Design of optimized universal influenza vaccines and their designs and uses

Abstract

To provide vaccine compositions and methods for producing universal influenza vaccines.SOLUTION: A composition for a universal influenza virus vaccine comprises at least two, preferably more than two, different influenza hemagglutinin (HA)-derived antigens. The HA proteins from which the antigens are derived have a hypervariable region located between conserved cysteines at positions 52 and 277, and the hypervariable region is deleted in the antigens. The at least two antigens each have a similarity with HA molecules of more than one influenza serotype in excess of 60, or 70, or 80, as calculated by the emboss explorer cons program.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the priority of Provisional Application No. 63 / 115,459, filed on November 18, 2020, is a non - provisional application, and is hereby incorporated by reference in its entirety into this specification.

[0002] Submission of Sequence Listing A sequence listing containing a file named Sequence_Listing, which is 18 kilobytes in size (measured in MS - Windows) and was created on November 18, 2021, is provided with this specification and is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to a composition containing an influenza HA - derived antigen having high similarity to more than one influenza A serotype. In another embodiment, the present disclosure relates to a composition for a universal influenza vaccine.

Background Art

[0004] Seasonal influenza, pandemic influenza, and highly pathogenic avian influenza (HP Al) originate from large reservoirs in birds and mammals. New influenza variants can rapidly recombine in unpredictable permutations. Since small genetic changes in HP Al can significantly increase its infectivity and enable efficient human - to - human transmission, pandemic spread can actually become a reality.

[0005] For example, H5N1 HP Al emerged in Asia in 2003 via bird-to-human infection. It spread worldwide, with Egypt being a hot spot. The original H5N1 HP Al strain in Asia was of clade 1, while the more recent Egyptian strains are of clade 2 (clade 2.2.1). The first H7N9 HP Al emerged in China in 2013, followed by further variants in 2016 and 2017. The US federal government maintains stockpiles of conventional egg-based H5N1 and H7N9 vaccines. Their immunogenicity, particularly that of the Sanofi-Pasteur clade 1 H5N1 vaccine, was significantly lower than that of standard seasonal influenza vaccines. Vaccines are administered in a prime-boost regimen, and the clade 2 H5N1 GSK vaccine is enhanced by an adjuvant. Other issues are the reduced level of heterologous protection between clade 1 and clade 2 H5N1 vaccines, and the transient nature of the immune response that can become ineffective within six months.

[0006] It has been proposed to address the unpredictable nature of influenza viruses using a broad - reactive vaccine, the so - called "universal" influenza vaccine. The most potent anti - influenza antibodies (Abs) bind to the head of the hemagglutinin (HA) and physically block the interaction with specific cell receptors. The high mutability of this HA domain provides specificity but limits cross - reactivity. Far rarer Abs recognize the highly conserved HA stem region. They bind broadly to several influenza A subtypes. Stem antibodies can act not by inhibiting cell receptor binding, but rather by inhibiting virus - membrane fusion, including ADCC of infected cells, and preventing virus entry into the nucleus. However, current two - antigen stem vaccines can deliver only a primary immunization. Such two - antigen stem vaccines are based on two different "conserved" antigens with relatively low similarity or "sequence overlap" with HA from different influenza serotypes. Thus, it may be necessary to boost the immune response induced by the two - molecule universal antigen with a vaccine based on a specific influenza strain. Some scientific evidence also suggests that broadly reactive Abs may have problems such as inducing disease - enhancing Abs that exacerbate subsequent influenza infections.

[0007] This current approach to immunoprophylaxis against seasonal and pandemic influenza as well as HPAI (and other emerging infectious threats) is severely lacking because existing solutions do not adequately address the following problems. (1) Titer: Current influenza vaccines, including stockpiled HPAI vaccines, provide only moderate transient immune protection. (2) Development speed: Current development and production schemes for influenza vaccines are slow and do not meet the challenges of rapidly emerging influenza (and other infectious threats). (3) Mutability of influenza strains: Influenza has the ability to rapidly change their antigenic composition, necessitating the continuous development of new vaccines to account for new strains and address new specificities.

[0008] It is desirable to develop a more consistent "universal" vaccine that uses conserved antigens derived from influenza hemagglutinin, maintains high similarity or conservation with proteins found in different serotypes of influenza, and induces a strong immune response.

[0009] Classical viral vaccines are produced as attenuated inactivated viruses or viral extracts using the limited technology of slowly growing fertilized eggs. More recent tissue culture techniques include virus growth and synthesis of specific antigens in cell culture broth, as in the case of influenza and ZIKV (Zika virus) vaccines. The low immunogenicity inherent in protein-based vaccines can be enhanced by the addition of adjuvants, which must be enhanced.

[0010] Rather than delivering the completed vaccine, gene immunization techniques cause the host to synthesize the antigen and present it to the immune system. These systems more closely mimic natural viral infection. Various strategies are being studied. Naked DNA vaccines are easy and quick to manufacture but require relatively high doses and special delivery systems to overcome their low immunogenicity. Gene vaccines often utilize engineered viruses such as Ad, vaccinia virus, vesicular stomatitis virus, yellow fever virus, and alphavirus. However, their underlying biology can limit their usefulness. They can be pathogenic, for example, as the 17D yellow fever virus strain, which is associated with rare but severe adverse reactions.

[0011] In addition to naked DNA, AAV, and RNA viruses as the basis for gene transfer, as well as vaccine vector platforms, adenovirus (Ad)-based approaches are also available. Ad-derived vectors have been proven to be benign, avoid integration into the host genome, and are essentially adjuvanted. A number of vaccines have been engineered based on early-generation (eg) Ad vectors minimally modified for replication deficiency. They have repeatedly demonstrated higher immunogenicity in head-to-head comparisons with other vaccine systems. Furthermore, they have also been shown to elicit a strong immune response against avian influenza and ZIKV. Importantly, in contrast to other vaccine systems, they provide sustained immune protection over a long period. Therefore, they have gathered new interest for vaccines against ZIKV, Ebola, tuberculosis, and malaria.

[0012] To overcome the limitations of early Ad systems, Ad vector systems integrating different strategies have been developed. The fully deleted (fd) Ad vector platform packages the vaccine genome into a human serotype with low morbidity, such as human serotype Ad6. Such vaccines are (fd) with all endogenous Ad genes completely deleted. fdAd vectors better focus the immune system on the vaccine antigen, minimize interference by anti-Ad immune responses, and enable prime-boost vaccination. fdAd vector systems using helper virus for capsid formation are associated with contamination by helper virus and replication-competent adenovirus (RCA). These impurities have the potential to induce a strong anti-Ad response. A new fdAd architecture was created, namely an fdAd vector that packages fdAd independently of helper virus (helper virus-independent, hi). These are fully deleted helper virus-independent Ad vectors (fdhiAd vectors).

