Oligomeric hemagglutinin antigens

Abstract

Recombinant, oligomerized, and scalably reproducible hemagglutinin (HA) antigens are provided for herein, which are induced into forming trimers by combining an HA domain with an oligomerization domain, for example a coronin domain, and optionally substituting one or more cysteine residues in the stalk portion of the HA protein to form disulfide bonds between, for example the three strands of a trimer, to stabilize the resulting HA protein.

Description

OLIGOMERIC HEMAGGLUTININ ANTIGENSFIELD OF INVENTION

[0001] The disclosures herein relate to influenza vaccines that comprise at least one hemagglutinin antigen (referred to as HA, or HA protein) polypeptide in oligomeric form, and conjugable with a virus, a virus particle, or other suitable carrier.BACKGROUND

[0002] Despite a prevalence of vaccines and vaccine administration, seasonal influenza is a recurring problem domestically and globally. In general, influenza vaccines developed from year to year are designed to be effective against specific influenza strains, but may be less effective against new or different strains that are associated with mutations in the surface glycoprotein hemagglutinin (HA).

[0003] The nomenclature of influenza viruses is based on their surface glycoproteins, specifically hemagglutinin (HA) and neuraminidase (NA). Influenzavirus A (Type A influenza) and Influenzavirus B (Type B influenza) are the more common causes of infection associated with seasonal outbreaks of influenza, or, with lesser frequency, pandemics. Influenzavirus A and Influenzavirus B, as well as Influenzavirus C, are in the orthomyxoviridae family of viruses. Influenza A viruses include at least 18 different hemagglutinin (HA) subtypes and at least 11 different neuraminidase (NA) subtypes. The HA subtypes are classified into group 1 (Hl , H2, H5, H6, H8, H9, Hl 1, Hl 2, Hl 3, Hl 6 and Hl 7) and group 2 (H3, H4, H7, H10, H14 and Hl 5), with RNA-protein complexes encoding the hemagglutinin, neuraminidase, and matrix proteins that are unique to each subtype. Influenza B viruses are more similar to A viruses, while influenza Cviruses have only one major surface glycoprotein, the hemagglutinin-esterase-fusion (HEF) protein, instead of HA and NA.

[0004] HA exists naturally as a homotrimer comprising an HA1 domain and an HA2 domain. Each monomer contains approximately 550 amino acids. Influenza viruses recognize N- acetylneuraminic (sialic) acid on the host cell surface. Upon recognition, the HA protein (or in the case of Influenza C viruses, the HEF protein) binds to sialic acid, and the virus is brought into the cell via a cellular process called endocytosis.

[0005] Antigenic drift is a gradual occurrence of mutations associated with continual replication in the surface proteins HA (hemagglutinin) and NA (neuraminidase) found in the globular head of these viruses. Occurrences of antigenic drift, particularly with respect to Type A and Type B influenzas, are one of the reasons that some influenza infections do not respond to strain-specific influenza vaccines.

[0006] The efficacy and potency of influenza vaccines are influenced by several factors discussed below. These include the vaccine delivery platform, adjuvant effects, and, as more relevant for the present disclosure, the extent of oligomerization (particularly trimerization) of HA components of the vaccine to enhance the potency of anti-HA antibody responses. Herein, “trimer” and related forms of the term are included within the meaning of “oligomer,” but not all oligomers are referring to the trimeric form.

[0007] HA monomers are poor immunogens as they lack proper antigenicity. By comparison, more complete antigenicity is achieved by trimeric antigens due to the conformational effect driven by trimerization. Therefore, key immunogenic epitopes of HA are only present (or completed) upon trimerization and needed to elicit a robust and natural immune response. Additionally, trimerization of recombinant HA antigens also shield the “silent faces” of the antigens that are notnaturally exposed, which, otherwise would lead to an antibody response to epitopes that are not naturally accessible on live native virus.

[0008] There also is a potential to further enhance one or more of these beneficial properties through the creation of disulfide bonds, which may be formed between strands of a short coiled coil (as further discussed below) or between neighboring antigenic units forming the oligomer. However, determining a proper or optimal location of disulfide bonds has proven difficult, particularly in the context of HA proteins, which consist of a number of different strains, with significant variations from one strain to another.

[0009] In addition, antigenic folding and conformational stability are important considerations when producing recombinant proteins for use as antigens. Conformational stability refers to the tendency of the three-dimensional protein to retain its folded tertiary structure under physical or chemical stresses, such as by pH changes or shifts in redox equilibrium. During purification of recombinant HA antigens, for example, the antigens are subjected to a wide array of stringent biochemical processes. Maintaining conformational stability throughout processing is a substantial factor in maintaining proper conformation of the antigen in the folded state and thereby eventually allowing suitably robust immunogenic reactions in a subject to whom a vaccine is administered. Also, after being taken up by a subject’s cells, the conformational stability of the antigen affects whether or not intra-cellular processing will result in the unfolding of the tertiary structure, which is likely followed by proteolytic degradation into simple and ineffective peptides.

[0010] Accordingly, oligomerization (particularly trimerization) and conformational stability of the folded state enhance the stability and presentation of HA proteins used in vaccines, and by extension enhance their stability and immunogenic effects upon a subject. In this regard, there is a significant need in the relevant field for recombinant antigens, and particularly HA proteins, thatform into stable trimers and retain conformational stability in the folded state, along with methods for their manufacture.SUMMARY OF EMBODIMENTS

[0011] In accordance with a broad range of embodiments herein, the present disclosure provides for antigenic compositions as well as formulations, compounds, conjugates, products (including vaccine products) that include such antigenic compositions, as well as methods of forming the same, as well as methods that induce immune response using such antigenic compositions that are described.

[0012] In some embodiments according to the present disclosure, a reproducible, recombinant and scalable HA is formed. The HA includes an oligomerization domain, which may be a coronin domain according to multiple embodiments disclosed herein, to induce formation of trimers during expression. In addition to other optional structural features, the inventive HA proteins may include ones in which disulfide bonds are formed by cysteine substitutions (meaning one or more cysteine residues are substituted as a single point mutation for one or more residues in the amino acid sequence), which enhance the stability of the HA stalk.

[0013] Structurally, short coiled coils are amino acid sequences arranged as a-helix strands that wind around each other. Coronin proteins are associated with the actin protein, and may be bacterially expressed. These proteins typically have a specific arrangement of hydrophobic core residues along with polar residues at specific locations that produce side chain interactions resulting in oligomerization. The actin-associated protein coronin 1 (herein referred to as “ccCORl”) is characterized by a well-conserved, distinct structural motif of short (generally, 50 amino acids or fewer) autonomous coiled-coil domains known to induce trimerization through the formation of three-stranded, parallel, and relatively thermodynamically stable coiled-coilstructures. In coronin proteins, the geometry of side chains on certain polar residues influences the oligomerization state and the orientation of the strands in these coiled coils in the oligomerized state. In some embodiments, as part of forming trimeric HA proteins, a cysteine residue is added at the C-terminus of at least one of the strands to facilitate a conjugation reaction with a virus particle.

[0014] When a statement contained in the present disclosure refers to an amino acid sequence, such statement or reference shall also include any nucleotide sequences encoding such amino acid sequence. In addition, some embodiments are described herein in relation to their sequence identity to a particular amino acid sequence. “Sequence identity” in connection with amino acids refers to a degree of identity of protein domains, which may be expressed as a percentage, between two sequences under comparison. Computer algorithms such as Basic Local Alignment Search Tool (BLAST) and the Expert Protein Analysis System (ExPASy) offer analytical tools known in the art for determining sequence identity. The BLAST suite of programs currently is available on various websites including the National Center for Biotechnology Information website.

[0015] Accordingly, multiple embodiments and alternatives within the scope of the present disclosure optionally provide for one or more of the use of oligomerization domains, particularly coronin trimerization domains; disulfide bonds between strands; enhanced conformational stability; and addition of a single cysteine residue at the C-terminus of the oligomerization domain of the antigen, which may facilitate conjugation with a virus particle.

[0016] In some embodiments according to the present invention, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence.

[0017] In some embodiments according to the present invention, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence of SEQ ID NO 1 and an oligomerization domain comprising a second amino acid sequence of SEQ ID NO 4 or SEQ ID NO 5, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence.

[0018] In some embodiments according to the present invention, there is provided herein a vaccine containing an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, further comprising a tobacco mosaic virus (TMV) wherein the polypeptide is conjugated with the TMV.

[0019] In some embodiments according to the present invention, there is provided herein a vaccine containing an HA polypeptide having an HA domain comprising a first amino acid sequence of SEQ ID NO 1 and an oligomerization domain comprising a second amino acid sequence of SEQ ID NO 4 or SEQ ID NO 5, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, further comprising a tobacco mosaic virus (TMV) wherein the polypeptide is conjugated with the TMV.

[0020] In some embodiments according to the present invention, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, for use in a vaccine.

[0021] In some embodiments according to the present invention, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence of SEQ ID NO 1 and an oligomerization domain comprising a second amino acid sequence of SEQ ID NO 4 or SEQ ID NO 5, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, for use in a vaccine.

[0022] In some embodiments according to the present invention, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, for use in the manufacture of a vaccine.

[0023] In some embodiments according to the present invention, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence of SEQ ID NO 1 and an oligomerization domain comprising a second amino acid sequence of SEQ ID NO 4 or SEQ ID NO 5, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, for use in the manufacture of a vaccine.

[0024] In some embodiments according to the present invention, there is provided herein a method of inducing an immune response in a subject, comprising administering to the subject a vaccine composition comprising an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence.

[0025] In some embodiments according to the present invention, there is provided herein a pharmaceutical composition comprising the HA polypeptide having an HA domain comprising afirst amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence.

[0026] In some embodiments according to the present invention, there is provided herein a method of manufacturing a vaccine comprising conjugating an HA polypeptide with a virus particle, wherein the HA polypeptide has an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence.BRIEF DESCRIPTION OF THE FIGURES

[0027] The patent or application file with respect to the present disclosure 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.

[0028] The drawings and embodiments described herein are illustrative of multiple alternative structures, aspects, and features of the multiple embodiments and alternatives disclosed herein, and they are not to be understood as limiting the scope of any of these embodiments and alternatives. It will be further understood that the drawing figures described and provided herein are not to scale, and that the embodiments are not limited to the precise arrangements, depictions, and instrumentalities shown.

[0029] Fig. 1A is an amino acid sequence of a representative influenza HA from a B / Phuket / 3073 / 2013 virus, also referred to as SEQ ID NO 1.

[0030] FIG. IB is a portion of the sequence provided in FIG. 1 A, representing the HA1 domain of B / Phuket / 3073 / 2013, also referred to as SEQ ID NO 2.

[0031] FIG. 1C is a portion of the sequence provided in FIG. 1A, representing the HA2 domain of B / Phuket / 3073 / 2013, also referred to as SEQ ID NO 3.

