Influenza vaccine

Abstract

Polypeptides, nucleic acid molecules, vectors, cells, fusion proteins, pharmaceutical compositions, combined preparations, and their use as vaccines against influenza are described, in which the polypeptide comprises a globular head domain of hemagglutinin subtype 5 (H5), and optionally a stem domain of hemagglutinin, and the polypeptide comprises an amino acid sequence having a deletion of an amino acid residue at a position corresponding to residue position 144 or 145 of the globular head domain of wild-type H5, or the polypeptide comprises an amino acid sequence having an E amino acid residue at a position corresponding to residue position 238 of the globular head domain of wild-type H5.

Description

[Technical field]

[0001] The present invention relates to nucleic acid molecules, polypeptides, vectors, cells, fusion proteins, pharmaceutical compositions, combined preparations and their use as vaccines against influenza. [Background technology]

[0002] Influenza is a highly contagious respiratory illness caused by influenza viruses that infect epithelial cells in the upper respiratory tract. The infection is characterized by the sudden onset of high fever, headache, muscle pain and fatigue, sore throat, cough and rhinitis. For the majority of cases, influenza lasts for a week and is usually limited to the upper respiratory tract. However, in medically vulnerable people (e.g., people over 65 years of age and those with certain chronic medical conditions), influenza can cause complications and even death. Approximately 9 to 45 million humans are infected. The WHO estimates that seasonal influenza causes 290,000 to 650,000 deaths each year due to respiratory disease alone. The development of an effective influenza vaccine is therefore important to the health of millions of people around the world.

[0003] The basic principle of a vaccine is to prepare the immune system to encounter a pathogen. Vaccines induce the human immune system to produce antibodies and T-cell responses, which help fight infection. Historically, once a pathogen was isolated and grown, it was mass-produced, killed or attenuated, and used as a vaccine. Later, recombinant genes from the isolated pathogen were used to generate recombinant proteins, which were mixed with adjuvants to stimulate an immune response. More recently, the pathogen genes were cloned into vector systems (attenuated bacterial or viral delivery systems) to express and deliver antigens in vivo. All of these strategies rely on pathogens isolated from past outbreaks to prevent future outbreaks. For pathogens that change significantly or only slowly, this traditional technique is effective. However, some pathogens tend to have an accelerating mutation rate, and previously generated antibodies do not always recognize evolved strains of the same pathogen. Newly emerged and re-emerged pathogens often evade the immune response by hiding or disguising their vulnerable antigens from the immune system.

[0004] Influenza is one of the best characterized re-emerging pathogens, with infections re-emerging in up to 100 million people worldwide each season. Influenza is a member of the family Orthomyxoviridae and has a single-stranded, negative-sense RNA genome. RNA viruses generally have a much higher mutation rate compared to DNA viruses because viral RNA polymerase lacks the proofreading ability of DNA polymerase. This contributes to antigenic drift, a continuous process of accumulation of mutations in the genome of an infectious agent that results in small changes in the antigens presented to the immune system of the host organism. Changes to antigenic regions of proteins on the influenza virion result in their evasion of the host immune system and potentially increased pathogenicity and infectivity. This is one reason why it is difficult to create an effective vaccine to prevent influenza. Influenza can undergo antigenic shifts, a process in which there are dramatic changes in the antigens presented on the influenza virus. Genetic segments from different subtypes of influenza can reassort and package into new virion particles that contain genetic information from both of the subtypes. This can give rise to viruses with antigenic features not previously seen in the human context, where we are immunologically naive. The new quasispecies of viruses can cause pandemics if neutralizing or inhibitory antibodies against the new influenza virus do not exist in the human population.

[0005] There are many types of influenza viruses, the most common in humans being influenza A, influenza B, and influenza C. Influenza A viruses infect a wide variety of birds and mammals, including humans, horses, marine mammals, pigs, ferrets, and chickens. In their natural reservoirs in waterfowl and bats, influenza A viruses show minimal evolution and cause subclinical disease; however, once they are transferred to different species, influenza A viruses can evolve rapidly as they adapt to their new hosts, possibly causing pandemics or epidemics of acute respiratory disease in poultry, lower animals, and humans. In animals, most influenza A viruses cause mild localized infections of the respiratory and intestinal tracts. However, highly pathogenic influenza A strains (e.g., some of the H5N1 subtype) can cause systemic infections in poultry as well as in overflowing human cases, which can have high mortality rates. Influenza B and C are restricted to human infection, but animal sources are unknown. Influenza B causes epidemic seasonal infections and has a pathogenicity similar to influenza A. Influenza C viruses are usually associated with very mild or asymptomatic infections in humans.

[0006] More than 100 years have passed since the devastating influenza pandemic of 1918, yet there is still no optimal prevention or treatment for influenza A and B. Although influenza viruses share some similarities in antigen presentation on their surface, the highly heterogeneous nature of these antigens presents considerable challenges in developing vaccines and treatments. Quadrivalent vaccines were widely provided during the 2019-2020 seasonal influenza epidemic. These conferred protection against two influenza A viruses and two influenza B viruses. However, the viruses evolve rapidly, and therefore it is crucial that influenza vaccines protect against many, if not all, potential influenza strains to prevent potential outbreaks of influenza that are unrecognized by the host immune system.

[0007] Influenza A has an outer envelope interspersed with three integral membrane proteins that coat the matrix protein (M1): hemagglutinin (HA); neuraminidase (NA); and a matrix ion channel (M2). The organization of influenza B is similar, with HA and NA interspersed throughout the lipid envelope, but in place of M2 are the NB and BM2 transmembrane ion channels.

[0008] Influenza A viruses are subtyped based on their combination of surface glycoproteins (GP), i.e., HA and NA. Influenza B viruses have much less antigenic variation than influenza A and are not subtyped. HA and NA are membrane-bound envelope GPs that are responsible for virus attachment, presentation of virus particles to cells, and release of virus particles from cells. They are the source of the major immunodominant epitopes for virus neutralization and protective immunity. Thus, both HA and NA proteins are considered the most important components of a prophylactic influenza vaccine. During HA-mediated entry, binding of GP to sialic acid-containing receptors on the host cell membrane initiates endocytosis of virions into cells. The low pH within the endosome induces a conformational change in HA to expose a hydrophobic region called the fusion peptide. The newly exposed fusion peptide then inserts into the endosomal membrane, thereby bringing the viral and endosomal membranes into immediate contact, allowing membrane fusion and virus entry into the cytoplasm. This release into the cytoplasm allows viral proteins and RNA molecules to enter the nucleus for viral transcription and subsequent replication. The transcribed positive stranded mRNA is exported from the nucleus and translated into viral proteins, and the replicated negative stranded RNA is exported from the nucleus and reassembles with newly synthesized viral proteins to form progeny viral particles. The virus buds from the apical cell membrane and combines with the host membrane to form virions that can infect another cell.

