA1 PVNP of the pandemic strain (P < 0.001) (FIG. 5, right panel). Certain levels of cross-reactivity among different subtypes were also observed. These data indicated that the PVNP-displayed HA1 antigens retain their authentic conformations and major antigenic features.

[0124] IgG responses of the S-HA1 PVNPs

[0125] After two or three immunizations with one of the four S-HA1 PVNPs using the S60 PVNP as a control, the HAl-specific antibody titers in mouse sera were determined by EIAs using homologous HA1 proteins as capture antigens. All four PVNPs induce similar HAl-specific IgG titers, ranging between 1800 and 2600 after two immunizations (FIG. 6, panel A), and between 40,000 and 64,000 after three immunizations (FIG. 6, panel B). While all these HAl-specific IgG titers were significantly higher than those elicited by the S60 PVNP (the negative control) (Ps < 0.05), the HAl-specific IgG titers induced by any of the four S-HA1 PVNPs after two or three immunizations were not statistically different from each other (Ps > 0.05).

[0126] Protective efficacy of the S-HA1 PVNP vaccine in mice [0127] All five groups of immunized mice were challenged with the mouse-adapted H1N1 influenza virus PR8 strain (A/Puerto Rico/8/1934/HlNl) at lethal dose, followed by daily monitoring of mouse appearance, survival rates and body weight losses for 10 days. It was found that the mice immunized the S-HA1 PR8 PVNP were fully (100 %) protected from mortality caused by the lethal challenge with the homologous H1N1 PR8 strain (FIG. 7A, P = 0.0004). In addition, the S-HA1 PDM PVNPs, containing the HA1 antigens from the 2009 pandemic strain, provided 50 % protection against mortality caused by the challenge with PR8 strain (P = 0.0452), despite the two strains circulating 75 years apart. In contrast, the other two S-HA1 PVNPs, containing the HA1 antigens from an H3N2 strain and an IBV virus (Yamagata lineage), respectively, did not confer significant protection to mice against challenge with the H1N1 PR8 strain (FIG. 7, panel C, Ps > 0.05), although the S-HA1 H3 PVNP group revealed 25 % decrease in the survival rate. It was noted that all challenged mice, including those vaccinated with the S- HA1 Hl PVNPs, experienced body weight loss starting as early as 2 days post challenge (DPC) and reached to the maximum weight loss at 5 or 6 DPC. Subsequently, the survival mice regained their body weight steadily and nearly fully recovered by 10 DPC (FIG. 7, panels B and D). It was observed that although the S-HA1 Hl PR8 PVNP-immunized mice had a 100 % survival rate, they showed 10% weight loss, along with hunched body posture and ruffled fur from DPC 4 to 7, indicating that the mice were infected with the challenged vims and experienced flu-like illness.

[0128] Discussion

[0129] This study demonstrates the effectiveness of the S-HA1 PVNP-based influenza vaccine candidates. In Applicant’s previous studies, the S-HA1 PVNPs containing HA1 antigens from an avian H7N9 influenza vims were generated and characterized. The S-HA1 H7 PVNPs were produced through the E. coli expression system as a soluble protein. The protein self-assembled into PVNPs mainly in a T = 1 icosahedral symmetry. The S-HA1 H7 PVNPs bound specifically to sialoglycans with 2,3-linked sialic acids, which are the host receptors of the avian influenza vims. Accordingly, the S-HA1 H7 PVNPs also hemagglutinated human red blood cells. The PVNPs were immunogenic, eliciting high titers of HAl-specific serum antibodies that inhibited the hemagglutination of the H7 hemagglutinin (HA).

[0130] On the other hand, given that the common influenza viruses causing seasonal influenza belong to H1N1 and H3N2 subtypes of lAVs, as well as IBVs, whether the S nanoparticle platform technology could be applied to these common influenza viruses was examined. For this reason, this study focused on 1) generating S-HA1 PVNPs displaying the HA1 antigens from those common influenza viruses, and 2) determining whether the S-HA1 PVNPs provide protection against influenza virus challenge. The data demonstrated clearly that multiple S-HA1 PVNPs can be produced, each displaying the HA1 antigens of the H1N1 subtype, the H3N2 subtype, and an IBV lineage. Importantly, the S-HA PVNP with HA1 antigens from the H1N1 PR8 strain provided 100% protection against mortality caused by a challenge with homologous PR8 strain (1934). Additionally, the S-HA PVNP displaying the HA1 antigens of the H1N1 pandemic strain (2009) provided 50% protection against mortality caused by a challenge with the PR8 strain, although the two strains circulating 75 years apart and belonging to two different evolutionary clades.