[0032] Fig. 2 A - Fig. 2E are schematic representations showing examples of HA constructs according to one or more embodiments disclosed herein for producing HA proteins, in some cases representing one or more of the following additional features as further described herein: one or two substituted cysteine residues in the HA stalk to induce disulfide bond formation; an oligomerization domain such as a trimerization domain; a short linker proximal to the C-Terminal containing a cysteine residue to facilitate conjugation with a virus or viral particle.

[0033] Fig. 3 is a ribbon diagram that shows an exemplary disulfide linked trimer for improved stability, according to one or more embodiments disclosed herein.

[0034] Fig. 4 A illustrates a modified tobacco mosaic virus prior to conjugation with at least one antigen.

[0035] Fig. 4B illustrates the virus of Fig. 4 A after conjugation with one or more antigens, according to one or more embodiments disclosed herein.

[0036] Figure 5A is a schematic diagram of a modified trimeric Anti-B / Phuket / 3073 / 2013 HA according to multiple embodiments herein.

[0037] Figure 5B is a schematic diagram of a monomeric Anti-B / Phuket / 3073 / 2013 HA.

[0038] Figure 5C is a schematic diagram of a modified trimeric Anti-A / Anhui / 1 / 2013 (H7N9)HA according to multiple embodiments herein.

[0039] Figure 5D is a schematic diagram of a monomeric Anti-A / Anhui / 1 / 2013 (H7N9) HA.

[0040] Figure 5E is a schematic diagram of a modified trimeric Anti-A / Victoria / 2570 / 2019 HA according to multiple embodiments herein.

[0041] Fig. 6A is a graph showing results of ELISA analyses conducted on two versions of a Phuket (anti-B HA) antigen, where the second version (V. 2) contained a substantially higher level of trimerization than the other (V. 1).

[0042] Fig. 6B is a graph showing results of a hemagglutination inhibition (HAI) assay conducted on the two versions of a Phuket (anti-B HA) antigen as shown in Fig. 6 A.

[0043] Fig. 6C is a graph showing results of a neutralization assay conducted on the two versions of a Phuket (anti-B HA) antigen as shown in Fig. 6 A.

[0044] Fig. 7A is a graph showing results of a hemagglutination inhibition (HAI) assay for two versions of an Anti-A / Anhui / 1 / 2013 (H7N9) virus hemagglutinin antigen compared to Placebo.

[0045] Fig. 7B is a graph showing results of a neutralization assay conducted on the two versions of the antigen referenced in connection with Fig. 7A compared to Placebo.

[0046] Fig. 8A provides a graph of weight changes in ferrets monitored for 10 days postchallenge following infection with influenza H7N9 virus.

[0047] Fig. 8B provides a graph of body temperature changes in ferrets monitored for 10 days post-challenge following infection with influenza H7N9 virus.

[0048] Fig. 8C shows results of an hemagglutination inhibition assay (HAI) based on A / Anhui / 1 / 2013 (H7N9) in serum samples of ferrets following inoculation with vaccine or Placebo according to a vaccine dosing regimen.

[0049] Fig. 8D shows results of a microneutralization assay (MNT) to measure the neutralizing antibody (nAb) titer in serum samples of ferrets following inoculation with vaccine or Placebo according to a vaccine dosing regimen.

[0050] Fig. 8E shows viral titers from nasal wash samples of vaccinated and saline control ferrets after challenge with A / Anhui / 1 / 2013 (H7N9) virus.

[0051] Fig. 9A shows results of an hemagglutination inhibition assay (HAI) for A / Anhui / 1 / 2013 (H7N9) based on serum samples of rabbits following inoculation with vaccine or Placebo according to a vaccine dosing regimen.

[0052] Fig. 9B shows results of a microneutralization assay (MNT) to measure the neutralizing antibody (nAb) titer for A / Anhui / 1 / 2013 (H7N9) based on serum samples of rabbits following inoculation with vaccine or Placebo according to a vaccine dosing regimen.

[0053] Fig. 10A shows results of a Hemagglutination inhibition assay (HAI) for A / Victoria / 2570 / 2019, based on serum samples in rabbits following inoculation with vaccine or Placebo according to a vaccine dosing regimen.

[0054] Fig. 10B shows ELISA measurement of GMT against Influenza Antigen for A / Victoria / 2570 / 2019, based on serum samples in rabbits following inoculation with vaccine or Placebo according to a vaccine dosing regimen.MULTIPLE EMBODIMENTS AND ALTERNATIVESHemagglutinin Polypeptide Compositions

[0055] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence.

[0056] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, further comprising a cysteinelocated at the C-terminus of the second amino acid sequence, preferably wherein the cysteine residue facilitates the conjugation with a virus particle.

[0057] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV).

[0058] Various methods can be employed to conjugate an antigen intermediate, for example but not limited to an HA, to a virus intermediate, for example but not limited to a virus particle (which as used herein includes viruses) modified at the N-terminus to provide an amine-containing lysine residue for reacting with and binding to the antigenic component. One such exemplary method includes sterilizing the virus intermediate, for example via UV irradiation, which also should inactivate the virion to a non-infectious state. The virus intermediate is then combined with an appropriate molar excess of Sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate (SMCC) and reacted for a sufficient period of time at room temperature.

[0059] In addition to preparing the virus intermediate, the antigen intermediate is prepared for conjugation by reducing in an appropriate buffer. The reduced antigen is sterilized prior to combining with the SMCC-labeled virus, and in some embodiments the two components are mixed at room temperature at a substantially neutral pH (e.g., 7.2) to achieve the conjugation reaction.

[0060] To facilitate conjugation using this SMCC method, antigen intermediates may be designed with a short linker proximal to the C-terminus, formed of the residues GSPPCP. The cysteine in this linker (i.e., a terminal cysteine) contains thiol functionality that chemically associates or covalently reacts with positively charged groups on the viral particle, such as foundin amine-containing lysine residues. In some embodiments, a molar concentration (e.g., 10 mM as a non-limiting example) of a reducing agent is used in preparing the antigen intermediate for conjugation, utilizing the terminal cysteine. The terminal cysteine should remain unpaired (i.e., without self-conjugating) so it can facilitate the conjugation between the antigen and the virus, while avoiding interference with the conformational folding of the oligomeric antigen. Also, in reducing the terminal cysteine, an appropriate reducing agent (including but not limited to, thiol reducing agents) and concentration should be selected to avoid affecting the reactivity of the substituted cysteines in the stalk, involved with creating disulfide bonds for intermolecular stability. Among other reducing agents known to those of ordinary skill in the art, betamercaptoethanol (BME), Tris (2-carboxyethyl) phosphine (TCEP), or Dithiothreitol (DTT) in phosphate-buffered saline (PBS) can be used for this purpose.

[0061] With respect to the aforementioned linker, the GSPP residues, like proline (P) located at the C-terminal side of cysteine, provides additional spacing and plays a role in preventing selfconjugation. These spacers, along with the coronin domain, serve to aid in the chemical association between cysteine and the positively charged viral particle surface, while positioning the terminal cysteine in a manner that prevents it from interacting with other cysteines in the molecule, which could interfere with folding. In some embodiments, a polyhistidine-tag (HHHHHH) with an endoplasmic reticulum (ER) retention sequence (DEL) are included in the antigen, in which case the aforementioned GSPPCP linker will be positioned relative to these as shown in SEQ ID NOS. 10 and 12.

[0062] During and after the conjugation reaction, the intermediates and the conjugate are kept sterile during remaining processing. In some embodiments, the SMCC-labeled virus and thereduced antigen are mixed at a 1 :1 ratio by weight, and the conjugates are further subject to sterilization and purification into a dosage form.

[0063] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands.

[0064] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, further comprising a cysteine located at the C-terminus of the second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands and wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV).

[0065] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence.

[0066] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising asecond amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1 and comprising a cysteine substitution at a position that may vary based on the HA as further described herein.

[0067] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1 and comprising a cysteine substitution as further described herein.

[0068] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1 and comprising a cysteine a substitution, wherein the substitution results in the formation of disulfide bonds in the HA stalk, preferably wherein the disulfide bonds enhance the stability of the HA stalk.

[0069] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, further comprising a cysteine located at the C-terminus of the second amino acid sequence, preferably wherein the cysteine residue facilitates the conjugation with a virus particle.

[0070] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV).

[0071] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the resulting amino acid sequence has at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO 8.

[0072] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, wherein the resulting amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO 10.

[0073] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the resulting amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO 12.

[0074] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, and the second amino acid has at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 4.

[0075] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, and the second amino acid has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 5.

[0076] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, and the amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 8.

[0077] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein 1 located at the C- terminus of the first amino acid sequence, and the amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 10.

[0078] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, and the amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 12.

[0079] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence and the second amino acid has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 5, further comprising a cysteine located at the C-terminus of the second amino acid sequence, preferably wherein the cysteine residue facilitates the conjugation with a virus particle.

[0080] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, and the second amino acid has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQID NO 4, wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV).

[0081] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, and the second amino acid has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 5, wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV).

[0082] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, wherein the second amino acid sequence is a modified version of SEQ ID NO 4 comprising amino acids, wherein the modification is a cysteine substitution at the terminal position of SEQ ID NO 4, wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV).

[0083] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, wherein the second amino acid sequence is a modified version of SEQ ID NO 4 comprising amino acids, wherein the modification is a cysteine substitution at the terminal position of SEQ ID NO 5, wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV).

[0084] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence comprises SEQ ID NO 1, and the second amino acid sequence comprises SEQ ID NO 4 or SEQ ID NO 5.

[0085] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence comprises SEQ ID NO 1, and the second amino acid sequence comprises SEQ ID NO 4 or SEQ ID NO 5, wherein the polypeptide is formed as a trimer comprising three strands.

[0086] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence.

[0087] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, .

[0088] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands.

[0089] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands.Vaccines and Pharmaceutical Compositions

[0090] In at least one embodiment, the HA polypeptide of any of the embodiments disclosed herein may be used in a vaccine.

[0091] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the second amino acid sequence, for use in a vaccine.

[0092] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a 1second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, further comprising a cysteine located at the C-terminus of the second amino acid sequence, preferably wherein the cysteine residue facilitates the conjugation with a virus particle, for use in a vaccine.

[0093] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV), for use in a vaccine.

[0094] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, for use in a vaccine.

[0095] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, further comprising a cysteine located at the C-terminus of the second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands and wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV), for use in a vaccine.

[0096] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, for use in a vaccine.

[0097] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1 and comprising a cysteine substitution at a position that may vary based on the HA, for use in a vaccine.

[0098] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1 and comprising a cysteine substitution as further described herein, for use in a vaccine.

[0099] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising asecond amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1 and comprising a cysteine a substitution, wherein the substitution results in the formation of disulfide bonds in the HA stalk, preferably wherein the disulfide bonds enhance the stability of the HA stalk, for use in a vaccine.