[0009] HA is present on the viral surface as a homotrimer, forming a cylindrically shaped molecule that protrudes outward from the virion and forms a type I transmembrane glycoprotein. Each monomer of an HA molecule consists of HA1 and HA2 regions linked by two disulfide bridges along with one HA0 polypeptide chain. Each HA0 polypeptide forms a globular head domain and a stem domain. The globular head domain contains the most dominant epitopes, while the stem domain has less dominant but important epitopes for broader antibody recognition. The amino acid sequences of these epitopes determine the binding affinity and specificity for the antibody. The globular head domain consists of a portion of HA1, including the receptor binding domain and the esterase domain, whereas the stem domain consists of a portion of HA1 and HA2. The amino acid residues of HA1 that form the globular head domain fold into a motif of eight canonical antiparallel β-sheets located in a shallow pocket at the distal tip that acts as a receptor binding site surrounded by antigenic sites. The remainder of the HA1 domain descends into a stem domain that contains primarily β-sheets. HA2 forms the majority of the stem domain and folds into a helical coiled-coil structure that forms the stem backbone. HA2 also contains a hydrophobic region required for membrane fusion, and a long helical strand and a short cytosolic tail that anchors it to the surface membrane.

[0010] Within influenza A, there are 18 different HA subtypes and 11 different NA subtypes. Theoretically, there are potentially 198 different influenza A subtype combinations, some of which may be virulent in humans and other animals. As a result, there is a significant concern that viruses derived from these subtypes may reassort with human-transmissible viruses and initiate the next pandemic. In recent years, avian viruses of the H5, H7, H9, and H10 subtypes have caused zoonotic infections with H5 and H7 viruses often causing severe disease. The 1997 H5N1 highly pathogenic Asian influenza (HPAI) outbreak resulted in the culling of the entire poultry population in Hong Kong. This panzootic also resulted in 860 confirmed cases and 454 deaths in humans, demonstrating the ability of avian-derived viruses to transmit to humans and cause high mortality. This HPAI of the H5N1 subtype is of particular concern due to its frequent re-emergence, its 60% mortality rate, and because it continues to evolve and diversify. The most recent influenza pandemic in 2009 was caused by a novel H1N1 influenza A virus that arose from the reassortment of circulating human influenza with human, swine, and avian influenza. This virus was significantly different from the H1N1 viruses circulating during the pandemic. As a result, few young people had any pre-existing immunity to the virus, and roughly one-third of people over 60 years of age had antibodies against it due to past exposure to similar H1N1 viruses. The CDC (Centers for Disease Control and Prevention) estimates that the total number of deaths caused by the 2009 outbreak worldwide ranged from 150,000 to 575,400. Influenza A evolves periodically in multiple species, and to prepare for this, the virus needs to be characterized and translated into an effective vaccine in a rapid manner.

[0011] Although they have less antigenic variation than influenza A viruses, influenza B viruses have recently emerged as two antigenically distinct lineages (B / Victoria / 2 / 1987-like and B / Yamagata / 16 / 1988-like), illustrating the fluidity with which influenza B can evolve and how it is now also essential to include both A and B viruses in seasonal influenza vaccinations.

[0012] There is a need to provide improved influenza vaccines that protect against many more influenza strains than current vaccines. In particular, there is a need to provide vaccines against influenza A and B viruses that protect against several influenza A and B variants. Specifically, there is a need to provide improved vaccines that induce a more broadly neutralizing immune response against influenza A H5 viruses. There is also a need to provide neutralizing antibody protection against the H1N1 subtype of influenza A.

[0013] In particular, new vaccine strategies are needed to 1) successfully combat vaccine evasion and 2) prevent the emergence and spread of new influenza pathogens in human populations. Envisioned herein is the use of large databases of different influenza virus sequences, not only from humans but also from animals, which are the source of new influenza virus reassortments that give rise to new human pathogens.

[0014] Influenza A H5 viruses Although most viral genes have been replaced through reassortment, resulting in many different genotypes, the specific H5 gene is still present in all influenza A isolates identified since its discovery in 1996. H5 therefore provides a stock against which evolving strains of influenza A can be effectively compared. The H5 HA clade nomenclature system was developed to compare the evolutionary patterns of this gene. Circulating H5N1 viruses are grouped into a number of viral clades based on characterization and sequence homology of the HA gene. The clades have one common ancestor where certain genetic changes occurred. As viruses within these clades continue to evolve, sub-lineages periodically emerge. Vaccines against influenza A H5 exist, but these vaccines either fail to induce neutralizing immune responses against key H5 clades or the affinity of the antigen for the neutralizing antibodies is suboptimal. The computationally optimised broadly reactive antigen (COBRA) Tier 2 vaccine design (Nunez et al, Vaccines, 2020, 38(4):830-839) is developed by consensus sequence alignment techniques using full-length sequences from H5N1 clade 2 infections isolated from both humans and birds. However, this design did not produce protection against the newer reassorted viruses across all H5N1 clades and subclades tested for hemagglutinin inhibition (HAI) antibodies or vaccines. The risk of humans becoming infected with avian influenza A (H5Nx), especially those of clade 2.3.4.4, is rising due to increased human-avian contact and poor biosafety practices.

[0015] Therefore, there is also a need to provide improved vaccines that induce a more broadly neutralizing immune response against influenza A H5 viruses. In particular, there is a need to provide vaccines that induce antibody responses that effectively neutralize influenza A clade 2.3.4.4. Summary of the Invention [Means for solving the problem]

[0016] Applicants have identified amino acid sequences and their encoding nucleic acid molecules that induce broadly neutralizing immune responses against important H5 clades of influenza A, including clade 2.3.4.4. Applicants have further identified amino acid sequences and their encoding nucleic acid molecules that are responsible for stabilizing the stem region of the H5 molecule in both the pre-fusion and post-fusion states.

[0017] An embodiment of H5 of the present invention is described below.

[0018] According to the invention, there is provided a method for the preparation of a globular hemagglutinin antibody comprising the globular head domain of hemagglutinin subtype 5 (H5), and optionally the stem domain of hemagglutinin, comprising the following amino acid residues at positions 156, 157, 171, 172, and 205 of the head domain: ·156: R; · 157: P or S, preferably P; 171: D or N; 172: T or A, preferably T; and 205: K or R, preferably K, An isolated polypeptide is provided having the formula:

[0019] Applicants have found that such polypeptides elicit broadly neutralizing antibody responses against a diverse panel of H5 influenza viruses, including viruses from different seasonal clades.

[0020] Optionally, a polypeptide of the invention comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along their entire length to the amino acid sequence of SEQ ID NO: 7, 8, 10, 11, 1, or 3.

[0021] Optionally, the polypeptides of the invention comprise the following amino acid residues at positions 156, 157, 171, 172, and 205 in the head domain: ·156: R; ·157: P; ·171: D; 172: T; and 205: K Includes.

[0022] Optionally, the polypeptide of the invention has an amino acid sequence of SEQ ID NO:7 or 8, or at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:7 or 8 and contains the following amino acid residues at positions corresponding to positions 156, 157, 171, 172, and 205 of SEQ ID NO:7 or 8: ·156: R; ·157: P; ·171: D; 172: T; and 205: K The amino acid sequence includes:

[0023] Optionally, a polypeptide of the invention comprises the amino acid sequence of SEQ ID NO: 7 (FLU_T3_HA_1) (see Example 4 below).