[0131] The observed protection conferred by the S-HA1 Hl PVNPs strongly suggests that the PVNP displayed HA1 antigens retain their authentic protective epitopes. This assumption was supported by the recognition of the S-HA1 PVNPs by antibodies against corresponding influenza viruses or their HAs. This hypothesis was also supported by the reactivity of the S-HA1 PVNP elicited antibodies with commercial HA1 proteins that were used as capture antigens in the EIAs to determine the HAl-specific IgG titers. These data are consistent with the previous observations that the E. coli-expressed S-HA1 H7 PVNP retains authentic glycan receptor binding specificity and hemagglutinates human erythrocytes [9]. Additionally, HA1 is known to be immunodominant, and antigenic supersites with limited variability have been identified at or near the conserved receptor binding sites on the HA1 antigens [5-8].

[0132] These antigenic supersites function as neutralizing [5-7] and/or protective [8] epitopes. These features of HA1 antigens may explain the observed protection and the cross-protection across two H1N1 clades, represented by the H1N1 PR8 and pandemic strains, respectively. On the other hand, the conserved antigenic supersites represent only a small portion of the HA1 antigen. Many epitopes on HA1 are known to be highly variable. Therefore, the HA1 molecule as a whole remains a highly variable antigen with limited cross-reactivity between evolutionarily diverse viruses. This was confirmed by the results of this study, which revealed low cross-reactivity of HA specific antibodies to the S-HA1 PVNPs displaying heterotypic HA Is and the absence of significant cross -protection against challenges with hetero-subtypic influenza viruses.

[0133] The S-HA1 PVNPs of an H7N9 avian influenza virus could be generated as a soluble protein in Applicant’s previous study [9]. However, all the S-HA1 PVNPs containing the HA1 antigens of the four common human influenza viruses respectively need to be produced using the denaturing and refolding approach. Fortunately, all the S-HA1 fusion proteins can be generated in large quantities and the refolded proteins self-assembled into typical PVNPs. As explained above, the data showed that the PVNPs bound influenza virus specific antibodies, the PVNP elicited antibodies recognized commercial HA1 proteins, and the S-HA1 Hl PVNP provides excellent protection against mortality caused by a challenge with H1N1 influenza virus. These data collectively indicate that the PVNP-displayed HA1 antigens retain authentic conformations, serving as solid evidence supporting the use of the S-HA1 PVNPs as promising influenza vaccine candidates.

[0134] The S-HA1 PVNPs produced via the denaturing/refolding approach in this study appeared to exhibit higher size variations compared to the S-HA H7 PVNPs, which were previously generated as a soluble protein [9]. In addition to the major PVNP populations at ~28 nm and ~ 35 nm, representing most likely the T = 1 and T = 3 icosahedrons, respectively, we also observed larger PVNPs at ~42 nm, likely corresponding to those in T = 4 icosahedral symmetry. All these PVNPs, varying in size, share a common organizational pattern consisting of a norovirus inner shell at the center and multiple trimeric surface protrusions made by the HA1 domains of influenza virus HAs. The polyvalent nature of the PVNPs and the preservation of the pathogen associated molecular patterns (PAMPs) of both the norovirus inner shells and the HA1 trimers of influenza viruses explain the strong immune response toward the HA1 antigens elicited by the S- HA1 PVNPs.

[0135] A potential question that might be raised is whether sequential immunizations with the S-HA1 PVNPs could lead to a selective response toward the norovirus S protein backbone, thereby reducing the response to the HA1 antigens. However, the results in this study do not align with that expectation. The data in FIG. 6 showed medium HAl-specific IgG titers ranging from 1:1800 to 1:2600 after the 2nd immunization. These titers increased significantly to 1:40,000 to 64,000 following the 3rd immunization, representing a 22- to 25 -fold increase compared to the 2nd immunization. A similar trend was observed in previous studies investigating PVNP-based vaccines using the same norovirus S protein backbone [11,16,25-27].

[0136] The bacterial system has the advantage of producing recombinant proteins in large quantities quickly and at a low cost. However, the native HA1 antigens are known for their glycosylation. In this case, the E. coll expression system, lacking the glycosylation feature, represents a disadvantage in producing the S-HA1 PVNPs. Viability of the S-HA1 PVNPs from the E. coli expression system as a low-cost vaccine candidate against influenza was assessed. The data provided supportive evidence toward this objective.