[0100] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, further comprising a cysteine located at the C-terminus of the second amino acid sequence, preferably wherein the cysteine residue facilitates the conjugation with a virus particle, for use in a vaccine.

[0101] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the HA polypeptide is conjugated to a tobacco mosaic virus (TMV), for use in a vaccine.

[0102] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the resulting amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO 8, for use in a vaccine.

[0103] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C- terminus of the first amino acid sequence, wherein the resulting amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO 10, for use in a vaccine.

[0104] In at least one embodiment, there is provided herein an HA polypeptide having an HA domain comprising a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the resulting amino acid sequence has at least 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO 12, for use in a vaccine.Nucleic Acids

[0105] In at least one embodiment, there is provided herein an isolated nucleic acid encoding the HA polypeptide of any of the embodiments disclosed herein.

[0106] In at least one embodiment, there is provided herein an isolated nucleic acid encoding an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence.

[0107] In at least one embodiment, there is provided herein an isolated nucleic acid encoding an HA polypeptide wherein the HA domain comprises a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1, wherein the modification is a cysteine substitution, wherein the second amino acid sequence is a modified version of SEQ ID NO 4, and which may further comprise a cysteine located at the C-terminus of the second amino acid sequence.

[0108] In at least one embodiment, there is provided herein an isolated nucleic acid encoding an HA polypeptide for wherein the HA domain comprises a first amino acid sequence, wherein 1the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, and an oligomerization domain comprising a second amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1, wherein the modification is a cysteine substitution, wherein the second amino acid sequence is a modified version of SEQ ID NO 5, and which may further comprise a cysteine located at the C-terminus of the second amino acid sequence.Methods

[0109] In at least one embodiment, there is provided herein a method of manufacturing a vaccine comprising conjugating an HA polypeptide, of any of the embodiments disclosed herein, with a virus particle.

[0110] In at least one embodiment, there is provided herein a method of manufacturing a vaccine comprising conjugating a tobacco mosaic virus (TMV) with an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence.

[0111] In at least one embodiment, there is provided herein a method of manufacturing a vaccine comprising conjugating a tobacco mosaic virus (TMV) with an HA polypeptide wherein the HA domain comprises a first amino acid sequence, wherein the first amino acid sequence hasat least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , and an oligomerization domain comprising a second amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1 and comprising a cysteine substitution resulting in the formation of one or more disulfide bonds in the HA stalk, wherein the second amino acid sequence is a modified version of SEQ ID NO 4, wherein the modification is a cysteine located at the C-terminus of the second amino acid sequence.

[0112] In at least one embodiment, there is provided herein a method of manufacturing a vaccine comprising conjugating a tobacco mosaic virus (TMV) with an HA polypeptide, wherein the HA domain comprises a first amino acid sequence, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , and an oligomerization domain comprising a second amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5, wherein the polypeptide is formed as a trimer comprising three strands, wherein the second amino acid sequence comprises a coronin 1 protein located at the C-terminus of the first amino acid sequence, wherein the first amino acid sequence of at least one of the strands is a modified version of SEQ ID NO 1 and comprising a cysteine substitution resulting in the formation of one or more disulfide bonds in the HA stalk, wherein the second amino acid sequence is a modified version of SEQ ID NO 5, wherein the modification is a cysteine located at the C-terminus of the second amino acid sequence.

[0113] In at least one embodiment, there is provided herein a method of inducing an immune response in a subject, comprising administering to the subject a composition comprising the HA polypeptide of any of the embodiments disclosed herein.

[0114] In at least one embodiment, there is provided herein a method of inducing an immune response in a subject, comprising administering to the subject a vaccine comprising the HA polypeptide of any of the embodiments disclosed herein.

[0115] In at least one embodiment, there is provided herein a method of inducing an immune response in a subject, comprising administering to the subject a composition comprising the nucleic acid of any of the embodiments disclosed herein.

[0116] In at least one embodiment, there is provided herein a method of inducing an immune response in a subject, comprising administering to the subject a vaccine comprising the nucleic acid of any of the embodiments disclosed herein.

[0117] In at least one embodiment, there is provided herein a HA polypeptide of any of the embodiments disclosed herein for use in a method of inducing an immune response in a subject, wherein the HA polypeptide induces an immune response against an antigen in the HA polypeptide in the subject.

[0118] In at least one embodiment, there is provided herein a vaccine comprising an HA polypeptide of any of the embodiments disclosed herein for use in a method of inducing an immune response in a subject, wherein the HA polypeptide induces an immune response against an antigen in the HA polypeptide in the subject.

[0119] In at least one embodiment, there is provided herein a nucleic acid encoding an HA polypeptide, of any of the embodiments disclosed herein, for use in a method of inducing animmune response in a subject, wherein the HA polypeptide induces an immune response against an antigen in the HA polypeptide in the subject.

[0120] In at least one embodiment, there is provided herein a vaccine comprising a nucleic acid encoding an HA polypeptide of any of the embodiments disclosed herein for use in a method of inducing an immune response in a subject, wherein the HA polypeptide induces an immune response against an antigen in the HA polypeptide in the subject.MULTIPLE EMBODIMENTS AND ALTERNATIVES

[0121] In some embodiments, oligomerized (e.g., trimerized) HA proteins are produced, which can bind with a viral carrier to form conjugates for use in vaccines. The inventive HA proteins exhibit improved presentation, conformational stability, and resulting immunogenic potency.

[0122] Fig. 1A, also referred to herein as SEQ ID NO 1 and produced at the end of this paragraph, is an amino acid sequence representative of an influenza HA from a B / Phuket / 3073 / 2013 virus. This sequence is exemplary of HA domains associated with the production of HA proteins (sometimes referred to as “HA polypeptides”) in accordance with present embodiments. The sequence in Fig. 1 A also is sometimes referred to as an HAO domain. In Fig. 1 A, the two residues “RG” - part of a longer portion comprising the residues KERGFF - are a cleavage site or loop, shown in bold letters (both in this sentence and in the figure).“(SEQ ID NO: 1)” HAO - B / Phuket:MGKMASLFATFLWLVSLSLASESSADRICTGITSSNSPHWKTATQGEVNVTGVIPLTT TPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSG CFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFA TMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIWDYMMQKPGKTGTIVYQRGVLLPQ KVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLK LANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVL LSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGE FSLPTFDSLNITAASLNDDGLDNHTHHHHHHDEL

[0123] FIG. IB, also referred to herein as SEQ ID NO 2, is an amino acid sequence of the HA1 domain of the B / Phuket / 3073 / 2013 virus. Fig. IB represents a portion of Fig. 1A (HAO domain).

[0124] “(SEQ ID NO: 2)” HA1 domainMGKMASLFATFLWLVSLSLASESSADRICTGITSSNSPHWKTATQGEVNVTGVIPLTT TPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFA TMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIWDYMMQKPGKTGTIVYQRGVLLPQ KVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKE

[0125] FIG. 1C, also referred to herein as SEQ ID NO 3, is the HA2 domain of the B / Phuket / 3073 / 2013 virus, and also represents a portion of Fig. 1A.

[0126] “(SEQ ID NO: 3)” HA2 domain:FFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELE VKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTHHHHHHDEL

[0127] The cleavage of HAO into the HA1 and HA2 domains occurs during expression, for example in the cells of a host plant, and is triggered by host cell proteases, with a likelihood of varying activity at this cleavage loop across different strains of HAs.

[0128] Fig. 2A - FIG. 2E are schematic representations showing examples of HA constructs for producing HA proteins. Each such antigen comprises an HA1 domain, which primarily is the globular head, and an HA2 domain, which primarily is the stalk of the HA protein. In this context, the term “domain” can refer either to an HA domain, or individual shorter domains comprising an HA domain, such as an HA1 domain and an HA2 domain, respectively. In other contexts as used herein, the term “domain” can refer to shorter amino acid sequences, such as coronin domains or other oligomerization domains discussed herein.

[0129] Fig. 2A is a schematic showing the globular head and the stalk of an HA protein, as might be contained in the aforementioned HAO, including signal peptide (SP) proximal to the N- terminal and polyhistidine-tag (HIS TAG) proximal to the C-terminal. Fig. 2B is a schematic similar to Fig. 2A, but modified by adding an oligomerization domain to facilitate the formation of oligomeric antigens, for example trimers. Fig. 2C is a schematic similar to Fig. 2A, but modified by adding a substituted cysteine (here, abbreviated according to its 3 -letter code, “Cys”) at the C- terminus to facilitate conjugation with a virus particle.

[0130] Fig. 2D indicates further modification using an oligomerization domain, such as but not limited to a coronin domain of SEQ ID NO 5, to induce trimerization, combined with a plurality of cysteine substitutions, one in the stalk to induce disulfide bond formation for added stability of the trimer, and one proximal to the C-terminus to facilitate conjugation with a virus particle. As previously noted, this cysteine being referred to as proximal to the C-terminus should not selfconjugate but rather should remain unpaired following the trimerization of the antigen, so as tofacilitate conjugation with the virus without inhibiting the antigen from remaining in the folded configuration.

[0131] Fig. 2E is similar to Figure 2D, but instead of one substituted cysteine it includes two substituted cysteines in the stalk, as representative of SEQ ID NOS. 10 and 12 discussed in Examples 5 and 8, respectively.Use of Oligomerization Domains

[0132] HA proteins presented to a cell in monomer form will likely be processed quickly and have a prohibitively short half-life following administration. Conversely, trimeric forms of such HA proteins retain stable conformation and are not easily degraded in processing and storage or following administration. Accordingly, in some embodiments, an HA domain is combined with a coronin domain, to express an HA protein. An exemplary peptide structure suitable for a coronin domain within the scope of present embodiments is a 37-residue coiled-coil domain of a human coronin protein, hCor-lA, referred to herein as SEQ ID NO 4:TPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQA

[0133] Oligomerization domains used in the present disclosure, such as coronin domains, may comprise hydrophobic core residues such as isoleucine and leucine, with polar residues like glutamine and threonine having reactive side chains. Another exemplary peptide structure within the scope of present embodiments is a 32-residue coiled-coil domain of a murine coronin protein, containing a motif at positions comprising polar residues at positions 21 - 26, Arg (R) -Glu (E), which is a substantial determinant of the peptide’s ability to form trimers. The sequence is sometimes referenced in the art as mCor-1 A, and it is referred to herein as SEQ ID NO 5 :VSRLEEDVRNLNAIVQKLQERLDRLEETVQAK

[0134] When a plurality of short coiled coils undergo trimerization, for example either the exemplary sequence of SEQ ID NO 4 or that of SEQ ID NO 5, antigenic components which are bound to each short coiled coil likewise interact with each other, thus producing the trimeric form of the influenza antigen.