[0024] Such polypeptides are particularly advantageous because they induce broadly neutralizing antibody responses against a diverse panel of H5 influenza viruses currently circulating in birds and humans, including H5 influenza viruses of clades 2.3.4 and 7.1 arising from the Goose Guangdong (A / Goose / Guangdong / 1 / 1996, GS / GD) lineage.

[0025] Optionally, the polypeptides of the invention comprise the following amino acid residues at positions 156, 157, 171, 172, and 205 in the head domain: ·156: R; ·157: P; ·171: N; 172: T; and 205: K, inclusive.

[0026] Optionally, a polypeptide of the invention has an amino acid sequence of SEQ ID NO: 10 or 11, or at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO: 10 or 11 and contains the following amino acid residues at positions corresponding to positions 156, 157, 171, 172, and 205 of SEQ ID NO: 10 or 11: ·156: R; ·157: P; ·171: N; 172: T; and 205: K The amino acid sequence includes:

[0027] Optionally, a polypeptide of the invention comprises the amino acid sequence of SEQ ID NO: 10 (FLU_T3_HA_2) (see Example 5 below).

[0028] Such polypeptides are particularly advantageous because they induce broadly neutralizing antibody responses against a diverse panel of H5 influenza viruses currently circulating in birds, including H5 influenza viruses of GS / GD clades 2.3.4 and 7.1.

[0029] Optionally, the polypeptides of the invention comprise the following amino acid residues at positions 156, 157, 171, 172, and 205 in the head domain: ·156: R; ·157: S; ·171: N; 172: A; and 205: R, Includes.

[0030] Optionally, a polypeptide of the invention has an amino acid sequence of SEQ ID NO:1 or 3, or at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:1 or 3, and contains the following amino acid residues at positions corresponding to positions 156, 157, 171, 172, and 205 of SEQ ID NO:1 or 3: ·156: R; ·157: S; ·171: N; 172: A; and 205: R The amino acid sequence includes:

[0031] Optionally, a polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:1 (FLU_T2_HA_1) (see Example 1 below).

[0032] Such polypeptides are particularly advantageous because they induce broadly neutralizing antibody responses against a diverse panel of H5 influenza viruses, including viruses of several different GS / GD clades.

[0033] Table 1 below summarizes the amino acid sequence differences at positions A-E of influenza hemagglutinin H5 for different embodiments of the present invention, and the differences at those positions compared to the prior art COBRA sequence. [Table 1]

[0034] Applicants have also designed additional amino acid sequences and their encoding nucleic acid molecules that induce broadly neutralizing immune responses against the important H5 clade of influenza A. These polypeptides are referred to herein as FLU_T3_HA_3, FLU_T3_HA_4, and FLU_T3_HA_5. Such polypeptides are particularly advantageous because they induce broadly neutralizing antibody responses against a diverse panel of H5 influenza viruses, as shown by the results described in Example 24 and FIG.

[0035] Figure 22 shows the amino acid sequences of FLU_T3_HA_3 (sequence number 27), FLU_T3_HA_4 (sequence number 35), and FLU_T3_HA_5 (sequence number 43) aligned with the amino acid sequences of FLU_T2_HA_1 (also referred to as FLU_T2_HA_9), FLU_T3_HA_1, and FLU_T3_HA_2, and the HA amino acid sequences of influenza A H5N1 strains A / whooper swan / Mongolia / 244 / 2005 (H5_WSN) (sequence number 64) and A / gyrfalcon / Washington / 41088-6 / 2014 (H5_GYR) (sequence number 65).

[0036] FIG. 21 summarizes the amino acid sequence differences at positions A through E of influenza hemagglutinin H5 for FLU_T2_HA_1 (also known as FLU_T2_HA_9), FLU_T3_HA_1, FLU_T3_HA_2, FLU_T3_HA_3, FLU_T3_HA_4, FLU_T3_HA_5.

[0037] There is also provided in accordance with the present invention an isolated polypeptide comprising a globular head domain of hemagglutinin subtype 5 (H5), and optionally a hemagglutinin stem domain, wherein the polypeptide comprises an amino acid sequence having a deletion of an amino acid residue at a position corresponding to residue position 144 or 145 of the globular head domain of wild-type H5.

[0038] Optionally, the polypeptide comprises an amino acid sequence having a deleted amino acid residue at a position corresponding to residue position 144 of the globular head domain of wild-type H5.

[0039] Optionally, the polypeptide comprises an amino acid sequence having a deleted amino acid residue at a position corresponding to residue position 145 of the globular head domain of wild-type H5.

[0040] Optionally, polypeptides of the invention having amino acid residues deleted at positions corresponding to residue positions 144 or 145 of the globular head domain of wild-type H5 comprise an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:3.

[0041] Optionally, an isolated polypeptide of the invention comprising a globular head domain of H5 retains at least some of the HA activity of the globular head domain of wild-type H5 (e.g., that of the H5 WSN isolate - SEQ ID NO:64).

[0042] HA activity can be determined, for example, by hemagglutination assay or by binding assay with sialic acid (SA). For a suitable hemagglutination assay, see Ustinov et al. (Biochemistry (Moscow), 2017, Vol. 82, No. 11, pp. 1234-1248: The Power and Limitations of Influenza Virus Hemagglutinin Assays). For a suitable binding assay, see Takemoto et al. (VIROLOGY 217, 452 - 458 (1996): A Surface Plasmon Resonance Assay for the Binding of Influenza Virus).

[0043] Influenza virions can agglutinate erythrocytes with the formation of a viscous gel. Agglutination occurs through the binding of virion-embedded HA to sialylated surface proteins of several erythrocytes at a time. The number of erythrocytes that are agglutinated is proportional to the HA content and can be used to estimate the functional activity of the protein itself. The classical procedure uses a 0.5-1.0% suspension of erythrocytes mixed with and incubated with a virus suspension, a negative control containing only erythrocytes, and a positive control containing erythrocytes and virions (Salk, JE (1944) A simplified procedure for titrating hemagglutinating capacity of influenza virus and the corresponding antibody, J. Immunol., 49, 87-98).

[0044] Hemagglutination tests can be performed not only on influenza virions, but also on isolated HA molecules if they are in the form of trimers that lead to the formation of a multicontact network. Thus, the HA ectodomain present solely in monomeric form does not agglutinate erythrocytes, whereas the oligomerization-prone HA1 (amino acids 1-330) does (Khurana, et al., (2010) Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic influenza virus, PLoS One, 5, e11548.). Removal of the N-terminal fragment of HA1 (amino acids 1-8) containing the oligomerization signal Ile-Cys-Ile leads to a complete loss of HA1 activity, whereas removal of the C-terminal part (amino acids 321-330), in contrast, stabilizes the trimers and promotes hemagglutination. The larger HA1 fragment (amino acids 1-104) is also capable of oligomerization but does not agglutinate erythrocytes due to the absence of the SA binding site.