[0137] In sum, the results of this study showed that the PVNP vaccines elicited high antibody responses, and these high antibody titers should also contribute to the observed protection against mortality caused by the influenza virus challenge. This study demonstrated the feasibility of using the norovirus S nanoparticle to generate S-HA1 PVNPs displaying the HA1 antigens of common influenza viruses, including those from H1N1 and H3N2 subtypes, as well as from the IBV lineage. The PVNPs elicited high titers of HAl-specific antibodies and provided protection against mortality and body weight loss caused by challenges with influenza viruses of homologous strains. The data provide a novel approach in generating S-HA1 PVNPs to display HA1 antigens from diverse influenza A and B viruses, offering a promising strategy to combat the deadly influenza disease. [0138] All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated. It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0139] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[0140] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

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VP8* domains of rotaviruses. Viruses 2021;13(l):72.

[0162] [22] Tan M, et al. Norovirus P particle, a novel platform for vaccine development and antibody production. J Virol 2011;85(2):753-64.

[0163] [23] Tan M, et al. Saliva as a source of reagent to study human susceptibility to avian influenza H7N9 virus infection. Emerging Microbes Infect 2018:7.

[0164] [24] Su S, et al. Epidemiology, evolution, and pathogenesis of H7N9 influenza viruses in five epidemic waves since 2013 in China. Trends Microbiol 2017;25(9):713-28.

[0165] [25] Xia M, et al. A viral protein 4-based trivalent nanoparticle vaccine elicited high and broad immune responses and protective immunity against the predominant rotaviruses. ACS Nano 2024;18(8):6673-89.

[0166] [26] Huang P, et al. A Pseudovirus nanoparticle displaying the vaccinia virus LI protein elicited high neutralizing antibody titers and provided complete protection to mice against mortality caused by a vaccinia virus challenge. Vaccines (Basel) 2024; 12 (8).

[0167] [27] Xia M, et al. Pseudovirus nanoparticles displaying plasmodium circumsporozoite proteins elicited high titers of sporozoite-binding antibody. Vaccines (Basel) 2023; 11(11).

[0168] Exemplary Combinations

[0169] The following examples relate to various non-exhaustive ways in which the teachings herein may be combined or applied. The following examples are not intended to restrict the coverage of any claims that may be presented at any time in this application or in subsequent filings of this application. No disclaimer is intended. The following examples are being provided for nothing more than merely illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the below examples. Therefore, none of the aspects or features referred to below should be deemed critical unless otherwise explicitly indicated as such at a later date by the inventors or by a successor in interest to the inventors. If any claims are presented in this application or in subsequent filings related to this application that include additional features beyond those referred to below, those additional features shall not be presumed to have been added for any reason relating to patentability.

[0170] Example 1.

[0171] A vaccine composition comprising a pseudovirus nanoparticle (PVNP) displaying a hemagglutinin HA1 antigen of an influenza virus, the PVNP comprising: a modified norovirus shell domain (“S domain”) comprising a sequence having at least 90% homology to SEQ ID NO: 1; and at least one HA1 domain of an influenza virus, the HA1 domain being operatively linked to the S domain.

[0172] Example 2.

[0173] The vaccine composition of example 1, the HA1 domain being Hl/pdm.

[0174] Example 3.

[0175] The vaccine composition of example 1, the HA1 domain being H1/PR8.

[0176] Example 4.

[0177] The vaccine composition of example 1, the HA1 domain being H3.

[0178] Example 5.

[0179] The vaccine composition of example 1, the HA1 domain being from the influenza B virus strain. [0180] Example 6.

[0181] The vaccine composition of any one of examples 1 through 5, the PVNP capable of self-assembling into an icosahedral structure selected from T=l, T=3, or T=4 symmetry under conditions suitable for nanoparticle formation.

[0182] Example 7.

[0183] The vaccine composition of any one of examples 1 through 6, wherein the PVNP exhibits immunogenicity and elicits an HA 1- specific immune response following administration to a subject.

[0184] Example 8.

[0185] The vaccine composition of any one of examples 1 through 6, the PVNP being a bivalent PVNP having at least two different HA1 antigens.

[0186] Example 9.

[0187] The vaccine composition of any one of examples 1 through 6, the PVNP being a trivalent PVNP having at least three different HA1 antigens.

[0188] Example 10.