[0013] The fdhiAd vaccine platform is constructed based on two independently modifiable components: (i) an fdAd vector genome module in which all endogenous Ad genes are deleted, and (ii) a non-packagable circular packaging expression plasmid that delivers the necessary Ad late genes in trans.

[0014] To obtain an fdAd vector genome with all endogenous Ad genes deleted, an fdAd vector-based module is assembled to accommodate different transgene constructs up to 33 kb in size. They "carry" the left and right ITRs and the packaging signals (Ψ) of different Ads.

[0015] The completed fdAd vector genome module is approximately 34 kb in size and is capsid-formed by co-transfection of the non-packagable packaging expression plasmid into host cells. The packaging expression plasmid provides in trans all Ad genes necessary for capsid assembly, replication of the fdAd vector genome module, and its incorporation into the capsid. Different circular packaging expression plasmids are engineered on a modified pBR322 backbone for human Ad serotypes C and B (serotype 35) capsids. They provide in trans the important late genes (L1, L2, L3, L4, L5) along with the early genes E2 and E4, and lack the packaging signal y and at least one ITR.

[0016] The fdAd technology has a large payload and can be utilized to deliver large transgene constructs. For example, it was possible to deliver the full-size human coagulation factor VIII cDNA together with the immunosuppressive gene CDS (12 kb in total) in a single fdAd vector. Both transgenes were efficiently expressed upon transduction into cells. Thus, the fdAd vector can be used as the basis for the production of an optimized universal influenza vaccine that delivers more than two conserved influenza antigen constructs.

[0017] Current universal influenza vaccines are constructed based on two conserved influenza hemagglutinin constructs. The initial design of the universal influenza vaccine followed the reactivity patterns of two antibodies, CR6261 and CR8020, which bind to epitopes of the alpha-helical structure in the stem region. Antibodies against this region neutralize virus particles. These antibodies appear to inhibit HA function by preventing the pH-induced conformational changes necessary for the full-length HA to fuse with the membrane. Since CR6261 and CR8020 are highly conserved regions for their functions, they neutralize a wide range of HA targets. CR6261 has been shown to neutralize HA in groups 1, subtypes 1, 2, 5, 6, 9, 13, and 16. CR8020 has been shown to bind to HA in groups 2, subtypes 3, 4, 7, 10, 14, and 15. Designing two conserved hemagglutinin antigens according to these two groups results in an average sequence identity of about 55%. Headless constructs are a promising approach to universal influenza vaccines, but technical problems have hindered the wider development of this approach. The low identity scores of the two antigen construct approaches result in a low-affinity immune response. Furthermore, increasing the number of antigen constructs to overcome low sequence redundancy makes this approach difficult and expensive. Headless HA cannot form viable influenza viruses that can grow in egg cultures to be inactivated for vaccines. Therefore, they have to be produced by alternative means.

[0018] The fdAd vector system overcomes these production problems. Headless HA does not need to function in influenza viruses or virus-like particles; they only need to be expressed in vivo in transduced cells. Furthermore, fdAd can easily deliver more than two HA constructs to their large payloads. The multivalency of the fdAd vaccine does not significantly increase the complexity of manipulation or production. Therefore, multivalent universal influenza vaccines can be produced cost-effectively.

Summary of the Invention

[0019] In one embodiment, the present disclosure provides a composition for a vaccine. According to an embodiment of the present disclosure, the composition for a vaccine includes at least two different influenza hemagglutinin (HA)-derived antigens, the HA protein from which the antigen is derived includes a hypervariable region, the hypervariable region is deleted from at least two different influenza HA-derived antigens, and each of the at least two different influenza HA-derived antigens has similarity with HA molecules of more than one influenza serotype exceeding at least 60 calculated by an emboss explorer program.

[0020] In one embodiment, the similarity is at least 70, or at least 80.

[0021] In one embodiment, the hypervariable region is replaced by a peptide linker in at least two different influenza HA-derived antigens.

[0022] In one embodiment, the at least two different influenza HA-derived antigens are proteins. In another embodiment, the at least two different influenza HA-derived antigens are either RNA or DNA encoding the HA protein from which the antigen is derived. In a further embodiment, either the RNA or the DNA is in a viral vector.

[0023] In one embodiment, the hypervariable region is located between the conserved cysteines at positions 52 and 277 using the amino acid numbering of influenza HA of serotype H3.

[0024] In another embodiment, the composition includes more than two different influenza HA-derived antigens.

[0025] In one embodiment, the present disclosure provides a method for manufacturing a universal influenza virus vaccine. According to an embodiment of the present disclosure, a method for manufacturing a universal influenza virus vaccine includes obtaining a first influenza HA-derived antigen having a similarity with HA molecules of a first plurality of influenza serotypes calculated by an Emboss Explorer program and exceeding at least 60, and obtaining a second influenza HA-derived antigen having a similarity with HA molecules of a second plurality of influenza serotypes calculated by the Emboss Explorer program and exceeding at least 60, wherein the first and second pluralities of influenza serotypes are composed of different serotypes.

[0026] In one embodiment, the first and second pluralities of influenza serotypes are composed of different non-overlapping serotypes.

[0027] In one embodiment, the HA proteins from which the first and second influenza HA-derived antigens are derived include hypervariable regions, and the hypervariable regions are deleted from the first and second influenza HA-derived antigens.

[0028] In one embodiment, the similarity is independently at least 70 or at least 80.

[0029] In one embodiment, the hypervariable regions are replaced by peptide linkers.

[0030] In one embodiment, the first and second influenza HA-derived antigens are proteins. In another embodiment, the first and second influenza HA-derived antigens are one of RNA and DNA encoding the HA protein from which the antigen is derived. In a further embodiment, the method includes encapsidating one of RNA and DNA encoding the HA protein into a viral vector.

[0031] In one embodiment, the method includes obtaining at least a third influenza HA-derived antigen having similarity to HA molecules of a third plurality of influenza serotypes, calculated by an Emboss Explorer program, that exceeds at least 60.