[0045] Optionally, an isolated polypeptide of the invention comprising an H5 globular head domain retains at least 25%, at least 50%, or at least 75% of the HA activity of a wild-type H5 globular head domain (e.g., an H5 WSN isolate - SEQ ID NO:64).

[0046] Optionally, an isolated polypeptide of the invention having a deletion of an amino acid residue at a position corresponding to residue position 144 or 145 of the globular head domain of wild-type H5 has the following amino acid residues at positions corresponding to residues 156, 157, 171, 172, and 205 of the globular head domain of wild-type H5: ·156: R; ·157: S; ·171: N; 172: A; and 205: R The amino acid sequence includes:

[0047] Optionally, an isolated polypeptide of the invention having an amino acid residue deleted at a position corresponding to residue position 144 or 145 of the globular head domain of wild-type H5 comprises the amino acid sequence of SEQ ID NO:27 or 29, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:27 or 29.

[0048] Optionally, an isolated polypeptide of the invention having a deletion of amino acid residues at positions corresponding to residue positions 144 or 145 of the globular head domain of wild-type H5 comprises the amino acid sequence of SEQ ID NO:29.

[0049] Optionally, an isolated polypeptide of the invention having a deletion of amino acid residues at positions corresponding to residue positions 144 or 145 of the globular head domain of wild-type H5 comprises the amino acid sequence of SEQ ID NO:27.

[0050] Optionally, an isolated polypeptide of the invention having a deletion of amino acid residues at positions corresponding to residue positions 144 or 145 of the globular head domain of wild-type H5 comprises a stem domain of hemagglutinin, the polypeptide having the following amino acid residues at positions corresponding to residue positions 416 and 434 of the wild-type H5 sequence: 416: F; and 434: F The amino acid sequence includes:

[0051] Optionally, the polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:27.

[0052] According to the invention there is provided an isolated polypeptide comprising a globular head domain of hemagglutinin subtype 5 (H5), and optionally a hemagglutinin stem domain, wherein the polypeptide comprises the following amino acid residues at positions corresponding to residue positions 148 and 149 of the globular head domain of wild type H5: ·148: V; 149: P Also provided is an isolated polypeptide comprising an amino acid sequence having the following structure:

[0053] Optionally, a polypeptide of the invention comprising an amino acid sequence having a V at a position corresponding to residue position 148 and a P at a position corresponding to residue position 149 may further comprise the following amino acid residue at a position corresponding to residue position 238 of the globular head domain of wild-type H5: 238: E The amino acid sequence includes:

[0054] Optionally, a polypeptide of the invention comprising an amino acid sequence having a V at a position corresponding to residue position 148 and a P at a position corresponding to residue position 149 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:3.

[0055] Optionally, an isolated polypeptide of the invention comprising a globular head domain of H5 retains at least some of the HA activity of the globular head domain of wild-type H5 (e.g., that of the H5 WSN isolate - SEQ ID NO:64).

[0056] Optionally, an isolated polypeptide of the invention comprising an H5 globular head domain retains at least 25%, at least 50%, or at least 75% of the HA activity of a wild-type H5 globular head domain (e.g., an H5 WSN isolate - SEQ ID NO:64).

[0057] Optionally, a polypeptide of the invention comprising an amino acid sequence having a V at residue position corresponding to 148 and a P at residue position corresponding to 149 has reduced affinity for its receptor compared to the globular head domain of wild-type H5.

[0058] Optionally, a polypeptide of the invention comprising an amino acid sequence having a V at a position corresponding to residue position 148 and a P at a position corresponding to residue position 149, can further comprise the following amino acid residues at positions corresponding to residues 156, 157, 171, 172, and 205 of the globular head domain of wild-type H5: ·156: R; ·157: S; ·171: N; 172: A; and 205: R The amino acid sequence includes:

[0059] Optionally, a polypeptide of the invention comprising an amino acid sequence having a V at a position corresponding to residue position 148 and a P at position 149 corresponding to residue position 149 comprises an amino acid sequence of SEQ ID NO:35 or 37, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:35 or 37.

[0060] Optionally, the isolated polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:37.

[0061] Optionally, the isolated polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:35.

[0062] Optionally, a polypeptide of the invention comprising an amino acid sequence having a V at a position corresponding to residue position 148 and a P at a position corresponding to residue position 149, the polypeptide comprising the stem domain of hemagglutinin, the polypeptide comprising the following amino acid residues at positions corresponding to residue positions 416 and 434 of the wild-type H5 sequence: 416: F; and 434: F The amino acid sequence includes:

[0063] Optionally, the polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:35.

[0064] 1. An isolated polypeptide comprising a globular head domain of hemagglutinin subtype 5 (H5), and optionally a hemagglutinin stem domain, wherein the polypeptide has the following amino acid residue at a position corresponding to residue position 238 of the globular head domain of wild-type H5: 238: E Also provided in accordance with the present invention is an isolated polypeptide comprising an amino acid sequence having the following structure:

[0065] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having an E residue at a position corresponding to residue position 238 of the globular head domain of wild-type H5, further comprising the following amino acid residues at positions corresponding to residue positions 148 and 149 of the globular head domain of wild-type H5: ·148: S; 149: S The amino acid sequence includes:

[0066] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having an E residue at a position corresponding to residue position 238 of the globular head domain of wild-type H5 comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:3.

[0067] Optionally, an isolated polypeptide of the invention comprising a globular head domain of H5 retains at least some of the HA activity of the globular head domain of wild-type H5 (e.g., that of the H5 WSN isolate - SEQ ID NO:64).

[0068] Optionally, an isolated polypeptide of the invention comprising an H5 globular head domain retains at least 25%, at least 50%, or at least 75% of the HA activity of a wild-type H5 globular head domain (e.g., an H5 WSN isolate - SEQ ID NO:64).

[0069] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having an E residue at a position corresponding to residue position 238 of the globular head domain of wild-type H5 has reduced affinity for its receptor compared to the globular head domain of wild-type H5.

[0070] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having an E residue at a position corresponding to residue position 238 of the globular head domain of wild-type H5, or having the following amino acid residues at positions corresponding to residues 156, 157, 171, 172, and 205 of the globular head domain of wild-type H5: ·156: R; ·157: S; ·171: N; 172: A; and 205: R The amino acid sequence includes:

[0071] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having an E residue at a position corresponding to residue position 238 of the globular head domain of wild-type H5 comprises the amino acid sequence of SEQ ID NO:43 or 45, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:43 or 45.

[0072] Optionally, the isolated polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:45.

[0073] Optionally, the isolated polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:43.

[0074] Optionally, the isolated polypeptide of the invention comprises the following amino acid residues at positions corresponding to residue positions 279 and 298 of the globular head domain of wild-type H5: 279 A; and 298 M The amino acid sequence includes:

[0075] Optionally, the polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to any of SEQ ID NOs: 27, 35, or 43.