[0189] The vaccine composition of any one of examples 1 through 9, the composition providing greater than 90% protection with an H1N1 PR8 strain, as measured by reduction in morbidity /mortality .

[0190] Example 11.

[0191] The vaccine composition of any one of examples 1 through 9, the composition providing 50% or more protection with an H1N1 strain, as measured by reduction in morbidity /mortality . [0192] Example 12.

[0193] The vaccine composition of any one of examples 1 through 11, the PVNP being formed via denaturation and refolding.

[0194] Example 13.

[0195] The vaccine composition of any one of examples 1 through 12, the PVNP having T=1 icosahedral symmetry, in which 60 identical subunits form a structure approximately 28 nm in diameter.

[0196] Example 14.

[0197] The vaccine composition of any one of examples 1 through 12, the PVNP having T=3 icosahedral symmetry, in which 180 identical subunits form a structure approximately 35 nm in diameter.

[0198] Example 15.

[0199] The vaccine composition of any one of examples 1 through 12, the PVNP having T=4 icosahedral symmetry, in which 240 identical subunits form a structure approximately 42 nm in diameter.

[0200] Example 16.

[0201] The vaccine composition of any one of examples 1 through 15, the S domain comprising a sequence having at least 95% sequence identity to SEQ ID NO: 1, wherein the S domain comprises a R69A mutation and a conserved hinge region.

[0202] Example 17. [0203] The vaccine composition of any one of examples 1 through 16, the S domain comprising a sequence having at least 99% sequence identity to SEQ ID NO: 1, wherein the S domain comprises a R69A mutation and a conserved hinge region.

[0204] Example 18.

[0205] The vaccine composition of any one of examples 1 through 17, the S domain comprising SEQ ID NO: 1.

[0206] Example 19.

[0207] The vaccine composition of any one of examples 1 through 18, wherein the vaccine composition elicits HA1 specific IgG and/or IgA specific antibodies in an individual administered the vaccine composition.

[0208] Example 20.

[0209] The vaccine composition of any one of examples 1 through 19, wherein the S domain is wildtypc at one or more of positions V57, Q58, or S136, and M140.

[0210] Example 21.

[0211] The vaccine composition of any one of examples 1 through 20, wherein the S domain is wildtype at least two of positions V57, Q58, or S 136, and M140.

[0212] Example 22.

[0213] The vaccine composition of any one of examples 1 through 21, wherein the S domain is wildtype at least three of positions V57, Q58, or S136, and M140.

[0214] Example 23. [0215] The vaccine composition of any one of examples 1 through 22, wherein the S domain is wildtype at each of positions V57, Q58, or S136, and M140.

[0216] Example 24.

[0217] The vaccine composition of any one of examples 1 through 23, wherein the HA1 antigen is derived from an H1N1 pandemic strain (Hl/pdm) and has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 7.

[0218] Example 25.

[0219] The vaccine composition of any one of examples 1 through 23, wherein the HA1 antigen is that of HA1 antigens from an H1N1 PR8 strain (H1/PR8) and has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 8.

[0220] Example 26.

[0221] The vaccine composition of any one of examples 1 through 23, wherein the HA1 antigen is that of HA1 antigens from an H3N2 influenza virus (H3) has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 9.

[0222] Example 27.

[0223] The vaccine composition of any one of examples 1 through 23, wherein the HA1 antigen is that of HA1 antigens from the influenza B virus (IBV) and has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 10.

[0224] Example 28.

[0225] The vaccine composition of any one of examples 1 through 27, wherein the PVNP composition elicits a specific IgG and IgA titer against the HA1 antigen in an individual administered the PVNP composition.

[0226] Example 29.

[0227] The vaccine composition of any one of examples 1 through 28, wherein the PVNP is produced using an Escherichia coli expression system.

[0228] Example 30.

[0229] The vaccine composition of any one of examples 1 through 29, wherein the PVNP comprises a C-terminal His tag.

[0230] Example 31.

[0231] A method of eliciting an immune response against an influenza virus in an individual in need thereof, comprising administering the vaccine composition of any one of examples 1 through 30, in an amount effective to elicit an immune response specific to the HA1 antigen in the individual.

[0232] Example 32.

[0233] The method of example 31 wherein the vaccine composition confers protection against mortality caused by influenza virus infection.

[0234] Example 33. [0235] The method of example 31 or 32, wherein the individual develops HA 1 -specific antibodies that recognize homologous and heterologous HA1 antigens of influenza viruses.

[0236] Example 34.

[0237] The method of any one of examples 31 through 33, the administering comprising administering at least 1 dose.