[0032] In one embodiment, the present disclosure provides a method of vaccinating an animal against at least two different influenza serotypes. According to an embodiment of the present disclosure, the method comprises a vaccine composition comprising at least two different influenza hemagglutinin (HA)-derived antigens, wherein the HA protein from which the antigen is derived comprises a hypervariable region, the hypervariable region is deleted from at least two different influenza HA-derived antigens, and each of the at least two different influenza HA-derived antigens has similarity to HA molecules of more than one influenza serotype, calculated by an Emboss Explorer program, that exceeds at least 60; and delivering the vaccine composition to an animal.

[0033] Brief Description of the Sequence Listing SEQ ID NO: 1 shows the consensus sequence of the red influenza serotype shown in FIG. 1.

[0034] SEQ ID NO: 2 shows the consensus sequence of the orange influenza serotype shown in FIG. 1.

[0035] SEQ ID NO: 3 shows the consensus sequence of the yellow influenza serotype shown in FIG. 1.

[0036] SEQ ID NO: 4 shows the consensus sequence of the green influenza serotype shown in FIG. 1.

[0037] SEQ ID NO: 5 shows the consensus sequence of the blue influenza serotype shown in FIG. 1.

[0038] SEQ ID NO: 6 shows the consensus sequence of the purple influenza serotype shown in FIG. 1.

[0039] The patent or application file includes at least one drawing created in color. Copies of this patent or patent application publication with color drawings are provided by the Patent Office upon payment of the claims and the required fees.

Brief Description of the Drawings

[0040]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0041] Before describing any embodiments of the present disclosure in detail, it is to be understood that the present disclosure is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting. The use of "including", "comprising", or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The use of "including essentially" and "consisting essentially of" and variations thereof herein is meant to encompass the items listed thereafter, as well as equivalents and additional items, provided that such equivalents and additional items do not materially change the overall characteristics, use, or manufacture. The use of "consisting of" and variations thereof herein is meant to encompass the items listed thereafter and only those items.

[0042] Referring to the drawings, throughout, like numerals refer to like elements. Terms such as first, second, etc. may be used herein to describe various elements, components, regions, and / or sections, but it is to be understood that these elements, components, regions, and / or sections are not to be limited by these terms. These terms are only used to distinguish one element, component, region, and / or section from another. Thus, a first element, component, region, or section may be referred to as a second element, component, region, or section without departing from the present disclosure.

[0043] The numerical ranges in the present disclosure are approximate values, and thus, unless otherwise indicated, values outside the ranges may be included. A numerical range includes, (unless otherwise specified), all values from and including the lower value and the higher value in increments of one unit, provided that there is at least a separation of two units between any lower value and any higher value. As an example, if a composition, physical or other property, such as the amount of a component by weight, is from 10 to 100, all individual values such as 10, 11, 12, etc., and sub-ranges such as 10 to 44, 55 to 70, 97 to 100, etc., are intended to be explicitly enumerated. For ranges that include explicit values (e.g., a range of 1, or 2, or 3 to 5, or 6, or 7), any sub-range between any two explicit values is included (e.g., the above range of 1 to 7 includes sub-ranges 1 to 2; 2 to 6; 5 to 7; 3 to 7; 5 to 6, etc.). For ranges that include values less than 1 or include fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered to be 0.0001, 0.001, 0.01, or 0.1. For ranges that include single-digit numbers less than 10 (e.g., 1 to 5), one unit is typically considered to be 0.1. These are merely examples of what is specifically intended, and all possible combinations of numerical values between the recited lowest and highest values should be considered to be explicitly stated in the present disclosure.

[0044] Spatial terms such as "beneath", "below", "lower", "above", "upper", etc. may be used in this specification to facilitate the description of the relationship between one element or feature and another element or feature, as shown in the figures. It will be understood that spatially relative terms are intended to encompass different orientations depending on the orientation in use or illustration. For example, if the device in the figure is turned over, an element described as "below" or "beneath" another element or feature will be oriented "above" the other element or feature. Thus, the exemplary term "below" can encompass both upward and downward orientations. The device may be oriented in other directions (rotated 90° or in other orientations), and the spatially relative descriptors used in this specification will be interpreted accordingly.

[0045] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. For example, when used in a phrase such as "A and / or B", the phrase "and / or" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" when used in a phrase such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In general, the nomenclature used herein, as well as laboratory procedures in cell culture, molecular genetics, nucleic acid chemistry and hybridization as described below, are well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and the culturing and transformation of microorganisms (e.g., electroporation, lipofection). In general, enzymatic reactions and purification steps are performed according to the manufacturer's specifications. Techniques and procedures are generally performed according to conventional methods in the art and various general references provided throughout this document (see generally, Sambrook et al. Molecular Cloning: a Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is hereby incorporated by reference). Units, prefixes, and symbols may be denoted in their SI accepted forms. Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' direction; amino acid sequences are written left to right in the amino to carboxyl orientation. Amino acids may be referred to herein by either the commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted one letter codes. Unless otherwise specified, software, electrical, and electronic terms used herein are as defined in The New' IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993).

[0047] The embodiments disclosed herein relate to the design, construction, and manufacture of multivalent universal influenza A vaccines.

[0048] As used throughout this disclosure, the following terms are to be understood to have the following meanings, unless otherwise indicated, and are more fully defined by reference to this specification as a whole.

[0049] As used herein, the terms "adenovirus," "adenovirus virion," and "adenovirus particle" include any virus that can be classified as an adenovirus, including any adenovirus that infects humans or animals, including all groups, subgroups, species, and serotypes.

[0050] As used herein, the term "adenovirus vector" includes any genetic construct or viral construct based on an adenovirus that is used to transfer genetic material. As used herein, the terms "deleted adenovirus" or "deleted adenovirus vector" include any adenovirus or adenovirus vector having one or more endogenous genes or gene fragments deleted therefrom. In contrast, as used herein, the terms "completely deleted adenovirus" and "completely deleted adenovirus vector" include any adenovirus and adenovirus vector in which all endogenous adenovirus genes and genetic material are deleted, except for the inverted terminal repeat sequences (ITRs) and the packaging signal (Ψ). As used herein, the term "adenovirus vector genome" includes the genetic material found in an adenovirus vector.

[0051] "Antigen" means a molecule containing one or more epitopes that stimulate the host immune system to cause a cellular antigen-specific immune response or a humoral antibody response. Thus, antigens include proteins, polypeptides, antigenic protein fragments, oligosaccharides, polysaccharides, and the like. Further, an antigen can be derived from any known virus, bacterium, parasite, plant protozoan or fungus, and can be an entire organism. This term also includes tumor antigens. Similarly, for example, oligonucleotides or polynucleotides that express an antigen in DNA immunization applications are also included in the definition of an antigen. Synthetic antigens, such as polyepitopes, contiguous epitopes, and other antigens of recombinant or synthetic origin (Bergmann et al. (1993) Eur. J. Immunol. 23:2777 - 2781; Bergmann et al. (1996) J. Immunol. 157:3242 - 3249; Suhrbier, A. (1997) Immunol. And Cell Biol. 75:402 - 408; Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, Jun 28 - Jul. 3, 1998) are also included.