[0076] The polypeptides of the invention may comprise any suitable hemagglutinin stem domain, including the stem domain of any suitable influenza hemagglutinin subtype, including non-H5 subtypes. Optionally, the stem domain is an H5 stem domain.

[0077] Optionally, the polypeptides of the invention comprise the following amino acid residues at positions 416 and 434 in the stem domain: 416: F; and 434: F Includes.

[0078] Optionally, the polypeptides of the invention can be of any of the following lengths: 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3000, 2000, 1500, 1000, 900, 800, 700, 600, 590, 580, 570, 560, 550, 540, 530 In some embodiments, the amino acid sequence may comprise up to 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, or 270 amino acid residues.

[0079] Applicants have also realized that polypeptides comprising fragments of the H5 globular head domain having amino acid residues from positions A-C may also elicit an antibody response against H5 influenza viruses. For example, such polypeptides may be used alone or grafted onto other HA subtype heads or other proteins (e.g., having similar folding motifs) to generate an appropriate antibody response.

[0080] Thus, according to the invention, the following amino acid sequence:

[0081] R(P / S)SFFRNVVWLIKKN(D / N)(T / A)YPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQT(K / R) (SEQ ID NO:13), or having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along the entire length of SEQ ID NO:13 and the following amino acid residues at positions corresponding to positions 1, 2, 16, 17, and 50 of SEQ ID NO:13: ·1: R; ·2: P or S, preferably P; 16: D or N; 17: T or A, preferably T; and 50: K or R, preferably K, Also provided is an isolated polypeptide comprising an amino acid sequence having the following structure:

[0082] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:13, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO:13 along its entire length, can comprise the following amino acid residues at positions 1, 2, 16, 17, and 50 of said amino acid sequence, or at positions corresponding to positions 1, 2, 16, 17, and 50 of SEQ ID NO:13: ·1: R; ·2: P; ·16: D; 17: T; and 50: K, Includes.

[0083] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:13, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO:13 along its entire length, can comprise the following amino acid residues at positions 1, 2, 16, 17, and 50 of said amino acid sequence, or at positions corresponding to positions 1, 2, 16, 17, and 50 of SEQ ID NO:13: ·1: R; ·2: P; ·16: N; 17: T; and 50: K, Includes.

[0084] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:13, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO:13 along its entire length, can comprise the following amino acid residues at positions 1, 2, 16, 17, and 50 of said amino acid sequence, or at positions corresponding to positions 1, 2, 16, 17, and 50 of SEQ ID NO:13: ·1: R; ·2: S; ·16: N; 17: A; and 50: R, Includes.

[0085] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:13, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:13, is up to 570, 560, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 amino acid residues in length.

[0086] According to the present invention, there is provided a method for the treatment of a leukemia comprising administering to a patient a leukemia or pediatric ... 148: F; and 166: F, Also provided is an isolated polypeptide comprising an amino acid sequence having the following structure:

[0087] Applicants have found that such polypeptides, when forming the stem region of a hemagglutinin molecule, stabilize the stem region in both the pre-fusion and post-fusion states. Such polypeptides may be provided, for example, with an H5 hemagglutinin head domain or a non-H5 head domain.

[0088] Optionally, a method of the present invention comprising the amino acid sequence of any of SEQ ID NOs: 5, 9, or 12, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NOs: 5, 9, or 12. Polypeptides may be of any of the following lengths: 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,500, 1,000, 900, 800, 700, 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370 , 360, 350, 340, 330, 320, 310, or up to 300 amino acid residues.

[0089] The polypeptide of the present invention may contain one or more conservative amino acid substitutions. Conservative amino acid substitutions are those substitutions that, when made, do not interfere much with the properties of the original protein, i.e., the structure and especially the function of the protein are preserved and not significantly changed by such substitutions. Examples of conservative substitutions are shown below: Original residue Conservative substitution Ala Ser Arg-Lys Asn Gln, His Asp Glu Cys Ser Gln Asn GluAsp His Asn, Gln Ile Leu, Val Leu Ile, Val LysArg, Gln Met Leu, Ile Phe, Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr, Trp, Phe Val Ile, Leu

[0090] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0091] Substitutions that are generally predicted to result in the greatest changes in protein properties may be non-conservative changes, such as (a) a hydrophilic residue (e.g., serine or threonine) is substituted for (or substituted with) a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) a cysteine ​​or proline is substituted for (or substituted with) any other residue; (c) a residue with an electropositive side chain (e.g., lysine, arginine, or histidine) is substituted for (or substituted with) an electronegative residue (e.g., glutamic acid or aspartic acid); or (d) a residue with a bulky side chain (e.g., phenylalanine) is substituted for (or substituted with) a residue without a side chain (e.g., glycine).

[0092] An isolated nucleic acid molecule encoding a polypeptide of the invention, or its complement, is also provided according to the invention.

[0093] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to a nucleic acid molecule of the present invention encoding a polypeptide of the present invention, or a complement thereof.

[0094] Optionally, a nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 2, 4, or 6, or a complement thereof.

[0095] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to SEQ ID NO:2, 4, or 6, or a complement thereof.

[0096] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:28, 30, 32, or 34, or comprising the nucleotide sequence of SEQ ID NO:32 and 34, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to SEQ ID NO:28, 30, 32, 34 or to SEQ ID NO:32 and 34, or a complement thereof.

[0097] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 28, 30, 32, or 34, or the nucleotide sequence of SEQ ID NO: 32 and 34, or complements thereof.

[0098] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO:28, or a complement thereof.

[0099] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO:36, 38, 40, or 42, or the nucleotide sequence of SEQ ID NO:40 and 42, or a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to SEQ ID NO:36, 38, 40, or 42 or to SEQ ID NO:40 and 42, or a complement thereof.

[0100] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 36, 38, 40, or 42, or the nucleotide sequence of SEQ ID NO: 40 and 42, or a complement thereof.

[0101] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 36, or a complement thereof.

[0102] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO:44, 46, 48, or 50, or the nucleotide sequence of SEQ ID NO:48 and 50, or a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:44, 46, 48, or 50 or to SEQ ID NO:48 and 50 over its entire length, or a complement thereof.

[0103] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 44, 46, 48, or 50, or the nucleotide sequence of SEQ ID NO: 48 and 50, or complements thereof.

[0104] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 44, or a complement thereof.

[0105] Optionally, an isolated nucleic acid molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:52, 54, 55, 56, or the nucleotide sequence of SEQ ID NO:52 and 54, or a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to SEQ ID NO:52, 54, 55, 56 or to SEQ ID NO:52 and 54, or a complement thereof.

[0106] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 52, 54, 55, or 56, or the nucleotide sequence of SEQ ID NO: 52 and 54, or a complement thereof.

[0107] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO:55, or a complement thereof.

[0108] Optionally, an isolated nucleic acid molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:58, 60, 61, 62, or the nucleotide sequence of SEQ ID NO:58 and 60, or a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to SEQ ID NO:58, 60, 61, 62 or to SEQ ID NO:58 and 60, or a complement thereof.

[0109] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 58, 60, 61, or 62, or comprises the nucleotide sequences of SEQ ID NOs: 58 and 60, or complements thereof.