[0135] The following examples will further describe and illustrate in a non-limiting manner a number of embodiments disclosed herein.Example 1 - Construct for Expressing HA with Coronin Trimerization Domain

[0136] As one non-limiting example for practicing multiple embodiments disclosed herein, a construct was designed and built to contain genes encoding the domains necessary to synthesize an HA polypeptide having an HAO domain as a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence. In the present example, the following amino acid sequences were combined for the synthesis of the aforementioned HA polypeptide:

[0137] a first amino acid sequence was SEQ ID NO1; which is combined with,

[0138] a second amino acid sequence was SEQ ID NO 4; resulting in,

[0139] an HA polypeptide having the follow amino acid sequence and referred to as:

[0140] “(SEQ ID NO: 6)” HA with trimerization domain amino acid sequence:DRICTGITSSNSPHWKTATQGEVNVTGVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNC TDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEKIRLS TQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFATMAWAVPKDNYKNATNPLTVEVPYI CTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDG GLPQSGRIWDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEFFGAIAGFL EGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTIS S QIELAVLLSNEGIINSEDEHLLALERKLKKMLGPS AVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHT TPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQA.

[0141] In the context of the present embodiments, the second amino acid sequence can be a coronin domain, which is located at the C-terminus of the first amino acid sequence.

[0142] In some embodiments, a plasmid is engineered and assembled to contain an expression construct of a recombinant HA for production in a host, for example iNicotiana benthatmana (Nb) plants. Likewise, an optional method for cloning the HA is accomplished using a TMV RNA- Based Overexpression (TRBO) vector, which as known in the art utilizes a 35S promotor-driven TMV replicon that lacks the TMV coat protein gene sequence, as described in John Lindbo, “TRBO: A High-Efficiency Tobacco Mosaic Virus RNA-Based Overexpression Vector,” Plant Physiol. Vol. 145, 2007. Such antigenic clones were used to transform Agrobacterium tumefaciens, again through methods known in the art, in preparation for infiltration into a host where expression occurs, e.g., Nb plants. It will be understood that the teachings herein are not limited to Nb plants, or plants generally, but rather may include non-plant hosts such as, without limitation, bacterial, algal, yeast, insect, or mammalian organisms. As illustrated and discussed further herein, the expression of an amino acid sequence in accordance with this example formed a trimeric HA polypeptide comprising an HA domain, having an HA1 domain then an HA2 domain fused with an oligomerization domain, the latter of which was a coiled-coil domain of a human co ronin protein, hCOR-lA. According to multiple embodiments described herein, the trimeric antigen formed in this example was conjugable with a TMV, and exhibited favorable potency.Example 2 - Alternative Construct for HA with Coronin Trimerization Domain

[0143] In SEQ ID NO 6, the second amino acid sequence comprises SEQ ID NO 4. However, other coronin sequences could be substituted for the second amino acid sequence, for example SEQ ID NO 5, resulting in

[0144] “(SEQ ID NO: 7)” HA with an alternative trimerization domain as disclosed in SEQ ID NO: 5:DRICTGITSSNSPHWKTATQGEVNVTGVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNC TDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEKIRLS TQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFATMAWAVPKDNYKNATNPLTVEVPYI CTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDG GLPQSGRIWDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCL HEEYGGLNI<SI<PYYTGI<HAI<AIGNC PIWVI<TPLI<LANGTI<YRPPAI<LLI<EFFGAIAGFL EGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSG AMDELHNEILELDEKVDDLRADTIS S QIELAVLLSNEGIINSEDEHLL A LERKLK KMLGPS AVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHT VSRLEEDVRNLNAIVQKLQERLDRLEETVQAK

[0145] HA proteins in accordance SEQ ID NO 7 were produced in the same manner as with Example 1. Again, the expression of the amino acid sequence in this example formed a trimeric HA polypeptide comprising an HA domain, having an HA1 domain then an HA2 domain fused with an oligomerization domain, the latter of which was a coiled-coil domain of a murine coronin protein, mCOR-lA. The trimeric antigen formed in this example was conjugable with a TMV, and exhibited favorable potency.Disulfide Bonds - Stabilizing Effects and Positioning

[0146] Fig. 3 is a ribbon diagram of a trimer having at least one disulfide bond within a region of the stalk indicated by a dashed-line circle in the figure, and denoted by a reference numeral, 300, for improved stability of the trimer. The reference numeral is not meant to signify any specific amino acid position in the sequence. The head of each strand, primarily the HA1 domain, contains a receptor binding site for attaching to and thereby gaining access to cells. The stalk of each strand, primarily the HA2 domain, comprises a-helices, the C-terminus of which can be joined to an oligomerization domain, such as a coiled-coil domain of a human coronin protein, thereby inducing trimerization. In Fig. 5A, substituted cysteines are represented at positions 46 and 419 of the Anti-B / Phuket / 3073 / 2013 Antigen. In other figures, as well as sequences contained herein, different HAs contain substituted cysteines at a different position(s) than for the Anti- B / Phuket / 3073 / 2013 Antigen. In some embodiments, this cysteine may be positioned at + / - 5 residues (i.e., plus or minus 5 positions) of one or both of the referenced positions of this antigen. In some embodiments, an additional cysteine residue is added at the C-terminus of one or more of the strands in each trimer.

[0147] Accordingly, Fig. 4A illustrates a modified tobacco mosaic virus prior to conjugation with at least one antigen. Additionally, Fig. 4B illustrates the virus of Fig. 4 A, after conjugation with one or more antigens, in which following conjugation the modified TMV is decorated with antigens and amenable to being produced as a vaccine.

[0148] Returning briefly to the antigen portion of these conjugates, an advantage of the use of disulfide bond(s) is to stabilize the trimeric structure to avoid or limit break down into monomers and subsequent degradation. Added stability also helps retain the folded state of the HA polypeptide, increasing its presentation within the subject after vaccine administration.

[0149] A challenge encountered in the relevant field is the proper number and location of disulfide bonds. One reason a consistent approach in this regard has proven difficult involves the variability of HA proteins from one strain to another.Example 3 - HA Construct - Coronin Domain. Disulfide Bonding

[0150] Again, by way of non-limiting examples, constructs were designed and built to contain genes encoding the domains necessary to synthesize an HA polypeptide having an HA domain as a first amino acid sequence, an oligomerization domain comprising a second amino acid sequence, and substituted cysteine residues forming disulfide bonds in the trimeric antigen produced using this construct.

[0151] In this regard, both trimeric and monomeric HA polypeptides were synthesized for use in the following examples. Figure 5A is a schematic diagram of a modified trimeric Anti- B / Phuket / 3073 / 2013 HA. Figure 5B is a schematic diagram of a monomeric Anti- B / Phuket / 3073 / 2013 HA, the performance of which was compared to the trimeric form mentioned in Fig. 5A. Figure 5C is a schematic diagram of a modified trimeric Anti-A / Anhui / 1 / 2013 (H7N9) HA. Figure 5D is a schematic diagram of a monomeric Anti-A / Anhui / 1 / 2013 (H7N9) HAc, , the performance of which was compared to the trimeric form mentioned in Fig. 5C. Figure 5E is a schematic diagram of a modified trimeric Anti-A / Victoria / 2570 / 2019 HA according to multiple embodiments herein. The trimeric antigens mentioned this paragraph are discussed in Examples 4-8 below, and demonstrated effective immunogenic responses indicating improved conformational stability and improved potency.Example 4 - Anti-B / Phuket / 3073 / 2013 Free Antigen Assays

[0152] To evaluate and quantify the effects of enhanced trimerization and of added disulfide bond(s) in certain inventive HA polypeptides according to present embodiments, animmunogenicity study was performed that tested the immune response of New Zealand White rabbits (NZW) to B / Phuket / 3073 / 2013 (Yam) HA free antigen with and without these modifications after two intramuscular doses.

[0153] In this example, a trimerized Anti-B / Phuket / 3073 / 2013 antigen having the following amino acid sequence was studied:

[0154] SEQ ID NO 8:MGKMASLF ATFLWLVSLSLASES S ADRICTGITS SNSPHWKT ACQGEVNVTGVIPLTT TPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSG CFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFA TMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIWDYMMQKPGKTGTIVYQRGVLLPQ KVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLK LANGTKYRPPAKLLKEETFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKS TQEAINCITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGE FSLPTFDSLNITAASLNDDGLDNHTGSGSVSRLEEDVRNLNAIVQKLQERLDRLEETVQA KGSHHHHHHDEL.

[0155] The amino acid sequence for this trimeric antigen is diagrammed schematically in Figure 5 A, wherein a cysteine has been substituted at positions 46 and 419, and the coronin domain of SEQ ID NO 5 has been inserted at positions 563 to 594. It will be further noted SEQ ID NO 8 does not include the residues KERGFF as a cleavage site; rather, the residues at this portion areKEETFF.

[0156] Also as examples of antigen design options for multiple embodiments herein, the N- terminus of SEQ ID NO 8 contains a signal peptide (MGKMASLFATFLVVLVSLSLASESSA), and proximal to the C-terminus contains a polyhistidine-tag plus ER retention sequence (HHHHHHDEL). However, it is noted that because free antigen was used in this example, the polyhistidine-tag would not be needed for detection of a successful conjugation.

[0157] Additional analysis related to this example was obtained through comparison of the trimerized antigen to a monomeric Anti-B / Phuket / 3073 / 2013 antigen, the latter having the following amino acid sequence:

[0158] SEQ ID NO 9:MGKMASLFATFLVVLVSLSLASESSADRICTGITSSNSPHWKTATQGEVNVTGVIPLTT TPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSG CFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFA TMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGDSNPQK FTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIWDYMMQKPGKTGTIVYQRGVLLPQ KVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLK LANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKS TQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVL LSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGE FSLPTFDSLNITAASLNDDGLDNHTHHHHHHDEL.

[0159] The amino acid sequence for the monomeric antigen is diagrammed schematically in Figure 5B, having no substituted cysteine nor added coronin domain.

[0160] A summary of the study design appears in Table 1 , below.

[0161] Table 1N = number of rabbits

[0162] Both versions of free antigen were delivered to test animals via intramuscular injection, in this case without use of a viral carrier. Each animal in the two groups received the same dose on the same days, and blood draws were obtained on the same day for each animal. The only variable was the structure of the antigen. Specifically, V.2 contained a coronin domain to enhance trimerization of the HA peptides comprising SEQ ID NO: 8. In addition, V.2 contained an additional cysteine residue at position 419 to further stabilize the trimer through disulfide bonding, as shown in Fig. 5A. Serum was collected from each animal once on Days 1 and 22 (prior to each dose administration) and once on Days 43, 64 and 84 and stored at < -65°C until immunogenicity analysis.

[0163] Immunogenicity assays performed, as identified in Table 1 , included ELISA analyses of anti-Phuket antibodies generated, hemagglutination inhibition (HAI) assay, and neutralization assay. The results of these studies are shown graphically in Figs. 6A (ELISA), 6B (HAI), and 6C (neutralization titers), respectively, and in general terms were carried out as follows.