[0052] A "coding sequence" or a sequence that "encodes" a selected polypeptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences (or "control elements"), is transcribed in vivo (in the case of DNA) and translated into a polypeptide (in the case of mRNA). The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence can be located 3' to the coding sequence. Transcription and translation of the coding sequence are typically regulated by "control elements" including, but not limited to, a transcription promoter, a transcription enhancer element, Shine and Delagamo sequences, a transcription termination signal, a polyadenylation sequence (located 3' to the translation stop codon), a sequence for optimization of translation initiation (located 5' to the coding sequence), and a translation termination sequence.

[0053] As used herein, the term "conserved antigen" refers to an antigen that exhibits similarity to gene variants expressed by viruses of more than one serotype.

[0054] As used herein, the term "conservative variant" of an amino acid residue reflects a change in the amino acid at a given position in a protein that includes amino acids having similar biochemical properties such as charge, hydrophobicity, and size.

[0055] The term "construct" refers to a genetic composition or at least one of the compositions according to the present disclosure as either an adenovirus genome or a packaging construct.

[0056] As used herein, the term "delete" or "deleted" means to erase, expunge, or remove.

[0057] As used herein, the terms "deleted Ad (virus) vector" and "gutted-", "mini-", "deleted-", "DELTA.", or "pseudo-vector" refer to a linear vector module having ITRs. These vectors can also encode several structural and / or non-structural gene sequences and / or one or more genes or transgenes of interest.

[0058] The term "express" refers to the transcription and / or translation of an endogenous gene, transgene, or coding region in a cell.

[0059] "Gene delivery vector", "GDV", "gene transfer vector", or "gene transfer vehicle" is a composition comprising a packaged vector module of the present disclosure.

[0060] As used herein, the term "gene expression construct" refers to a promoter, at least a fragment of a gene of interest, and a polyadenylation signal sequence. The vector modules of the present disclosure may include a gene expression construct.

[0061] As used herein, the terms "gene of interest", "GOI", and "transgene" refer to a gene encoding a gene whose function may be medically important and which may not be a natural flavivirus gene. The gene of interest may be a gene that exerts its effect at the RNA or protein level. Examples of genes of interest include, but are not limited to, therapeutic genes, immunomodulatory genes, viral genes, bacterial genes, protein-producing genes, inhibitory RNAs or proteins, and regulatory proteins.

[0062] "Gene sequence" refers to the order of nucleotides. The gene sequence may be adjustable. Regulation of gene expression can be achieved by one of (1) changes in gene structure: site-specific recombinases (e.g., Cre based on the Cre-loxP system) can activate gene expression by removing inserted sequences between the promoter and the gene; (2) changes in transcription: either by induction (coverage) or reduction of inhibition; (3) changes in mRNA stability by specific sequences incorporated into mRNA or siRNA; and (4) changes in translation by sequences in mRNA. Deleted adenoviruses are also called "high-capacity" adenoviruses. These deleted adenoviruses can accommodate gene sequences up to 33 kb.

[0063] The term "headless hemagglutinin" refers to a hemagglutinin construct consisting of the hemagglutinin stem.

[0064] The term "hemagglutinin stem" or "stem region" refers to a structural component of the influenza hemagglutinin protein that is relatively invariant and does not include the gene hypervariable region of influenza hemagglutinin.

[0065] The term "heterologous" is used for any combination of DNA sequences that are not normally closely related in nature.

[0066] The term "homology" refers to the existence of a common ancestor between a pair of structures or genes.

[0067] A "host cell" or "packaging cell" is a cell that can package an adenovirus or adenovirus vector genome or modified genome to produce viral particles. It can be engineered to provide a defective gene product or its equivalent. Thus, a packaging cell can package an adenovirus genome into adenovirus particles. Production of such particles requires that the genome be replicated and that the proteins necessary to assemble infectious virus be produced. The particles may also require specific proteins necessary for the maturation of the viral particles. Such proteins can be provided by the vector, the packaging construct, or the packaging cell. Exemplary host cells (HC) that can be used to generate packaging cell lines according to the present disclosure include, but are not limited to, A549, HeLa, MRC5, W138, CHO cells, Vero cells, human embryonic retinal cells, or any eukaryotic cell, so long as the host cell permits the growth of adenovirus. Some host cell lines include adipocytes, chondrocytes, epithelial cells, fibroblasts, glioblastoma, hepatocytes, keratinocytes, leukemia, lymphoblastoid cells, monocytes, macrophages, myoblasts, and neurons. Other cell types include, but are not limited to, cells derived from primary cell cultures, such as human primary prostate cells, human embryonic retinal cells, human stem cells. Diploid and aneuploid cell lines of eukaryotes are included within the scope of the present disclosure. A packaging cell must be able to express the products of the different constructs described herein at levels suitable for their products in order to generate a high titer stock of recombinant virus vector.

[0068] An "immune response" is an acquired immune response, such as a cellular or humoral immune response.

[0069] In the context of the present disclosure, an "immunomodulatory molecule" is a polypeptide molecule that modulates, i.e., increases or decreases, the cellular and / or humoral host immune response to target cells in an antigen-specific manner, preferably decreasing the host immune response. Generally, according to the teachings of the present disclosure, the immunomodulatory molecule(s) associate with the target cell surface membrane after expression from the GDV described herein, for example, are inserted into the cell surface membrane or bind covalently or non-covalently to the cell surface membrane.

[0070] As used herein, the terms "influenza virus", "influenza virion", and "influenza particle" include any virus that can be classified as an influenza virus, including any influenza virus that infects humans or animals, including all groups, subgroups, and serotypes.

[0071] As used herein, the term "introduce" or "transfection" refers to the delivery of an expression vector into a host cell. The vector can be introduced into the cell by transfection, which typically involves physical means (e.g., calcium phosphate transfection, electroporation, microinjection or lipofection); infection (typically referring to introduction by an infectious agent, i.e., a virus); or transduction (typically referring to stable infection of a cell by a virus or the transfer of genetic material from one microorganism to another by a viral agent (e.g., bacteriophage)), meaning the insertion of heterologous DNA or RNA into the cell. The vector can be a plasmid, virus or other vehicle.