[0110] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO:61, or a complement thereof.

[0111] Optionally, an isolated nucleic acid molecule of the present invention comprises a messenger RNA (mRNA) molecule.

[0112] The term "broadly neutralising immune response" is used herein with respect to influenza A to include an immune response elicited in a subject that is sufficient to inhibit (i.e. reduce), neutralise or prevent infection and / or progression of infection with at least three antigenically distinct clades of influenza A. Optionally, the broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection and / or progression of infection with different H5 clades of influenza A. Optionally, advantageously, the different clades include clades 2.3.4 and / or 7.1. Optionally, the different clades include clade 2.3.4.4.

[0113] Additional H5 embodiments of the present invention:

[0114] Applicants have also designed additional amino acid sequences and their encoding nucleic acid molecules that induce broadly neutralizing immune responses against the important H5 clade of influenza A. Polypeptides comprising such amino acid sequences are referred to herein as FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3 polypeptides. Such polypeptides are particularly advantageous because they induce broadly neutralizing antibody responses against a diverse panel of H5 clade 2.3.4.4 influenza viruses, as discussed below.

[0115] Clade 2.3.4.4 Applicants have designed additional amino acid sequences and their encoding nucleic acid sequences that induce a broadly neutralizing immune response against clade 2.3.4.4 strains of influenza A. These polypeptides are referred to herein as FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3. Such polypeptides are particularly advantageous because they induce broadly neutralizing antibody responses against a diverse panel of clade 2.3.4.4 influenza viruses, as shown by the results described in Figures 29-34 and Example 34.

[0116] FIG. 25 summarizes the novel differences in amino acid sequence for the new H5 designs FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3.

[0117] Figure 28 shows the amino acid sequences of FLU_T4_HA_1 (SEQ ID NO: 71), FLU_T4_HA_2 (SEQ ID NO: 80), and FLU_T4_HA_3 (SEQ ID NO: 89) aligned with the amino acid sequences of previously designed tier 3 (T3) H5 sequences and the H5 amino acid sequences of influenza A H5 strains. The residue positions in the alignment correspond to the residue positions of A / Sichuan / 26221 / 2014.

[0118] 29-34 show neutralization assays in mice immunized with Tier 4 (T4) vaccine candidates, previously designed sequences, or wild-type strains against the challenge strain. Each of FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3 induces a similar neutralization response against the homologous H5 strain as the challenge strain, and induces a high response against the heterologous strain.

[0119] Table 2 below summarizes the amino acid residue differences between the H5 A / Sichuan / 2014 isolate and the tier 4 (T4) H5 design of the present invention. [Table 2]

[0120] FLU_T4_HA_1 polypeptides and encoding nucleic acid molecules. According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 71 (FLU_T4_HA_1: amino acid sequence of HA0).

[0121] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 71 (FLU_T4_HA_1: amino acid sequence of HA0) or its complement.

[0122] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 71 (amino acid sequence of FLU_T4_HA_1: HA0) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 72 (nucleic acid sequence of FLU_T4_HA_1: HA0), or its complement.

[0123] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 72 (FLU_T4_HA_1: nucleic acid sequence of HA0) or its complement.

[0124] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 73 (FLU_T4_HA_1: amino acid sequence of the head region).

[0125] According to the present invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 73 (FLU_T4_HA_1: amino acid sequence of the head region) or its complement.

[0126] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 73 (FLU_T4_HA_1:head region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 74 (FLU_T4_HA_1:head region nucleic acid sequence), or a complement thereof.

[0127] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 74 (FLU_T4_HA_1: head region nucleic acid sequence) or a complement thereof.

[0128] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 75 (FLU_T4_HA_1: amino acid sequence of the first stem region).

[0129] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 75 (FLU_T4_HA_1: amino acid sequence of the first stem region) or its complement.

[0130] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 75 (FLU_T4_HA_1: amino acid sequence of the first stem region) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 76 (FLU_T4_HA_1: nucleic acid sequence of the first stem region) over its entire length, or is a complement thereof.

[0131] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 76 (FLU_T4_HA_1: nucleic acid sequence of the first stem region) or a complement thereof.

[0132] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 77 (FLU_T4_HA_1: amino acid sequence of the second stem region).

[0133] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 77 (FLU_T4_HA_1: amino acid sequence of the second stem region) or its complement.

[0134] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 77 (FLU_T4_HA_1: amino acid sequence of the second stem region) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 78 (FLU_T4_HA_1: nucleic acid sequence of the second stem region) over its entire length, or a complement thereof.

[0135] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 78 (FLU_T4_HA_1: nucleic acid sequence of the second stem region) or a complement thereof.

[0136] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:79 (pEVAC-FLU_T4_HA_1), or a complement thereof.

[0137] FLU_T4_HA_2 polypeptides and encoding nucleic acid molecules. According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 80 (amino acid sequence of FLU_T4_HA_2: HA0).

[0138] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 80 (FLU_T4_HA_2: amino acid sequence of HA0) or its complement.

[0139] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 80 (amino acid sequence of FLU_T4_HA_2: HA0) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 81 (nucleic acid sequence of FLU_T4_HA_2: HA0), or its complement.

[0140] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 81 (nucleic acid sequence of FLU_T4_HA_2: HA0) or its complement.

[0141] According to the invention there is also provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:80 (FLU_T4_HA_2: amino acid sequence of HA0), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:80 and including amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5) or amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO:100 (A / Sichuan / 2014 H5).

[0142] Optionally, the polypeptide is an amino acid residue E at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), at a position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue T at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); the amino acid residue R at the position corresponding to amino acid residue 344 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or At a position corresponding to amino acid residue 345 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), Further includes:

[0143] According to the present invention, there is provided an amino acid sequence of SEQ ID NO: 80 (FLU_T4_HA_2: amino acid sequence of HA0), or a sequence of SEQ ID NO: 80 having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto along its entire length and including the amino acid residue F at the position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5) or the amino acid residue F at the position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5). In one embodiment, an isolated nucleic acid molecule is provided comprising a nucleotide sequence, or a complement thereof, that encodes an amino acid sequence comprising amino acid residue E at a position corresponding to amino acid residue 238 of H5).

[0144] According to the invention there is also provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:80 (FLU_T4_HA_2: amino acid sequence of HA0), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:80 and comprising amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5) and amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO:100 (A / Sichuan / 2014 H5).

[0145] Optionally, the polypeptide is an amino acid residue E at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), at a position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue T at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); the amino acid residue R at the position corresponding to amino acid residue 344 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or At a position corresponding to amino acid residue 345 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), Further includes:

[0146] According to the present invention, there is provided an amino acid sequence of SEQ ID NO: 80 (FLU_T4_HA_2: amino acid sequence of HA0), or a sequence of SEQ ID NO: 80 having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto along its entire length, and including the amino acid residue F at the position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5) and the amino acid residue S of SEQ ID NO: 100 (A / Sichuan / 2014 H6). In one embodiment, an isolated nucleic acid molecule is provided comprising a nucleotide sequence, or a complement thereof, that encodes an amino acid sequence comprising amino acid residue E at a position corresponding to amino acid residue 238 of H5).