[0164] Anti-HA ELISA: serum samples were analyzed for total rabbit IgG to B / Phuket / 3073 / 2013 (Yam) HA. Serum samples were thawed, and duplicate samples were plated in B / Phuket / 3073 / 2013 (Yam) antigen-coated 96-well ELISA plates. Captured anti -HA IgGs were detected by the addition of a conjugated anti-rabbit IgG. ELIS As were developed by the sequential addition of anti-rabbit IgG conjugated to horseradishperoxidase (HRP) and 3, 3’5,5’- tetramethylbenzidine (TMB) Peroxidase Substrate. Reactions were stopped by the addition of a IM phosphoric acid stopping solution. Plates were read at 450 and 650 nm on a commercially available microplate reader.

[0165] Influenza hemagglutinin inhibition (HAI) assay: nonspecific inhibitors (agglutinins) were removed from serum samples enzymatically. The samples were then serially diluted against a defined amount of influenza virus and mixed with Turkey red blood cells. Sera was tested against influenza virus: B / Phuket / 3073 / 2013, with antibody titers defined by the lowest serum dilution causing complete inhibition of red blood cell agglutination.

[0166] Neutralization assay: study serum samples were inactivated and underwent an initial dilution with PBS. The inactivated sera were serially diluted and then incubated with the standardized virus, B / Phuket / 3073 / 2013. Aliquots of serum / virus mixture were loaded into respective wells of a 96-well plate over a monolayer of mammalian cells for culturing and then incubated in a CO2 incubator. After incubation, inoculate from each well was removed and an emulsifying overlay was applied to every well. Overlaid plates were incubated in a carbon dioxideincubator overnight. After incubation, the cells were fixed in paraformaldehyde (PF A) and then washed and stained with primary antibody; anti-influenza B nucleoprotein antibody; followed by a conventional anti-mouse IgG, and the reaction was stopped with water and allowed to dry protected from light. Plaques were then visualized and enumerated using a commercially available enzyme-linked immune absorbent spot reader.

[0167] According to Fig. 6A, both treatment groups demonstrated an increase in antiPhuket IgG titers that peaked on Day 43, followed by decreasing levels over Days 64 and 84, respectively. Averaging the results with each group, both groups had the highest titers on Day 43, with the substantially more trimeric Phuket V.2 Free Antigen (represented by triangles in the upper curve shown in the graphs) generating higher titer levels. Thus, Phuket V.2 Free Antigen was significantly more effective and longer lasting compared to Phuket V.1 Free Antigen (represented by circles in the lower curve in the graphs) on Day 43 and Day 64.

[0168] Similar differences are observable in Fig. 6B with regard to the HAI assays, in which for the examples described geometric mean titers were recorded as the reciprocal of the highest serum dilution that exhibits complete inhibition of hemagglutination. Results through day 22 showed a GMT below the level of detection (i.e., < 10). Animals that received Phuket V.1 Free Antigen showed GMT values <10 across all days, with the exception of one animal (KY081), which expressed detectable levels of titers on days 43-84. Conversely, all animals that were administered Phuket V.2 Free Antigen had detectable titers on Day 43, Day 64, and Day 84 with significantly higher peak titers at Day 43 compared to Phuket V.l Free Antigen treated animals. As with the ELISA analysis, peak titers were detected for Phuket V.2 Free Antigen treated animals on Day 43, followed by decreasing levels over Days 64 and 84, respectively.

[0169] In a neutralization study (Fig. 6C), again through day 22, there was a GMT below the level of detection (i.e., < 100). Also consistent with the other two studies, lower levels of titers were observed in animals that were administered Phuket V.1 Free Antigen. Conversely, titer levels were significantly higher in Phuket V.2 Free Antigen treated animals on each of Days 43, 64, and 84 compared to Phuket V.1 Free Antigen treated animals. As with the other two studies, peak titers were detected for Phuket V.2 Free Antigen treated animals on Day 43, followed by a decreased level on Day 64 then a slight increase between Days 64 and 84.

[0170] Accordingly, Phuket V.2 Free Antigen performed consistently better than Phuket V.1 Free Antigen throughout the study, with the former maintaining significantly higher titers by comparison, and showing significantly more effective and longer lasting results of the two versions studied. As Table 1 indicates, the comparative study did not introduce variables related to concentration, dosing, or blood draws, indicating that enhanced trimerization and stabilization through disulfide bonds for Phuket V.2 Free Antigen account for the improved performance.

[0171] A significant consideration stems from the variability between influenza strains, particularly in the HA1 domain primarily comprising the head of these viruses. While some of the general teachings provided herein also can be applied to more than one influenza virus strain, in view of the variability that exists, in some cases there will be a need or a benefit to varying the amount or location of disulfide bonds to achieve even further improvements in the results.Example 5 - Anti-A / Anhui / 1 / 2013 (H7N9) Rabbit Immunogenicity Study

[0172] In this example, a trimerized Anti-A / Anhui / 1 / 2013 antigen having the following amino acid sequence was studied:

[0173] SEQ ID NO 10:MGKMASLFATFLVVLVSLSLASESSADKICLGHHAVSNGTKVNTLTERGVEWNATETVERTNIPRICSKGKKTVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEECLRQILRESGGIDKEAMGFTYSGIRTNGATSACRRSGSSFYAEMKWLLSNTDNAAF PQMTKSYKNTRKSPALIVWGIHHSVSTAEQTKLYGSGNKLVTVGSSNYQQSFVPSPGAR PQVNGQSGRIDFHWLMLNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQVDANCEG DCYHSGGTIISNLPFQNIDSRAVGKCPRYVKQRSLLLATGMKNVPEIPKGETLFGAIAGFI ENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNE FNEVEKCIGNVINWTRDSITEVWSYNAELLVAMENQHTIDLADSEMDKLYERVKRQLR ENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAMQNRIQIDPVKLVSRLEEDV RNLNAIVQKLQERLDRLEETVQAKGSPPCPHHHHHHDEL

[0174] The amino acid sequence for this trimeric antigen is diagrammed schematically in Figure 5C, wherein a cysteine has been substituted at positions 123 and 423, and a coronin domain according to SEQ ID NO 5 has been inserted at positions 526 to 557. In some embodiments, the substituted cysteine is positioned at + / - 5 residues of one or both of the referenced positions with respect to the Anti-A / Anhui / 1 / 2013 antigen.

[0175] Additional analysis related to this example was through comparison of the trimerized antigen to monomeric Anti-A / Anhui / 1 / 2013 antigen, the latter having the following amino acid sequence:

[0176] SEQ ID NO 11 :MGKMASLFATFLVVLVSLSLASESSADKICLGHHAVSNGTKVNTLTERGVEWNATET VERTNIPRICSKGKKTVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFV NEEALRQILRESGGIDKEAMGFTYSGIRTNGATSACRRSGSSFYAEMKWLLSNTDNAAF PQMTKSYKNTRKSPALIVWGIHHSVSTAEQTKLYGSGNKLVTVGSSNYQQSFVPSPGARPQVNGQSGRIDFHWLMLNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQVDANCEG DCYHSGGTIISNLPFQNIDSRAVGKCPRYVKQRSLLLATGMKNVPEIPKGETLFGAIAGFI ENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNE FNEVEKQIGNVINWTRDSITEVWSYNAELLVAMENQHTIDLADSEMDKLYERVKRQLR ENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAMQNRIQIDPVKLVPRGSSGH HHHHHDEL

[0177] The amino acid sequence of the monomeric antigen is diagrammed schematically in Figure 5D, with no substituted cysteine nor added coronin domain.

[0178] Assays were performed on two versions of antigen administered to test animals (rabbits), and these were compared to Placebo. In this way, the beneficial effects of enhanced trimerization and of added disulfide bond(s) on one version of antigen studied were able to be evaluated and quantified. A summary of the study design appears in Table 2, below.

[0179] Table 2* influenza virus H7N9 Anhui 2013 hemagglutinin antigen

[0180] The two versions of antigen and Placebo were delivered to animals in the respective groups via intramuscular injection, some as free antigen and some with use of a viral carrier, at different doses, with blood draws obtained on the same day for each animal as shown in Table 2. Again, the main variable between the two treatment groups was the structure of the antigen. Specifically, FLU-H7263 contained a coronin domain to enhance trimerization of the H7 peptides, and FLU-H7263 contained a substituted cysteine residue at positions 123 and 423 to further stabilize the trimer through disulfide bonding, as shown in Fig. 5C. In some embodiments, this cysteine is positioned at + / - 5 residues of the referenced positions.

[0181] Further with respect to the current example, serum was collected from each animal once on Days -2 and 19 (prior to each dose administration) and once on Days 42, 63 and 84 and stored at < -65°C.

[0182] Assays performed on both non-Placebo versions of antigen identified in Table 2 included hemagglutination inhibition (HAI) assays and neutralization assays over the Anti- A / Anhui / 1 / 2013 (H7N9) virus hemagglutinin antigens. Results of these studies are shown graphically in Figs. 7A (HAI), and 7B (neutralization titers), respectively.

[0183] According to HAI assay results shown in Fig. 7A, all samples across the Placebo and treatment groups for Day -2 and Day 19 had a GMT <10 (below level of detection). Now turning to the treatment groups, animals receiving the antigen non-trimerized AFLU-H7073, GMTs (geometric mean titer) began to increase on Day 42 until Day 63, followed by decrease to Day 84, albeit at significantly lower levels on each test day compared to FLU-H7263. In turn, FLU-H7263(trimerized and stabilized with disulfide bond through cysteine substitution) demonstrated a substantially higher increase in GMTs on Day 42 that then decreased over Days 63 and 84. While some response was detected for all three groups after Day 21, FLU-H7263 was significantly more effective compared to FLU-H7073 on Days 42 and Day 63, at least.

[0184] These data, particularly as quantified with respect to comparative results for FLU- 117263, demonstrate that trimerizing the Anti-A / Anhui / 1 / 2013 (H7N9) Virus hemagglutinin antigen (FLU-H7263) showed increased immunogenicity on Days 42 and 63 and was more effective compared to monomeric FLU-H7073. This likely is due to increased in vivo stability and increased antigen authenticity. These data also indicate that lower doses of trimeric FLU-H726 induced superior immune responses compared to higher doses of monomeric FLU-H7073.