[0072] The term "linear DNA" refers to an uncircularized DNA molecule. The term "linear RNA" refers to an uncircularized RNA molecule.

[0073] As used herein, the term "naturally" refers to that which is found in nature; wild type; essentially or in essence.

[0074] The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single-stranded or double-stranded form, and includes known analogs having the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to natural nucleotides, unless otherwise particularly limited. A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous. However, an enhancer does not need to be contiguous. The linkage is achieved by ligation at convenient restriction enzyme recognition sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0075] As used herein, the term "non-structural gene" refers to a group of genes present in the adenovirus genome. These genes encode "non-structural genes".

[0076] The term "packaging construct" or "packaging expression plasmid" refers to an engineered plasmid construct of a circular double-stranded DNA molecule, the DNA molecule containing at least a subset of adenovirus structural or non-structural genes under the control of a promoter. The "packaging construct" does not contain more than one ITR or genetic information than is necessary to allow for infection, independent viral replication to produce viral particles, and / or efficient packaging of this genetic material into viral particles.

[0077] A cell that is "permissive" supports the replication of the virus.

[0078] As used herein, the term "plasmid" refers to an extrachromosomal DNA molecule separate from chromosomal DNA that can replicate independently of chromosomal DNA. Often, it is circular and double-stranded.

[0079] The term "polylinker" refers to a short stretch of artificially synthesized DNA that possesses several unique restriction enzyme recognition sites that allow for the easy insertion of any promoter or DNA segment.

[0080] The term "promoter" means a regulatory region of DNA that promotes the transcription of a specific gene. A promoter usually contains a TATA box that can direct RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site of a specific coding sequence. A promoter may further contain other recognition sequences, called upstream promoter elements, generally located upstream or 5' of the TATA box, which affect the rate of transcription initiation. A "constitutive promoter" refers to a promoter that allows for continuous transcription of its associated gene in many cell types. An "inducible promoter system" refers to a system that uses regulators (including small molecules such as tetracycline, peptide hormones and steroid hormones, neurotransmitters, and environmental factors such as heat and osmolality) to induce or silence genes. Such systems are "analogous" in the sense that their responses are graded and dependent on the concentration of the regulator. Also, such systems are reversible by removal of the regulator. The activity of these promoters is induced by the presence or absence of biological or abiotic factors. Inducible promoters are powerful tools in genetic engineering because they can turn the expression of genes operably linked to them on or off at specific stages of the development of an organism or a particular tissue.

[0081] As used herein, the term "propagate" or "propagated" refers to reproduction, multiplication, or other increase in number, amount, or degree by any process.

[0082] As used herein, the term "purify" refers to a process of purifying or freeing substantially little, little, substantially all, or all foreign, exogenous, or undesirable elements.

[0083] "Regulatory sequence", "regulatory region", or "regulatory element" refers to a promoter, enhancer, or other segment of DNA to which a regulatory protein, such as a transcription factor, binds preferentially. They control gene expression and thus protein expression.

[0084] As used herein, the term "recombinase" refers to an enzyme that catalyzes genetic recombination. Recombinase enzymes catalyze the exchange of short DNA segments between two long DNA strands, particularly the exchange of homologous regions between paired maternal and paternal chromosomes.

[0085] "Restriction enzyme" or "restriction endonuclease" is an enzyme that cleaves double-stranded DNA.

[0086] The term "restriction enzyme recognition site" or "restriction recognition site" refers to a specific sequence of nucleotides recognized by a restriction enzyme as the site at which to cleave a DNA molecule. These sites are generally, but not always, palindromic (since restriction enzymes usually bind as homodimers), and a particular enzyme can cleave between two nucleotides either within or near its recognition site.

[0087] The term "replicate" or "replicating" means, for example, but not limited to, creating identical copies of an object such as a virus particle.

[0088] As used herein, the term "replication-deficient" refers to a characteristic of a virus that cannot replicate in a natural environment. A replication-deficient virus is a virus that has deleted one or more genes essential for its replication, for example, but not limited to, a virus with a deleted E1 gene. Replication-deficient viruses can be propagated in the laboratory in cell lines that express the deleted gene.

[0089] As used herein, the term "similarity" or "sequence similarity" refers to a measure of the empirical relationship between protein sequences. The similarity scores used herein approximate the evolutionary distance between amino acid residues within a given protein and thus represent similarity and / or identity.

[0090] As used herein, the term "structural gene" refers to a group of genes present in the adenovirus genome that form the viral capsid.

[0091] The term "stuffer" refers to a DNA or RNA sequence that is inserted into another DNA or RNA sequence to increase its size. A stuffer fragment usually does not encode any protein and does not contain regulatory elements for gene expression such as transcriptional enhancers or promoters.

[0092] As used herein, the term "target" or "targeted" refers to a biological entity such as, but not limited to, a protein, cell, organ, or nucleic acid whose activity can be modified by an external stimulus. Depending on the nature of the stimulus, there may be no direct change to the target, or a conformational change of the target may be induced.

[0093] The term "transfection" refers to the introduction of cellular genetic material as DNA or RNA (e.g., the introduction of an isolated nucleic acid molecule or a construct of the present disclosure). As used herein, the term "transduction" refers to the introduction of DNA or by using the GDV of the present disclosure into cellular DNA. The GDV of the present disclosure can be transduced into target cells.

[0094] As used herein, the term "universal influenza vaccine" refers to an influenza vaccine that carries an antigen capable of inducing an immune response against more than one serotype or several influenza serotypes of influenza virus in humans and animals.

[0095] As used herein, the term "untranslated region" refers to the RNA portion that does not encode a protein.

[0096] The term "vector" refers to a nucleic acid that is used for the infection of a host cell and into which a polynucleotide can be inserted. A vector is often a replicon. An expression vector enables the transcription of the nucleic acid inserted therein. Some common vectors include, but are not limited to, plasmids, cosmids, viruses, phages, recombinant expression cassettes, and transposons. The term "vector" can also refer to an element that aids in the transfer of a gene from one location to another.

[0097] The term "vector module" refers to an adenoviral gene composition packaged into an adenoviral virion.

[0098] As used herein, the terms "viral DNA" or "viral RNA" refer to the DNA or RNA sequences found in viral particles.

[0099] As used herein, "viral genome" is the entirety of the DNA or RNA found in viral particles and containing all the elements necessary for viral replication. The genome is replicated and transmitted to viral progeny in each cycle of viral replication.