[0147] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 82 (FLU_T4_HA_2: amino acid sequence of the head region).

[0148] According to the present invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 82 (FLU_T4_HA_2: amino acid sequence of the head region) or its complement.

[0149] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 82 (FLU_T4_HA_2:head region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 83 (FLU_T4_HA_2:head region nucleic acid sequence), or the complement thereof.

[0150] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 83 (FLU_T4_HA_2: head region nucleic acid sequence) or a complement thereof.

[0151] According to the invention there is also provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:82 (FLU_T4_HA_2: amino acid sequence of the head region), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:82 and including amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5) or amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO:100 (A / Sichuan / 2014 H5).

[0152] Optionally, the polypeptide is an amino acid residue E at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), the amino acid residue A at the position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or At a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), Further includes:

[0153] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:82 (FLU_T4_HA_2: amino acid sequence of the head region), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:82 and including amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5) or amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO:100 (A / Sichuan / 2014 H5), or a complement thereof.

[0154] According to the invention there is also provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:82 (FLU_T4_HA_2: amino acid sequence of the head region), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:82 and comprising amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5) and amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO:100 (A / Sichuan / 2014 H5).

[0155] Optionally, the polypeptide is an amino acid residue E at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), the amino acid residue A at the position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or At a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), Further includes:

[0156] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:82 (FLU_T4_HA_2: amino acid sequence of the head region), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:82 and including amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5) and amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO:100 (A / Sichuan / 2014 H5), or a complement thereof.

[0157] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 84 (FLU_T4_HA_2: amino acid sequence of the first stem region).

[0158] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 84 (FLU_T4_HA_2: amino acid sequence of the first stem region) or its complement.

[0159] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 84 (FLU_T4_HA_2: amino acid sequence of the first stem region) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 85 (FLU_T4_HA_2: nucleic acid sequence of the first stem region) over its entire length, or a complement thereof.

[0160] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 85 (FLU_T4_HA_2: nucleic acid sequence of the first stem region) or a complement thereof.

[0161] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 86 (FLU_T4_HA_2: amino acid sequence of the second stem region).

[0162] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 86 (FLU_T4_HA_2: amino acid sequence of the second stem region) or its complement.

[0163] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 86 (FLU_T4_HA_2: amino acid sequence of the second stem region) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 87 (FLU_T4_HA_2: nucleic acid sequence of the second stem region) over its entire length, or is the complement thereof.

[0164] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 87 (FLU_T4_HA_2: nucleic acid sequence of the second stem region) or a complement thereof.

[0165] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:88 (pEVAC-FLU_T4_HA_2), or a complement thereof.

[0166] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of any of the above-described FLU_T4_HA_2 polypeptides of the present invention, or a complement thereof.

[0167] FLU_T4_HA_3 polypeptides and encoding nucleic acid molecules. According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 89 (amino acid sequence of FLU_T4_HA_3: HA0).

[0168] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:89 (amino acid sequence of FLU_T4_HA_3: HA0), or a complement thereof.

[0169] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 89 (amino acid sequence of FLU_T4_HA_3: HA0) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 90 (nucleic acid sequence of FLU_T4_HA_3: HA0), or its complement.

[0170] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 90 (nucleic acid sequence of FLU_T4_HA_3: HA0) or its complement.

[0171] According to the invention, there is provided a method for the preparation of a medicament for ... Also provided is an isolated polypeptide comprising an amino acid sequence comprising an amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO:100 (A / Sichuan / 2014 H5), or an amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO:100 (A / Sichuan / 2014 H5).

[0172] Optionally, the polypeptide is an amino acid residue T at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue Q at a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); the amino acid residue R at the position corresponding to amino acid residue 344 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or At a position corresponding to amino acid residue 345 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), Further includes:

[0173] According to the present invention, there is provided an HA0 polypeptide having an amino acid sequence of SEQ ID NO: 89 (FLU_T4_HA_3: amino acid sequence of HA0), or a sequence of SEQ ID NO: 89 having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto along its entire length, and comprising an amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue H at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue H at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue I ... N at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue V at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A In one embodiment, an isolated nucleic acid molecule is provided comprising a nucleotide sequence, or a complement thereof, encoding an amino acid sequence comprising an amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), or an amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5).

[0174] According to the invention, there is provided a method for the preparation of a medicament for ... Also provided is an isolated polypeptide comprising an amino acid sequence comprising an amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO:100 (A / Sichuan / 2014 H5) and an amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO:100 (A / Sichuan / 2014 H5).

[0175] Optionally, the polypeptide is an amino acid residue T at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue Q at a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); the amino acid residue R at the position corresponding to amino acid residue 344 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or At a position corresponding to amino acid residue 345 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), Further includes:

[0176] According to the present invention, there is provided an HA0 polypeptide having an amino acid sequence of SEQ ID NO: 89 (FLU_T4_HA_3: amino acid sequence of HA0), or a sequence of SEQ ID NO: 89 having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto along its entire length, and comprising an amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue H at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue H at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue I ... N at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue V at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A In one embodiment, an isolated nucleic acid molecule is provided comprising a nucleotide sequence, or a complement thereof, encoding an amino acid sequence comprising an amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO:100 (A / Sichuan / 2014 H5) and an amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO:100 (A / Sichuan / 2014 H5).

[0177] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 91 (FLU_T4_HA_3: amino acid sequence of the head region).

[0178] According to the present invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:91 (FLU_T4_HA_3: amino acid sequence of the head region) or its complement.

[0179] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 91 (FLU_T4_HA_3:head region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 92 (FLU_T4_HA_3:head region nucleic acid sequence), or the complement thereof.

[0180] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 92 (FLU_T4_HA_3: head region nucleic acid sequence) or a complement thereof.

[0181] According to the present invention, there is provided an antibody having an amino acid sequence of SEQ ID NO:91 (FLU_T4_HA_3: amino acid sequence of the head region), or a sequence of SEQ ID NO:91 having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto along its entire length, and including the amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5), the amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO:100 (A / Sichuan / 2014 H5), the amino acid residue H ... Also provided is an isolated polypeptide comprising an amino acid sequence comprising amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5) or amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5).

[0182] Optionally, the polypeptide is an amino acid residue T at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or At a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), Further includes:

[0183] According to the present invention, there is provided an antibody having an amino acid sequence of SEQ ID NO:91 (FLU_T4_HA_3: amino acid sequence of the head region), or a sequence of SEQ ID NO:91 having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto along its entire length, and including the amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5), the amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO:100 (A / Sichuan / 2014 H5), the amino acid residue H ... In one embodiment, an isolated nucleic acid molecule is provided comprising a nucleotide sequence, or a complement thereof, encoding an amino acid sequence comprising an amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), or an amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5).