[0238] Example 35.

[0239] The method of any one of examples 31 through 34, the administering comprising administering at least 2 doses.

[0240] Example 36.

[0241] The method of any one of examples 31 through 35, the administering comprising administering at least 3 doses.

[0242] Example 37.

[0243] The method of any one of examples 31 through 36, wherein the vaccine composition is administered intramuscularly, subcutaneously, intradermally, or intranasally.

[0244] Example 38.

[0245] An S-HA1 fusion protein comprising a sequence having at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0246] Example 39. [0247] A cell system for producing a PVNP composition comprising Chinese Hamster Ovary (CHO) cells capable of expressing a fusion protein comprising a modified S domain and an HA1 antigen.

[0248] Example 40.

[0249] The cell system of example 39, the fusion protein comprising SEQ ID NO: 5.

[0250] Example 41.

[0251] A method of producing a vaccine composition, comprising: expressing a fusion protein comprising a norovirus shell (S) domain and an HA1 domain of an influenza vims in an Escherichia coli expression system; purifying the fusion protein via affinity chromatography; refolding the fusion protein in a refolding buffer to allow self-assembly into PVNPs; and formulating the PVNPs into a vaccine composition suitable for administration to a subject.

[0252] Example 42.

[0253] A method of making the PVNP of any preceding example, comprising: expressing a fusion protein comprising a norovirus shell (S) domain and an HA1 domain of an influenza vims in Escherichia coli, wherein the fusion protein forms insoluble inclusion bodies to produce a solubilized protein; solubilizing the inclusion bodies using a chemical denaturant; purifying the solubilized protein; and refolding the purified protein by removing the denaturant through buffer exchange, dilution, or dialysis.

[0254] Example 43.

[0255] The method of example 42 wherein the chemical denaturant is selected from one or both of guanidine hydrochloride and urea.

[0256] Example 44. [0257] The method of example 42 or 43 wherein the purifying is carried out using chromatography

Claims

CLAIMS What is claimed is:

1. A vaccine composition comprising a pseudovirus nanoparticle (PVNP) displaying a hemagglutinin HA1 antigen of an influenza virus, the PVNP comprising:

(a) a modified norovirus shell domain (“S domain”) comprising a sequence having at least 90% homology to SEQ ID NO: 1; and

(b) at least one HA1 domain of an influenza virus, the HA1 domain being operatively linked to the S domain.

2. The vaccine composition of claim 1, the HA1 domain being Hl/pdm.

3. The vaccine composition of claim 1, the HA1 domain being H1/PR8.

4. The vaccine composition of claim 1, the HA1 domain being H3.

5. The vaccine composition of claim 1, the HA1 domain being from the influenza

B virus strain.

6. The vaccine composition of any one of claims 1 through 5, the PVNP capable of self-assembling into an icosahedral structure selected from T=l, T=3, or T=4 symmetry under conditions suitable for nanoparticle formation.

7. The vaccine composition of any one of claims 1 through 6, wherein the PVNP exhibits immunogenicity and elicits an HA 1- specific immune response following administration to a subject.

8. The vaccine composition of any one of claims 1 through 6, the PVNP being a bivalent PVNP having at least two different HA1 antigens.

9. The vaccine composition of any one of claims 1 through 6, the PVNP being a trivalent PVNP having at least three different HA1 antigens.

10. The vaccine composition of any one of claims 1 through 9, the composition providing greater than 90% protection with an H1N1 PR8 strain, as measured by reduction in morbidity /mortality .

11. The vaccine composition of any one of claims 1 through 9, the composition providing 50% or more protection with an H1N1 strain, as measured by reduction in morbidity/mortality .

12. The vaccine composition of any one of claims 1 through 11, the PVNP being formed via denaturation and refolding.

13. The vaccine composition of any one of claims 1 through 12, the PVNP having T=1 icosahedral symmetry, in which 60 identical subunits form a structure approximately 28 nm in diameter.

14. The vaccine composition of any one of claims 1 through 12, the PVNP having T=3 icosahedral symmetry, in which 180 identical subunits form a structure approximately 35 nm in diameter.

15. The vaccine composition of any one of claims 1 through 12, the PVNP having T=4 icosahedral symmetry, in which 240 identical subunits form a structure approximately 42 nm in diameter.

16. The vaccine composition of any one of claims 1 through 15, the S domain comprising a sequence having at least 95% sequence identity to SEQ ID NO: 1, wherein the S domain comprises a R69A mutation and a conserved hinge region.