[0100] As used herein, the term "virion" refers to a viral particle. Each virion consists of genetic material within a protective protein capsid.

[0101] As used herein, the term "wild-type" refers to the typical form of an organism, strain, gene, protein, nucleic acid, or characteristic that occurs in nature. Wild-type refers to the most common phenotype in a natural population. The terms "wild-type" and "naturally occurring" are used interchangeably.

[0102] According to embodiments of the present disclosure, a universal influenza vaccine is provided.

[0103] In one embodiment, the universal influenza vaccine is a multimeric vaccine comprising a set of antigens, the set of antigens comprising at least two, preferably more than two, different influenza hemagglutinin (HA)-derived antigens. Hemagglutinin is a glycoprotein found on the surface of the influenza virus and is essential for the infectivity of the virus.

[0104] The antigen derived from influenza HA can be a DNA sequence or an RNA sequence encoding all or part of influenza HA, or a protein sequence against all or part of influenza HA itself.

[0105] Influenza HA is numbered using the amino acid numbering of influenza hemagglutinin of serotype H3 and has a hypervariable region located between the conserved cysteines at positions 52 and 277. In one embodiment, the DNA, RNA or protein sequence of the antigen corresponding to the hypervariable region is deleted.

[0106] In one embodiment, the HA hypervariable region is replaced with a peptide linker (or a DNA or RNA sequence corresponding to the peptide linker) to increase the stability and cell surface expression of the headless hemagglutinin construct. An exemplary non-limiting peptide linker is GGGGS-GGGGS-GGGGS-GGGGS ((GGGGS)4).

[0107] In one embodiment, the influenza HA-derived antigen is based on a headless influenza hemagglutinin protein.

[0108] In one embodiment, at least two, preferably more than two different influenza HA-derived antigens are composed of sequences (whether DNA, RNA or protein) having similarity with HA sequences (DNA, RNA or protein) of more than one influenza serotype that exceed a similarity score of 60, or 65, or 70, or 75, or 80, or 85, or 90 as calculated by an emboss explorer program. The similarity score is based on the presence of identical amino acid residues (or their respective coding DNA or RNA) at corresponding positions of different HAs, and the presence of conservative variants of amino acid residues (or their respective coding DNA or RNA) at corresponding positions of different HAs. FIG. 1 provides a visual illustration of influenza HA and similarity scores by aligning and grouping HAs according to sequence identity in the HA stem region.

[0109] In one embodiment, a representative hemagglutinin of a particular serotype of influenza A is used in the alignment of different hemagglutinins. Exemplary representative influenza A serotypes include, but are not limited to, the following: H1: A / Califomia / 48 / 2017 (H1N1) H2: A / Moscow / 1019 / 1965 (H2N2) H3: A / Washington / 16 / 2017 (H3N2) No 8 H4: A / duck / Guangdong / DGQTSJ147P / 2015 (H4N8) H5: A / Cygnus olor / Belgium / 1567 / 2017 (H5N8) H6: A / green-winged teal / Tennessee / 17OS0651 / 2017 (H6N1) H7: A / Guangdong / HP001 / 2017 (H7N9) H8: American black duck / Illinois / 4119 / 2009 (H8N4) H9: A / Japanese Quail / Vietnam / 4 / 2009 (H9N2) H10:A / American black duck / Alberta / 118 / 2016(H10N7) H11:A / duck / Memphis / 546 / 1974 H12:American black duck / New Brunswick / 00998 / 2010(H12N6) H13:American white pelican / Minnesota / Sg-0611 / 2008(H13N9) H14:A / Northem shoveler / Missouri / 16OS6248 / 2016(H14N7) H15:A / duck / Bangladesh / 24704 / 2015(H15N9) H16:A / glaucous-Owinged gull / Southcentral Alaska / 16MB03160 / 2016(H16N3) H17:A|H17N10|09 / 2010|A / little_yellow_shouldered_bat / Guatemala / 060 / 2010 H18:4|HA|A|A / dark_fhrit_eating_bat / Bolivia / PBV780_781 / 2011

[0110] In another embodiment, the similarity scores used herein represent an approximation (and thus similarity and / or identity) of the evolutionary distance between amino acid residues within a given protein. Based on that data, phylogenetic trees of the different hemagglutinin serotypes are created, showing the evolutionary connections and distances of the different hemagglutinins as phylogenetic trees (Figure 2).

[0111] In another embodiment, several conserved headless hemagglutinin protein constructs are designed based on these similarity scores. An exemplary "peptide sequence" of the conserved construct is shown in Figure 3, and the sequence similarity of these sequences to influenza A hemagglutinin is shown in Figure 4.

[0112] As described herein, a universal influenza virus vaccine comprises a set of antigens composed of at least two, or preferably more than two, different influenza hemagglutinin (HA)-derived antigens. In one embodiment, at least two, preferably more than two, different influenza HA-derived antigens are selected from the sequences provided in Figure 3, or at least two, preferably more than two, conserved headless hemagglutinin protein constructs selected from the sequences conHAl, conCHA2, conHA3, conH4, conH5 and conH6. In one embodiment, at least two, or preferably more than two, antigens are provided to humans and / or animals in the form of a vaccine.

[0113] In one embodiment, at least two, or preferably more than two, influenza HA-derived antigens are provided as proteins, combined with virus-like particles, delivered by nanoparticles, delivered by emulsions, their sequences are incorporated into DNA or RNA vaccines as transgenes, or their sequences are incorporated into viral vectors. Figure 5 is a schematic diagram of a viral vector model.

[0114] In another embodiment, the vaccines of the present disclosure deliver influenza HA-derived antigens in a suitable pharmaceutical carrier formulated using standard methods of vaccine formulation, in the presence or absence of immunostimulatory moieties such as, but not limited to, vaccine adjuvants and interleukins.

[0115] In another embodiment, one or more influenza HA-derived antigen constructs are delivered as a vaccine to humans and animals via different routes of administration including, but not limited to, intramuscular, intranasal, intradermal, subcutaneous, oral, rectal or intratracheal.

[0116] In another embodiment, one or more influenza HA-derived antigen constructs are delivered as a transgene construct in an adenoviral vector.

[0117] In another embodiment, one or more influenza HA-derived antigen constructs are delivered as a transgene construct in a completely deleted adenovirus vector, e.g., but not limited to, a completely deleted helper virus-independent adenovirus vector.