[0185] Regarding the neutralization assay (Fig. 7B), both treatment groups demonstrated peak neutralization GMTs on Day 42 that decreased over Days 63 and 84. On Day 42, at least, FLU- 117263 GMTs measured significantly higher compared to Placebo, in contrast to more modest increase for FLU-H7073 compared to Placebo. On Day 63, FLU-H7263 still registered significantly higher than Placebo, while FLU-H7073 shows no discernible increase over Placebo on this day. On Days 63 and 84, high standard deviations were noted with FLU-H7263, but even so GMTs were noticeably higher on those days for FLU-H7263 compared to the other groups.Expression., Purification and Conjugation of HA proteins for Use as Vaccines

[0186] Regarding the embodiments disclosed herein, there are known methods and alternatives for recombinantly manufacturing and purifying HA proteins; for manufacturing, purifying, and rendering inactive a suitable viral carrier for said antigens; and for conjugating an antigen with a viral carrier, resulting in conjugated and purified virus particles decorated with antigens for use as vaccines. Several such methods and alternatives are disclosed in United States Patent No.US11696948B2, “Vaccines formed by virus and antigen conjugation,” July 11, 2023, the contents of which are fully incorporated by reference herein. In some embodiments in accordance with the present disclosure, both HA proteins and viruses are transiently expressed in the cells of a host, for example Nb plants, then harvested for further downstream processing. Conjugation reactions wherein soluble forms of HA proteins as antigens bind to a viral carrier - e.g., a virus particle or a virus such as but not limited to a tobacco mosaic virus (TMV) - can be carried out over a range of virus-to-antigen weight ratios, using one or more various known reagents and buffers, and under a range of process controls (e.g., pH, molarity, reaction time, filtration) which are suitable to achieve the conjugation reaction as known in the art. In some embodiments, an added cysteine is positioned at the C-terminus of a coronin domain found on at least one of the strands. This placement helps limit or avoid interaction with other cysteine residues contained in an HA polypeptide in accordance with present embodiments. This terminal cysteine reacts with one or more residues found on surface coat proteins of a viral carrier such as TMV. For example, in some embodiments, lysine residues on surface coat proteins of a modified TMV bind to this terminal cysteine during conjugation. Thus, in some embodiments surface residues found on the coat proteins of such a virus particle are modified at the N-terminus to provide an amine-containing lysine residue for reacting with and binding to the antigenic component.

[0187] Various optional approaches can be used to document conjugation between HA polypeptides provided herein with a carrier virus particle, such as but not limited to TMV. In an exemplary approach, through known methods a label is bound to the aforementioned terminus, e.g., a terminal cysteine, of an HA polypeptide. The label, for example a polyhistidine tag, is detectable to serve as an indicator of successful conjugation between HA polypeptides and viral components.

[0188] Following successful conjugation and other processing as may be desirable or necessary, vaccines based on these conjugates can be produced, diluted to target concentrations, buffered, and stored for later use following conjugation. Alternatively, purified intermediates of the antigenic and virus particle can be produced and stored separately for a period of time prior to being conjugated. Many options exist, which are well known in the art, for administering one or more vaccines in accordance with present embodiments. These include, but are not necessarily limited to, delivery in the form of unit doses provided to a subject, such as but not limited to administration by syringe or spray through routes that include, but are not limited to, subcutaneous, intramuscular, intradermal administration, and nasal, as well as administration orally by mouth and / or topically, to the extent clinically indicated.Example 6 - Anti-A / Anhui / 1 / 2013 (H7N9) Ferret Challenge Study

[0189] Example 6 describes a study evaluating efficacy of recombinantly expressed influenza vaccines according to present embodiments, of varying doses and comprising the trimeric Anti- A / Anhui / 1 / 2013 antigen of Example 5, SEQ ID NO: 10. In this study, which ferrets were challenged by influenza infection using A / Anhui / 1 / 2013 (H7N9) virus in a pulmonary infection / inflammation model. All animals met the health requirements of the Testing Facility based on the daily observations and were within the age range as stated in the study protocol. The first and last day of immunization occurred on study day 0 and study day 21, respectively. The influenza challenge occurred on study day 42, and the last bleed / end of in-life occurred on study day 65.

[0190] Thirty-six (36) Specific-pathogen-free male ferrets, 4-6 months old, were selected for this study. All selected animals met the protocol inclusion requirements. Table 3 identifies theanimal enrollment and study design of Example 6 with respect to the 6 groups of animals (6 animals per group).Table 31Cytosine phosphoguanine (CpG) dose level: 0.05 mg / DoseSpecies / Type / Gender: Ferret / Specific-pathogen -free / Male

[0191] The animals received 15 or 45 pg of vaccine with or without CpG adjuvant. Doses were calculated based on the HA concentration and diluted in vehicle to the desired dose. The control group received vehicle only (Placebo). All ferrets received 2 doses of vaccine, 3 weeks apart (on study days 0 and 21) and administered in 0.5 mL volume by intramuscular injection. During thevaccination phase there were no notable adverse reactions to the vaccines. Serum samples were collected at 14, 21, 28, 36 and 63 days after the first dose.

[0192] The challenge experiment was performed on study day 42, i.e., 21 days after the second dose. Ferrets were anesthetized with isoflurane and inoculated intranasally with 1 mL of 1 x 106TCID50 / animal of wild-type A / Anhui / 1 / 2013 (H7N9) virus. Signs of infection, weight, and temperature were monitored for 10 days after the challenge. Post-challenge, the presence of clinical signs (Figs. 8 A and 8B) indicates that all animals were infected with influenza H7N9 virus. Body temperatures elevation and body weight loss during the on-set of infection was the typical timing for onset of clinical symptoms after influenza challenge in ferrets. Post-challenge, the clinical signs of disease were reduced in vaccinated groups, including transiently elevated temperature and weight loss.

[0193] On days 2, 4, 6, and 8 after virus inoculation, ferrets were anesthetized with ketamine, and nasal wash samples were collected in 1 mL of phosphate-buffered saline (PBS).

[0194] Seroconversion of challenged and contact animals as described below was assessed using the hemagglutination inhibition (HAI) assay. Whole blood was collected, and the serum was separated by centrifugation at 290 g for 10 minutes. Serum was then diluted 1 :4 with receptor destroying enzyme (RDE) (Denka-Seiken, Tokyo, Japan) and incubated for 18 hours at 37°C. Next, RDE was heat inactivated by incubation at 56°C for 30 minutes. HI assays were performed in U bottom 96-well plates (Corning, Tewksbury, MA) by diluting 50 pL RDE-treated sera 1 :2 in PBS, incubating with 25 pL 4 hemagglutinating units (HAU) of A / Anhui / 1 / 2013 (H7N9) at room temperature for 1 hour, followed by a 30-min incubation with 0.5% chicken red blood cells at room temperature.

[0195] The microneutralization assay as described below was used to measure the neutralizing antibody (nAb) titer in animal sera. The collected sera were inactivated at 56°C for 30 minutes. Sera were serially diluted two-fold from 1 :40 to 1 :5120 in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 4% BSA (Sigma Aldrich, St. Louis, MO, USA), 2% antibiotic antimycotic mixture (Gibco), and 1 pg / ml TPCK-treated trypsin (Worthington Biochemical, Lakewood, NJ, USA), then mixed with equal volume of 100 tissue culture infectious dose (TCID50 / mL) of A / Anhui / 1 / 2013 (H7N9) and incubated for 1 hr at 37°C. A total volume of 100 pL of the virus-sera mix was inoculated in duplicate to Madin -Darby canine kidney (MDCK) cells in a 96-well tissue culture plates. After 1 hr of incubation at 37°C, the inoculums were removed and 200 pL infection medium were added. The plates were then incubated for three more days at 37°C in 5% CO2 in a humidified incubator. A virus back-titration was performed without immune serum to confirm TCID50 viral titer used. Virus hemagglutination activity was then tested in 0.5% chicken red blood cells (RBCs). The absence of hemagglutination was considered a positive test result for antibodies to the virus. A titer of >40 was considered positive.

[0196] As shown in Lig.8A, body weights were taken daily after A / Anhui / 1 / 2013 (H7N9) virus intranasal inoculation (challenge) on study day 42. No animals reached 20% or more of body weight loss. However, challenged animals lost weight as soon as 1 day post challenge indicative of disease onset / seventy. In LLU-H7263 PA (45ug), FLU-H7263-NP (15ug) and FLU-H7263- NP+CpG (45ug) groups, vaccination significantly prevented disease related body weight loss compared to Placebo.

[0197] As shown in Fig.8B, the body temperature of the ferrets was measured prior to challenge and monitored for up to 10 days after the challenge. Mean temperature per group is shown foreach group and normalized to temperature on the day of infection (day 0). An evident rise in body temperature was observed in the control group on study day 1-4, the typical timing for onset clinical signs after influenza challenge in ferrets. In corroboration with the body weight loss data, statistically significant effects of FLU-H7263-NP-H1S (45 pg), FLU-H7263-NP (45 pg), and FLU- 117263 -NP+CpG (45 pg) vaccine administration on body temperature was detected 3 and / or 4 days after challenge.

[0198] Blood was collected from each ferret on study Days 14, 21, 28, 36, and 63, processed to serum, and analyzed by HAI and MNT assays. Fig. 8A provides a graph of weight changes in ferrets monitored for 10 days post-challenge following infection with influenza H7N9 virus. Fig. 8B provides a graph of body temperature changes in ferrets monitored for 10 days post-challenge following infection with influenza H7N9 virus. Data from HAI and microneutralization (MNT) assays are found in Figs. 8C and 8D, respectively. Fig. 8C shows hemagglutination inhibition assay (HAI) quantification of geometric mean antibody titers (GMT) to A / Anhui / 1 / 2013 (H7N9) in serum samples. HAI titers were recorded as the reciprocal of the highest serum dilution that exhibits complete inhibition of hemagglutination. Fig. 8D shows MNT assay quantification of geometric mean titers (GMT) to A / Anhui / 1 / 2013 (H7N9) in serum samples. MNT titers were recorded as the reciprocal of the highest serum dilution at which virus infection was blocked.

[0199] As shown in Figs. 8C and 8D, all vaccinated animals had the presence of anti-influenza Antigen H7N9-specific antibodies except for the Placebo group. Pre-challenge, peak titers for all assays were reported on Day 28 for all vaccinated groups. The HAI geometric mean titers (GMT) of vaccinated groups ranged from 15.9-403.2 on Day 28 with the highest HAI GMT observed in Group FLU-H7263 -NP+CpG (45 pg). Table 4 provides a Two-way ANOVA multiple comparisonanalysis based on the results of the HAI assay, comparing the means of each column with the mean of the control column for the days listed.Table 4

[0200] The Microneutralization (MNT) GMT of vaccinated groups ranged from 17.8 - 570.2 on Day 28 with the highest MNT GMT also observed in Group FLU-H7263-NP+CpG (45 pg). Further, on study Days 28, and / or 36 Groups FLU-H7263-NP-His (45 pg) and FLU-H7263- NP+CpG (45 pg) had significantly elevated HAI titers compared to Placebo group. Correspondingly, on study days 21 and 28 Group FLU-H7263-NP+CpG (45 pg) had significantlyelevated MNT titers compared to Placebo group. Also, on study days 14 and 21, before the second vaccination, only FLU-H7263-NP+CpG (45 pg) treated ferrets had detectable HAI and MNT titers, in contrast to all other groups. Table 5 provides a Two-way ANOVA multiple comparison analysis based on the results of the MNT assay, comparing the means of each column with the mean of the control column for the days listed.Table 5

[0201] For analysis of viral load, nasal swabs were taken after challenge on Days 2, 4, 6, and8. Peak infectious virus was detected in all groups 2 days after challenge which subsequentlydeclined to undetectable levels by Day 8 post-challenge. On Day 4 post-challenge, FLU-H7263- NP (45 pg) and FLU-H7263-NP+CpG (45 pg) vaccinated animals had significantly reduced viral load. In the latter group, only 2 of 6 animals had detectable viral load. By Day 6 after challenge, all vaccinated groups had a significantly reduced viral load as determined by the nasal wash viral titers compared to the Placebo group.