17. The vaccine composition of any one of claims 1 through 16, the S domain comprising a sequence having at least 99% sequence identity to SEQ ID NO: 1, wherein the S domain comprises a R69A mutation and a conserved hinge region.

18. The vaccine composition of any one of claims 1 through 17, the S domain comprising SEQ ID NO: 1.

19. The vaccine composition of any one of claims 1 through 18, wherein the vaccine composition elicits HA1 specific IgG and/or IgA specific antibodies in an individual administered the vaccine composition.

20. The vaccine composition of any one of claims 1 through 19, wherein the S domain is wildtype at one or more of positions V57, Q58, or S136, and M140.

21. The vaccine composition of any one of claims 1 through 20, wherein the S domain is wildtype at least two of positions V57, Q58, or S 136, and M140.

22. The vaccine composition of any one of claims 1 through 21, wherein the S domain is wildtype at least three of positions V57, Q58, or S136, and M140.

23. The vaccine composition of any one of claims 1 through 22, wherein the S domain is wildtype at each of positions V57, Q58, or S136, and M140.

24. The vaccine composition of any one of claims 1 through 23, wherein the HA1 antigen is derived from an H1N1 pandemic strain (Hl/pdm) and has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 7.

25. The vaccine composition of any one of claims 1 through 23, wherein the HA1 antigen is that of HA1 antigens from an H1N1 PR8 strain (H1/PR8) and has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 8.

26. The vaccine composition of any one of claims 1 through 23, wherein the HA1 antigen is that of HA1 antigens from an H3N2 influenza virus (H3) has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 9.

27. The vaccine composition of any one of claims 1 through 23, wherein the HA1 antigen is that of HA1 antigens from the influenza B virus (IBV) and has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 10.

28. The vaccine composition of any one of claims 1 through 27, wherein the PVNP composition elicits a specific IgG and IgA titer against the HA1 antigen in an individual administered the PVNP composition.

29. The vaccine composition of any one of claims 1 through 28, wherein the PVNP is produced using an Escherichia coli expression system.

30. The vaccine composition of any one of claims 1 through 29, wherein the PVNP comprises a C-terminal His tag.

31. A method of eliciting an immune response against an influenza virus in an individual in need thereof, comprising administering the vaccine composition of any one of claims 1 through 30, in an amount effective to elicit an immune response specific to the HA1 antigen in the individual.

32. The method of claim 31 wherein the vaccine composition confers protection against mortality caused by influenza virus infection.

33. The method of claim 31 or 32, wherein the individual develops HAl-specific antibodies that recognize homologous and heterologous HA1 antigens of influenza viruses.

34. The method of any one of claims 31 through 33, the administering comprising administering at least 1 dose.

35. The method of any one of claims 31 through 34, the administering comprising administering at least 2 doses.

36. The method of any one of claims 31 through 35, the administering comprising administering at least 3 doses.

37. The method of any one of claims 31 through 36, wherein the vaccine composition is administered intramuscularly, subcutaneously, intradermally, or intranasally.

38. An S-HA1 fusion protein comprising a sequence having at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

39. A cell system for producing a PVNP composition comprising Chinese Hamster Ovary (CHO) cells capable of expressing a fusion protein comprising a modified S domain and an HA1 antigen.

40. The cell system of claim 39, the fusion protein comprising SEQ ID NO: 5.

41. A method of producing a vaccine composition, comprising:

(a) expressing a fusion protein comprising a norovirus shell (S) domain and an HA1 domain of an influenza virus in an Escherichia coli expression system; (b) purifying the fusion protein via affinity chromatography;

(c) refolding the fusion protein in a refolding buffer to allow self-assembly into PVNPs; and

(d) formulating the PVNPs into a vaccine composition suitable for administration to a subject.

42. A method of making the PVNP of any preceding claim, comprising:

(a) expressing a fusion protein comprising a norovirus shell (S) domain and an HA1 domain of an influenza virus in Escherichia coli, wherein the fusion protein forms insoluble inclusion bodies;

(b) solubilizing the inclusion bodies using a chemical denaturant to produce a solubilized protein;

(c) purifying the solubilized protein; and

(d) refolding the purified protein by removing the denaturant through buffer exchange, dilution, or dialysis.

43. The method of claim 42 wherein the chemical denaturant is selected from one or both of guanidine hydrochloride and urea.

44. The method of claim 42 or 43 wherein the purifying is carried out using chromatography