[0118] In another embodiment, one or more influenza HA-derived antigen constructs contain a promoter and a polyadenylation site. In another embodiment, the transgene construct is linked by an internal ribosome entry site. In another embodiment, the transgene construct encodes more than one influenza HA-derived antigen construct linked by an internal ribosome entry site. In another embodiment, the transgene construct encodes more than one influenza HA-derived antigen construct linked by a linker self-cleaving enzyme or digestible by a proteolytic enzyme.

[0119] In another embodiment, the transgene construct of the influenza HA-derived antigen construct is encoded within a vector, e.g., but not limited to, an adenovirus vector, as a replication-competent RNA, e.g., but not limited to, an alphavirus replicon.

[0120] In one embodiment, a method of manufacturing a universal influenza vaccine is provided. In an embodiment, the method comprises obtaining at least two, preferably more than two, different influenza HA-derived antigens having similarity with HA molecules of at least 60, or 65, or 70, or 75, or 80, or 85, or more than one influenza serotype exceeding a similarity score of 90. Next, the at least two, preferably more than two, different influenza HA-derived antigens are provided as a DNA sequence encoding the HA antigen, an RNA sequence encoding the HA antigen, or a protein sequence for inclusion in a vaccine composition. The at least two, preferably more than two, influenza HA-derived antigens may be according to any embodiment or combination of embodiments provided herein. The at least two, preferably more than two, different influenza HA-derived antigens may be provided in the form of a viral vector for inclusion in a vaccine composition.

[0121] In one embodiment, a method of vaccinating an animal (e.g., including humans) against at least two, preferably more than two, influenza A subtypes comprises providing a vaccine composition comprising at least two, preferably more than two, different influenza HA-derived protein antigens.

Examples

[0122] Example 1 - Design of Influenza HA-Derived Antigens Arrange the protein sequences of examples of protein sequences of influenza HAs of different serotypes. The protein sequences used in the examples are derived from the following influenza A subtypes. H1: A / Califomia / 48 / 2017 (H1N1) H2: A / Moscow / 1019 / 1965 (H2N2) H3: A- / Washington / 16 / 2017 (H3N2) No 8 H4: A / duck / Guangdong / DGQTSJ147P / 2015 (H4N8) H5:A / Cygnus olor / Belgium / 1567 / 2017(H5N8) H6:A / green-winged teal / Tennessee / 17OS0651 / 2017(H6N1) H7:A / Guangdong / HP001 / 2017(H7N9) H8:American black duck / Illinois / 4119 / 2009(H8N4) H9:A / Japanese Quail / Vietnam / 4 / 2009(H9N2) H10:A / American black duck / Alberta / 118 / 2016(H10N7) H11:A / duck / Memphis / 546 / 1974 H12:American black duck / New Brunswick / 00998 / 2010(H12N6) H13:American white pelican / Minnesota / Sg-0611 / 2008(H13N9) H14:A / Northem shoveler / Missouri / 16OS6248 / 2016(H14N7) H15:A / duck / Bangladesh / 24704 / 2015(H15N9) H16:A / glaucous-Owinged gull / Southcentral Alaska / 16MB03160 / 2016(H16N3) H17:A|H17N10|09 / 2010|A / little_yellow_shouldered_bat / Guatemala / 060 / 2010 H18:4|HA|A|A / dark_fhut_eating_bat / Bolivia / PBV780_781 / 2011

[0123] Based on the analysis of the similarity of different HA serotypes, as shown in Figures 1 and 2, the serotypes are classified into six similarity groups. The HA sequences are downloaded from fludg.org. Incomplete sequences are removed for analysis. The remaining sequences are aligned using the MAFFT online server version 7: https: / / mafft.cbrc.jp / alignment / server / large.html?aug31. The following settings are used for the analysis. i.FFT-NS-2 ii.Memory usage: normal iii.Amino acid sequence scoring matrix: BLOSUM 62 iv.Gap opening penalty: 5

[0124] Based on this alignment, the HA serotypes are classified into the following groups. H-I (red): having HA serotypes H7, H10, H15 H-II (orange) having HA serotypes H3, H4, H14 H-III (yellow) having HA serotypes H1, H2, H5, H6 H-IV (green) having HA serotypes H8, H9, H12 H-V (blue) having HA serotypes H11, H13, H15 H-VI (purple) having HA serotypes H17, H18

[0125] In these alignments, as shown in Figure 1, the identity rate of amino acid residues within each HA protein of different HA groups exceeds a value of 63.7% identity for the HA proteins analyzed.

[0126] Example 2 - Sequences of Influenza HA-Derived Antigens Based on the analysis of Example 1, consensus proteins of different HA serotypes are developed for the conserved regions of HA. The consensus sequences for each HA serotype group H1 to H18 are derived from the EMBOSS Explorer Con: http: / / www.bioinformatics.nl / cgi-bin / emboss / cons. The following settings are used: i. Set multiple checks to 1 ii. Required identity number at position: 1

[0127] In step 1, consensus sequences for each of the 18 HA serotypes are developed. The sequences are found at fludb.org.

[0128] The following number of sequences are used to derive each HA serotype consensus sequence. H1: 22868 H2: 622 H3: 25600 H4: 1932 H5: 5694 H6: 1745 H7: 2743 H8: 150 H9: 3885 H10: 1236 H11: 670 H12: 210 H13: 6856 H14: 31 H15: 21 H16: 268 H17: 3 H18: 2

[0129] Step 1: Once the HA consensus sequences are identified, the headless versions of the consensus sequences for each of the six groups are determined as follows. (a) The 18 representative consensus sequences are aligned with ClustalW in BioEdit. (b) The universally conserved cysteine is identified at position 60 as the residue immediately preceding the deletion. (c) Identify the universally conserved CxxxC near positions 287 - 290 (about 100 aa from the universally conserved GLFGAIA sequence) and use this as the end of the headless deletion. (d) Delete the region between the two cysteines in (c) and replace it with GGGG or (GGGGS) n (n≧1)

[0130] In Figure 3, the size protein consensus sequences are listed with monomeric peptide linkers.

[0131] Example 3 - Similarity of Conserved HA Region Consensus Sequences Align the six protein consensus sequences shown in Figure 3 with the HA protein sequences of the HA serotypes assigned to each consensus sequence. The percentage of similarity is calculated taking into account amino acid identity and the similarity of conservative amino acid changes at given positions in the amino acid residues. The scores are calculated using the EMBOSS Explorer Con program and are provided in Figure 4. Note that all scores exceed 83.