[0202] Furthermore, it was noted that no virus was detectable in FLU-H7263-NP-His (45 pg), FLU-H7263-NP (15 pg), FLU-H7263-NP (45 pg), or FLU-H7263 -NP+CpG (45 pg) treated animals 6 days post challenge. By comparison, 5 of 6 Placebo group animals and 1 of 6 FLU- 117263 FA (45 pg) treated animals still had detectable viral load. Fig. 8E shows viral titers from nasal wash samples of vaccinated and saline control ferrets after challenge with A / Anhui / 1 / 2013 (H7N9) virus, over 8 days. Viral titers are presented as TCID (tissue culture infectious dose-50) means and standard deviations. Table 6 provides a Two-way ANOVA multiple comparison analysis based on the results of this assay, comparing the means of each column with the mean of the control column for the days listed.Table 6

[0203] Accordingly, Example 6 showed that all immunized animals had anti-H7N9 antibody titers prior to challenge as indicated by HAI and MNT assay results. At all timepoints, FLU- H7263-NP+CpG (45) treated animals showed the highest immune responses. The Viral load analysis post-challenge from the nasal wash viral titers demonstrated that all vaccinated animals cleared virus faster than non-treated animals. Furthermore, animals immunized with FLU-H7263- NP (45) and FLU-H7263-NP+CpG (45) cleared virus more rapidly compared to all other groups.Example 7 - Anti-A / Anhui / 1 / 2013 (H7N9)

[0204] To evaluate and quantify the effects of enhanced trimerization and of added disulfide bond(s) in certain inventive HA polypeptides, according to present embodiments, immune responses to Anti-A / Anhui / 1 / 2013 (H7N9) HA free antigen were studied in test animals (rabbits). The antigens tested included one with the modifications (trimerized form, with added disulfide bond(s)), and one in monomeric form without these modifications. A summary of the study design appears in Table 7, below.

[0205] Table 7

[0206] #influenza virus H7N9 Anhui 2013 hemagglutinin antigen

[0207] *Body wt taken on Study Day 83 to determine drug dosages for Study Day 84

[0208] The H7073 identifiers were A22FLU016, pKB073, and the sequence for this monomeric antigen is that listed in Example 5, SEQ ID NO 11. The H7263 identifiers were A22FLU014, pKBP263, and the sequence for this trimeric antigen is that listed in Example 5, SEQ ID NO 10.

[0209] Both monomeric and trimeric versions of free antigen were delivered to test animals via intramuscular injection. As reflected in Figs. 9A and 9B, each animal in the FLU-H7073 group received a 15 pg dose, while each animal in the FLU-H7263 group received a 5.5 pg dose. Blood draws were obtained on the same day for each animal in all the groups, again with the only variable being the structure of the antigen. Specifically, FLU-H7263 contained a coronin domain to enhance trimerization of the HA peptides comprising SEQ ID NO: 10. Also, FLU-H7263 contained an additional cysteine residue at position 423 to further stabilize the trimer through disulfide bonding, as shown in Fig. 5C. Serum was collected from each animal once on Days -2 and 19 (prior to each dose administration) and once on Days 42, 63 and 84 and stored at < -65°C.

[0210] Assays performed included hemagglutination inhibition (HAI) assay and neutralization assay. The results of these studies are shown graphically in Figs. 9A (HAI) and 9B (neutralization titers), respectively, and in general terms were carried out in the same manner as Example 4, indicating the following results.

[0211] All samples across all Groups for Day -2 and Day 19 had a GMT <10 (below level of detection). Monomeric FLU-H7073 GMTs (geometric mean titer) began to increase on Day 42 until Day 63, decreasing on Day 84. Trimeric FLU-H7263 demonstrated an increase in GMTs on Day 42 that then decreased over Days 63 and 84.

[0212] While a response was detected for all groups, Fig. 9A indicates that FLU-H7263 was significantly more effective compared to FLU-H7073 on Days 42 and Day 63. Likewise, in Fig. 9B both treatment groups demonstrated peak microneutralization GMTs on Day 42 that decreased over Days 63 and 84. On Day 42, FLU-H7263 GMTs were significantly higher compared to Placebo, in contrast to FLU-H7073 which registered only slightly higher than Placebo. In regards to the data reflected in both figures, higher standard deviations are noted, but it can be observed that GMTs were substantially higher for FLU-H7263 in both cases.

[0213] Accordingly, these data demonstrate that trimerizing the H7N9 Anhui 2013 Virus hemagglutinin antigen (FLU-H7263) increased its immunogenicity, likely due to increased in vivo stability as well as increased antigen authenticity. Additionally, these data establish the dosesparing effects of trimeric FLU-H7263 compared to monomeric FLU-H7073, as the former was dosed ~3 fold less than FLU-H7073 and induced superior immune responses as indicated in Figs. 9 A and 9B.Example 8 - Anti-A / Victona / 2570 / 2019 (HIND

[0214] In this example, a trimerized Anti-A / Victoria / 2570 / 2019 antigen having the following amino acid sequence was studied:

[0215] SEQ ID NO 12:MGKMASLFATFLWLVSLSLASESSADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLL EDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYP GDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDNGVTAACPHAGAKSFYKNLIWLV KKGKSYPKINQTYINDKGKEVLVLWGIHHPPTIADQQSLYQNEDAYVFVGTSRYSKKFK PEIATRPKVRDREGRMNYYWTLVEPGDKITFEATGNLVAPRYAFTMERDAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSETLFCAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQF TAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKCLYEK VRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLV PRGSSGVSRLEEDVRNLNAIVQKLQERLDRLEETVQAKGSPPCPHHHHHHDEL.

[0216] The amino acid sequence is diagrammed schematically in Figure 5E, wherein a cysteine has been substituted at positions 357 and 423, and the coronin domain of SEQ ID NO 5 has been inserted at positions 539 to 570. In some embodiments, the substituted cysteines are positioned at + / - 5 residues of one or both of the referenced positions with respect to the Anti- A / Victoria / 2570 / 2019 antigen.

[0217] A dose-response study was performed on rabbits using this Anti-A / Victoria / 2570 / 2019 antigen conjugated with a viral carrier. A summary of the study design, in view of different concentrations of the same antigen compared to Placebo, appears in Table 8, below.

[0218] Table 8

[0219] For non-Placebo groups, the vaccine antigen was the same (except for concentration) and was identified as FLU-VT280-NP SMCC

[0220] Blood was collected from each rabbit on study Days 1, 22, 43, 64 and 84 and processed to serum. Serum samples from Days 1, 22, 43, 64 and 84 were analyzed via HAI assay. All vaccinated animals had the presence of anti-influenza Antigen A / Victoria-specific antibodies except for Placebo.

[0221] Fig. 10A shows the results of a Hemagglutination inhibition assay (HAI) as indicated by quantification of geometric mean antibody titers (GMT) to A / Victoria / 2570 / 2019 in serum samples. Fig. 10B shows ELISA measurement of GMT against Influenza Antigen A / Victoria / 2570 / 2019. The results of these studies are shown graphically in these figures, and in general terms were carried out in the same manner as Example 4.

[0222] With respect to Fig. 10A, the HAI geometric mean titers (GMT) of immunized groups ranged from 183-1114 on Day 43. Peak titers were observed on Day 43 or 64 for all immunized groups. An HA related dose-response was observed on study Days 22, 43, 65, and 83 between FLU-VT280-NP SMCC 1.0 pg HA, FLU-VT280-NP SMCC 3.0 pg HA, FLU-VT280-NP SMCC 8.0 pg HA, and FLU-VT280-NP SMCC 75.0 pg HA, according to HAI titers. The Placebo Group also was compared to adjuvanted and non-adjuvanted groups FLU-VT280-NP SMCC 3.0 pg (non- adjuvanted group) and FLU-VT280-NP SMCC 3.0 pg + CpG (adjuvanted group), both of which were dosed on Day 43. The comparison indicated that the FLU-VT280-NP SMCC 3.0 pg + CpG adjuvanted group had significantly enhanced immune responses compared to Placebo, whereas the FLU-VT280-NP SMCC 3.0 pg non-adjuvanted group did not.

[0223] Turning to Fig. 10B, blood was collected from each rabbit on study Days 1, 22, 43, 64 and 84 and processed to serum. Serum samples from Days 1, 22, 43, 64 and 84 were analyzed via ELISA. All vaccinated animals had the presence of anti-influenza Antigen A / Victoria-specific antibodies except for Placebo. The anti-HA GMT of immunized groups ranged from 32,959.7-396,816.0 on Day 43. Peak titers were observed on Day 43 or 64 for all immunized groups. An HA related dose-response was observed on study Days 22, 43, 65, and 83 between FLU-VT280- NP SMCC 1.0 pg HA, FLU-VT280-NP SMCC 3.0 pg HA, FLU-VT280-NP SMCC 8.0 pg HA, and FLU-VT280-NP SMCC 75.0 pg HA according to anti-influenza Antigen A / Victoria specific titers. The Placebo Group also was compared to adjuvanted and non-adjuvanted groups FLU- VT280-NP SMCC 3.0 pg (non-adjuvanted group) and FLU-VT280-NP SMCC 3.0 pg + CpG (adjuvanted group), both of which were dosed on Day 43. Again, the comparison indicated that the FLU-VT280-NP SMCC 3.0 pg + CpG adjuvanted group had significantly enhanced immune responses compared to Placebo, whereas the FLU-VT280-NP SMCC 3.0 pg non-adjuvanted group did not.

[0224] Examples 4-8 have been directed to the Anti-B / Phuket / 3073 / 2013, Anti- A / Anhui / 1 / 2013 (H7N9), and Anti-A / Victoria / 2570 / 2019 (H1N1) antigens. Further, it will be appreciated that the current teachings, including substituted cysteine residue(s) for disulfide bonding, addition of an oligomerization domain such as a coronin domain, and / or addition of a cysteine residue at the C-terminus also can be applied to other HAs.

[0225] In view of the above, the following are provided as a non-limiting representation of embodiments included herein.

[0226] Embodiment A is an HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence includes a coronin 1 protein located at or proximal to the C- terminus of the first amino acid sequence.