[0132] Example 4 - Design and Construction of a Completely Deleted Ad Virus Vector Carrying Transgenes for Six Conserved Universal Influenza HA - Derived Antigen Constructs The fdAd vector has all endogenous ad genes deleted. The space within the Ad genome can accommodate transgene constructs up to 33 kb in length. The fdAd vector genome carries only Ad sequences corresponding to the adenovirus inverted terminal repeat (ITR) and Ad packaging signals such as adenovirus Ψ.

[0133] The fdAd viral vector genome is packaged into an adenovirus capsid in a host or packaging cell that has the deleted adenovirus genes necessary for genome and capsid formation by a second gene construct. The second gene construct can be provided by, but is not limited to, a helper virus construct or a packaging expression plasmid.

[0134] In one example, once linearized, the fdAd vector genome is co-transfected with a packaging expression plasmid that delivers the gene encoding the Ad capsid of human serotype 6 into cells of a host cell, such as, but not limited to, cells of the human fetal kidney cell line HEK-293, as shown in FIG. 5. The capsid-formed adenovirus vector once released from the cells is purified and used, for example, for vaccination.

[0135] In one example, fdAd carries a transgene construct encoding six consensus sequences. Three transgene expression cassettes are produced with the following composition: Number 1: cytomegalovirus promoter enhancer sequence, followed by a transgene encoding the H-I (red) consensus protein, followed by an internal ribosome entry site, followed by a transgene encoding the H-II (orange) consensus protein, followed by a polyadenylation site. Number 2: cytomegalovirus promoter enhancer sequence, followed by a transgene encoding the H-III (yellow) consensus protein, followed by an internal ribosome entry site, followed by a transgene encoding the H-IV (green) consensus protein, followed by a polyadenylation site. Number 3: cytomegalovirus promoter enhancer sequence, followed by a transgene encoding the H-V (blue) consensus protein, followed by an internal ribosome entry site, followed by a transgene encoding the H-Vl (purple) consensus protein, followed by a polyadenylation site.

[0136] Transfer the three transgene constructs, numbers 1, 2, and 3, into an adenovirus vector genome that is completely deleted.

[0137] Example 5 - Protocol for immunizing humans or animals with an fdAd vector carrying the transgene of six conserved universal headless HA protein constructs An fdAd vector carrying the transgene of the HA protein conserved in six consensus is capsid-formed into the Ad transcript as described in Example 4. This is suspended in a physiological solution and used to immunize humans and / or animals. For this purpose, it is delivered to the recipient by injection via, but not limited to, intramuscular, intradermal, subcutaneous routes, or by other routes such as, but not limited to, intranasal or oral routes.

[0138] Both humoral and cellular immune responses are induced in humans and animals exposed to this adenovirus vector. These immune responses protect humans and animals from the attack of influenza viruses belonging to members of any influenza A serotype.

[0139] Although multiple embodiments of the universal influenza vaccine and related methods have been described in detail herein, it should be apparent that modifications and variations of them are possible and that all of them fall within the true spirit and scope of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desirable to limit the invention to the exact construction and operation illustrated and described, and accordingly, reliance may be placed on all suitable modifications and equivalents falling within the scope of the present disclosure.

Claims

1. A vaccine composition comprising: A vaccine composition comprising at least two different influenza hemagglutinin (HA) derived antigens, the HA proteins from which the antigens are derived comprising a hypervariable region, the hypervariable region being deleted from the at least two different influenza HA derived antigens, and each of the at least two different influenza HA derived antigens having similarity to the HA molecules of more than one influenza serotype, greater than at least 60, as calculated by the EMBOSS Explorer Con program.

2. The composition of claim 1 , wherein the similarity is at least 70.

3. The composition of claim 1 , wherein the similarity is at least 80.

4. 4. The composition of claim 1, wherein the hypervariable region is replaced by a peptide linker in at least two different influenza HA-derived antigens.

5. 5. The composition of claim 1, wherein the at least two different influenza HA-derived antigens are proteins.

6. 5. The composition of claim 1, wherein the at least two different influenza HA-derived antigens are one of RNA and DNA encoding the HA protein from which the antigens are derived.

7. The composition of claim 6 , wherein one of the RNA and the DNA is in a viral vector.

8. 8. The composition of any one of claims 1 to 7, wherein the hypervariable region is located between the conserved cysteines at positions 52 and 277 using the amino acid numbering of influenza HA of serotype H3.

9. 9. The composition of claim 1 , comprising more than two different influenza HA-derived antigens.

10. 1. A method for producing a universal influenza virus vaccine, comprising: obtaining a first influenza HA-derived antigen having a similarity to at least more than 60 HA molecules of a first plurality of influenza serotypes as calculated by the EMBOS Explorer Con program; obtaining a second influenza HA-derived antigen having a similarity to at least more than 60 HA molecules of a second plurality of influenza serotypes as calculated by the EMBOS Explorer Con program; The method, wherein the first and second plurality of influenza serotypes are comprised of different serotypes.

11. 11. The method of claim 10, wherein the first and second plurality of influenza serotypes are comprised of different, non-overlapping serotypes.

12. 12. The method of claim 10, wherein the HA protein from which the first and second influenza HA-derived antigens are derived comprises a hypervariable region, and the hypervariable region is deleted from the first and second influenza HA-derived antigens.

13. 13. The method of claim 10, wherein the similarity is, independently, at least 70.

14. 13. The method of claim 10, wherein the similarity is, independently, at least 80.

15. 15. The method of any one of claims 10 to 14, wherein the hypervariable region is replaced by a peptide linker.

16. 16. The method of any one of claims 10 to 15, wherein the first and second influenza HA-derived antigens are proteins.

17. 16. The method of any one of claims 10 to 15, wherein the first and second influenza HA-derived antigens are one of RNA and DNA encoding the HA protein from which the antigens are derived.

18. 20. The method of claim 17, comprising encapsidating one of RNA and DNA encoding the HA protein into a viral vector.

19. 19. The method of any one of claims 10 to 18, comprising obtaining at least a third influenza HA-derived antigen having similarity to at least 60 HA molecules of a third plurality of influenza serotypes as calculated by the EMBOSS EXPLORER CON program.

20. 1. A method of vaccinating an animal against at least two different influenza serotypes, comprising: A method comprising: providing a vaccine composition comprising at least two different influenza hemagglutinin (HA) derived antigens, wherein the HA proteins from which the antigens are derived comprise a hypervariable region, and the hypervariable region is deleted from the at least two different influenza HA derived antigens, and each of the at least two different influenza HA derived antigens has similarity to HA molecules of more than one influenza serotype, at least more than 60, as calculated by the EMBOSS Explorer Con program; and delivering the vaccine composition to an animal.

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