[0227] Embodiment B is the HA polpeptide of Embodiment A, having a cysteine positioned proximal to the C-terminal of the second amino acid sequence.

[0228] Embodiment C is the HA polypeptide of Embodiment A, having at least one substituted cysteine positioned in a stalk portion of the HA polypeptide.

[0229] Embodiment D is the HA polypeptide of Embodiment A, having two substituted cysteines positioned in a stalk portion of the HA polypeptide.

[0230] Embodiment E is the HA polypeptide of Embodiment A, wherein the first amino acid sequence comprises SEQ ID NO 8, wherein the amino acid sequence has substituted cysteines at positions 46 and 419.

[0231] Embodiment F is the HA polypeptide of Embodiment A, wherein the amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO 8, wherein the amino acid sequence has substituted cysteines at position 46 + / - 5 positions and at position 419 + / - 5 positions.

[0232] Embodiment G is the HA polypeptide of Embodiment A, wherein the first amino acid sequence comprises SEQ ID NO 10, wherein the amino acid sequence has substituted cysteines at positions 123 and 423.

[0233] Embodiment H is the HA polypeptide of Embodiment A, wherein the amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO 10, wherein the amino acid sequence has substituted cysteines at position 123 + / - 5 positions and at position 423 + / - 5 positions.

[0234] Embodiment I is the HA polypeptide of Embodiment A, wherein the first amino acid sequence comprises SEQ ID NO 12, wherein the amino acid sequence has substituted cysteines at positions 357 and 423.

[0235] Embodiment J is the HA polypeptide of Embodiment A, wherein the amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO 12,wherein the amino acid sequence has substituted cysteines at position 357 + / - 5 positions and at position 423 + / - 5 positions.

[0236] Embodiment K is the HA polypeptide of Embodiment A, wherein the second amino acid sequence comprises SEQ ID NO 5.

[0237] Embodiment L is the HA polypeptide of any of Embodiments A-K, wherein the first amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the HA polypeptide recited therein.

[0238] Embodiment M is the HA polypeptide of any of Embodiments A-L, wherein the polypeptide is formed as a trimer comprising three strands, and the second amino acid sequence comprises SEQ ID NO 4 or SEQ ID NO 5.

[0239] Embodiment N is the HA polypeptide of any of Embodiments A-M, wherein the cysteine substitution results in the formation of disulfide bonds in the HA stalk.

[0240] Embodiment O is the HA polypeptide of Embodiment N wherein the disulfide bonds enhance the stability of the HA stalk.

[0241] Embodiment P is the HA polypeptide of Embodiment B wherein the cysteine facilitates the conjugation of the HA polypeptide with a virus particle.

[0242] Embodiment Q is a vaccine containing the HA polypeptide of any of Embodiments A- P, further comprising a tobacco mosaic virus (TMV) wherein the polypeptide is conjugated with the TMV.

[0243] Embodiment R is the HA polypeptide of any of Embodiments A-P, for use in a vaccine.

[0244] Embodiment S is the HA polypeptide of Embodiment N, for use in the manufacture of a vaccine.

[0245] Embodiment T is an isolated nucleic acid encoding the HA polypeptide of any one of Embodiments A-P, R, or S.

[0246] Embodiment U is a method of inducing an immune response in a subject, comprising administering to the subject a vaccine composition comprising the HA polypeptide of any Embodiments A-P, R, or S, wherein the HA polypeptide induces an immune response against an antigen in the HA polypeptide in the subject.

[0247] Embodiment V is an HA polypeptide as recited in any of Embodiments A-P, R, or S, the vaccine of Embodiment Q, or the isolated nucleic acid of Embodiment T, for use in a method of inducing an immune response in a subject, wherein the HA polypeptide induces an immune response against an antigen in the HA polypeptide in the subject.

[0248] Embodiment W is a pharmaceutical composition comprising an HA polypeptide as recited in any of Embodiments A-P, R, or S, the vaccine of Embodiment Q or the nucleic acid of Embodiment T and a pharmaceutically acceptable carrier.

[0249] Embodiment X is the pharmaceutical composition of Embodiment W, wherein the pharmaceutical composition further comprises an adjuvant.

[0250] Embodiment Y is a method of manufacturing a vaccine comprising conjugating an HA polypeptide as recited in any one of Embodiments A-P, R, or S with a virus particle.

[0251] Embodiment Z is the method of Embodiment Y wherein the virus particle is a tobacco mosaic virus (TMV).

[0252] It will be understood that the embodiments described herein are not limited in their application to the details of the teachings and descriptions set forth, or as illustrated in the accompanying figures. Rather, it will be understood that the present embodiments and alternatives, as described and claimed herein, are capable of being practiced or carried out invarious ways. Also, it is to be understood that words and phrases used herein are for the purpose of description and should not be regarded as limiting. The use herein of “including,” “comprising,” “e.g.,” “containing,” or “having” and variations of those words is meant to encompass the items listed thereafter, and equivalents of those, as well as additional items.

[0253] Accordingly, the foregoing descriptions of several embodiments and alternatives are meant to illustrate, rather than to serve as limits on the scope of what has been disclosed herein. The descriptions herein are not intended to be exhaustive, nor are they meant to limit the understanding of the embodiments to the precise forms disclosed. It will be understood by those having ordinary skill in the art that modifications and variations of these embodiments are reasonably possible in light of the above teachings and descriptions.SEQUENCE LISTINGSEQ ID NO Description1 HAO - B / PhuketMGKMASLF ATFLVVLVSLSLASES S AS SNSPHWKTATQGEVNVTGVIPLTTTPTKS YFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFATMAWAVPI<DNYI<NATNPLTVEVPYICTEGEDQITVWGFHSDNI<TQMI<SLYGDSNPQI<FTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIWDYMMQKPGKTGTTVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQI ELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAA GTFNAGEFSLPTFDSLNITAASLNDDGLDNHTHHHHHHDEL2 HA1 - B / PhuketDRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFATMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRTWDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKE3 HA2 - B / PhuketMGKMASLF ATFLVVLVSLSLASES SAFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTHHHHHHDEL4 hCOR-lA: TPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQA5 mCOR-lA: VSRLEEDVRNLNAIVQKLQERLDRLEETVQAK6 HAl+HA2+hcor-l ADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFATMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIWDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADC LHEEYGGLNI<SI<PYYTGI<HAI<AIGNC PIWVI<TPLI<LANGTI<YRPPAI<LLI<EFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQA7 HAl+HA2+mcor-lADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFATMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIWDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKEFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTVSRLEEDVRNLNAIVQKLQERLDRLEETVQAK8 Anti-B / Phuket / 3073 / 2013 Antigen - TrimericMGKMASLFATFLWLVSLSLASESSADRICTGITSSNSPHWKTACQGEVNVTGVIPLT TTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVT SGCFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKI GFFATMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGD SNPQKFTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIWDYMMQKPGKTGTIVYQ RGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNC PIWVKTPLKLANGTKYRPPAKLLKEETFFGAIAGFLEGGWEGMIAGWHGYTSHGAHG VAVAADLKSTQEAINCITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTC LDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTGSGSVSRLEEDVRNLNAIVQK LQERLDRLEETVQAKGSHHHHHHDEL.9 Anti-B / Phuket / 3073 / 2013 Antigen - MonomericMGKMASLF ATFLWLVSLSLASES S ADRICTGIT S SNSPHWKTATQGEVNVTGVIPLT TTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVT SGCFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKI GFFATMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGD SNPQKFT S S ANGVTTHYVSQIGDFPDQTEDGGLPQS GRIWD YMMQKPGKTGTIVYQ RGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNC PIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAH GVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTHHHHHHDEL10 Anti-A / Anhui / 1 / 2013 (H7N9) - TrimericMGKMASLFATFLWLVSLSLASESSADKICLGHHAVSNGTKVNTLTERGVEWNATE TVERTNIPRICSKGKKTVDLGQCGLLGTTTGPPQCDQFLEFSADLIIERREGSDVCYPGK FVNEECLRQILRESGGIDKEAMGFTYSGIRTNGATSACRRSGSSFYAEMKWLLSNTDN AAFPQMTKSYKNTRKSPALIVWGIHHSVSTAEQTKLYGSGNKLVTVGSSNYQQSFVP SPGARPQVNGQSGRIDFHWLMLNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQV DANCEGDCYHSGGTIISNLPFQNIDSRAVGKCPRYVKQRSLLLATGMKNVPEIPKGET LFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKT NQQFELIDNEFNEVEKCIGNVINWTRDSITEVWSYNAELLVAMENQHTIDLADSEMD KLYERVKRQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAMQNRIQI DPVKLVSRLEEDVRNLNAIVQKLQERLDRLEETVQAKGSPPCPHHHHHHDEL11 Anti-A / Anhui / 1 / 2013 (H7N9) - MonomericMGKMASLFATFLWLVSLSLASESSADKICLGHHAVSNGTKVNTLTERGVEWNATE TVERTNIPRICSKGKKTVDLGQCGLLGTTTGPPQCDQFLEFSADLIIERREGSDVCYPGK FVNEEALRQILRESGGIDKEAMGFTYSGIRTNGATSACRRSGSSFYAEMKWLLSNTDN AAFPQMTKSYKNTRKSPALIVWGIHHSVSTAEQTKLYGSGNKLVTVGSSNYQQSFVP SPGARPQVNGQSGRIDFHWLMLNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQV DANCEGDCYHSGGTIISNLPFQNIDSRAVGKCPRYVKQRSLLLATGMKNVPEIPKGET LFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKT NQQFELIDNEFNEVEKQIGNVINWTRDSITEVWSYNAELLVAMENQHTIDLADSEMD KLYERVKRQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAMQNRIQI DPVKLVPRGSSGHHHHHHDEL12 Anti-A / Victoria / 2570 / 2019 (H1N1)MGKMASLFATFLVVLVSLSLASESSADTLCIGYHANNSTDTVDTVLEKNVTVTHSVN LLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGT CYPGDFINYEELREQLS S VS SFERFEIFPKTS S WPNHDSDNGVTAACPHAGAKSF YKNL IWLVKKGKSYPKINQTYINDKGKEVLVLWGIHHPPTIADQQSLYQNEDAYVFVGTSR YSKKFKPEIATRPKVRDREGRMNYYWTLVEPGDKITFEATGNLVAPRYAFTMERDAG SGniSDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPS IQSETLFCAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVN SVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDY HDSNVKCLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEA KLNREEIDGVKLVPRGSSGVSRLEEDVRNLNAIVQKLQERLDRLEETVQAKGSPPCPH HHHHHDEL

Claims

CLAIMSWhat is claimed includes but may not be limited to the following:

1. An HA polypeptide having an HA domain comprising a first amino acid sequence and an oligomerization domain comprising a second amino acid sequence, wherein the second amino acid sequence includes a coronin 1 protein located at or proximal to the C- terminus of the first amino acid sequence.73

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