[0184] According to the present invention, there is provided an antibody having an amino acid sequence of SEQ ID NO:91 (FLU_T4_HA_3: amino acid sequence of the head region), or a sequence of SEQ ID NO:91 having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto along its entire length, and including the amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5), the amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO:100 (A / Sichuan / 2014 H5), the amino acid residue H ... Also provided is an isolated polypeptide comprising an amino acid sequence comprising an amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO:100 (A / Sichuan / 2014 H5) and an amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO:100 (A / Sichuan / 2014 H5).

[0185] Optionally, the polypeptide is an amino acid residue T at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or At a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), Further includes:

[0186] According to the present invention, there is provided an antibody having an amino acid sequence of SEQ ID NO:91 (FLU_T4_HA_3: amino acid sequence of the head region), or a sequence of SEQ ID NO:91 having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto along its entire length, and including the amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5), the amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO:100 (A / Sichuan / 2014 H5), the amino acid residue H ... In one embodiment, an isolated nucleic acid molecule is provided comprising a nucleotide sequence, or a complement thereof, encoding an amino acid sequence comprising an amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO:100 (A / Sichuan / 2014 H5) and an amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO:100 (A / Sichuan / 2014 H5).

[0187] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 93 (FLU_T4_HA_3: amino acid sequence of the first stem region).

[0188] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 93 (FLU_T4_HA_3: amino acid sequence of the first stem region) or its complement.

[0189] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 93 (FLU_T4_HA_3: amino acid sequence of the first stem region) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 94 (FLU_T4_HA_3: nucleic acid sequence of the first stem region) over its entire length, or a complement thereof.

[0190] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 94 (FLU_T4_HA_3: nucleic acid sequence of the first stem region) or a complement thereof.

[0191] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 95 (FLU_T4_HA_3: amino acid sequence of the second stem region).

[0192] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 95 (FLU_T4_HA_3: amino acid sequence of the second stem region) or its complement.

[0193] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 95 (FLU_T4_HA_3: amino acid sequence of the second stem region) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 96 (FLU_T4_HA_3: nucleic acid sequence of the second stem region) over its entire length, or a complement thereof.

[0194] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 96 (FLU_T4_HA_3: nucleic acid sequence of the second stem region) or a complement thereof.

[0195] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:97 (pEVAC-FLU_T4_HA_3), or a complement thereof.

[0196] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of any of the above-described FLU_T4_HA_3 polypeptides of the present invention, or a complement thereof.

[0197] M2 The extracellular domain of M2 has been identified as being largely invariant across all influenza A strains, and this represents a potential solution to the problem of creating a universal influenza A vaccine that would induce broad-spectrum protection against all influenza A infections.

[0198] Applicants have identified amino acid sequences and their encoding nucleic acid molecules that induce a broadly neutralizing immune response against influenza A M2.

[0199] An embodiment of M2 of the present invention is described below.

[0200] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:14, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO:14 along its entire length.

[0201] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:15, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15 over its entire length, or a complement thereof.

[0202] Neuraminidase Applicants have also identified amino acid sequences, and their encoding nucleic acid molecules, that contain epitopes of neuraminidase that are conserved by several different influenza subtypes.

[0203] Neuraminidase embodiments of the invention are described below.

[0204] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:16, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO:16 along its entire length.

[0205] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:18, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO:18 along its entire length.

[0206] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:17, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17 over its entire length, or a complement thereof.

[0207] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:19, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:19 over its entire length, or a complement thereof.

[0208] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 98 (the amino acid sequence of FLU_T3_NA_3).

[0209] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:98 (amino acid sequence of FLU_T3_NA_3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO:98 along its entire length.

[0210] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 98 (the amino acid sequence of FLU_T3_NA_3), or a complement thereof.

[0211] Optionally, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 98 (amino acid sequence of FLU_T3_NA_3) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 99 (nucleic acid sequence of FLU_T3_NA_3), or its complement.

[0212] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 99 (the nucleic acid sequence of FLU_T3_NA_3) or a complement thereof.

[0213] Influenza A H1 Applicants have also designed amino acid sequences and their encoding nucleic acid molecules that can be used in vaccines to induce broad H1 immunity and protection against diverse strains of influenza A. The designed amino acid sequences are hereinafter referred to as FLU_T2_HA_3_I3 and FLU_T2_HA_4.

[0214] An embodiment of H1 of the present invention is described below.

[0215] FLU_T2_HA_3_I3: An embodiment of FLU_T2_HA_3_I3 of the present invention is described below.

[0216] Also provided in accordance with the present invention is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:22 (FLU_T2_HA_3_I3).

[0217] Also provided in accordance with the present invention is an isolated polynucleotide comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 22 (FLU_T2_HA_3_I3), or its complement.

[0218] The nucleotide sequence may comprise the sequence of SEQ ID NO: 23 or its complement.

[0219] Also provided in accordance with the present invention is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:22 (FLU_T2_HA_3_I3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:22.

[0220] Also provided in accordance with the present invention is an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:23, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to SEQ ID NO:23, or a complement thereof.

[0221] FLU_T2_HA_4: An embodiment of FLU_T2_HA_4 of the present invention is described below.

[0222] Also provided in accordance with the present invention is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:68 (FLU_T2_HA_4).

[0223] Also provided in accordance with the present invention is an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:69 (FLU_T2_HA_4), or a complement thereof.

[0224] The nucleotide sequence may comprise the sequence of SEQ ID NO:69 or its complement.

[0225] Also provided in accordance with the present invention is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:68 (FLU_T2_HA_4), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:68.

[0226] Also provided in accordance with the present invention is an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:69, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to SEQ ID NO:69, or a complement thereof.

[0227] The H5 and H1 embodiments of the invention are hereinafter collectively referred to as the HA embodiments.

[0228] Combination vaccines To prevent vaccine evasion more effectively, a vaccine is provided that has a combination of two or more (preferably three or more) evolutionarily constrained and computationally designed viral antigen targets, where each antigen target is designed to provide maximum breadth of vaccine protection independently. The vaccine of the present invention can include ancestral antigen-based designs of HA, NA and M2 (either alone or in combination). In addition, combinations of modified HA and NA antigen structures that are not found to be widely spread in humans as natural combinations are provided (e.g., group 1 HA (e.g., H1N1 or H3N2) combined with group 2 NA, which are not found to be widely spread and co-evolved together).

[0229] The polypeptides or nucleic acid molecules of the invention may be combined in any suitable combination (e.g., the HA and / or M2 and / or neuraminidase embodiments of the invention, the H5 and / or M2 and / or neuraminidase embodiments of the invention, the H1 and / or M2 and / or neuraminidase embodiments of the invention, or the FLU_T2_HA_3_I3 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention, or the FLU_T2_HA_4 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention) to provide an influenza vaccine that protects against many more influenza strains than current vaccines. In some embodiments, such combination vaccines protect against several influenza A and B variants (especially those including M2 embodiments, since M2 is better conserved between influenza A and B).

[0230] Optionally, one embodiment of each different category of embodiments is used in combination, for example, an HA embodiment (H5 or H1) and / or an M2 embodiment and / or a neuraminidase embodiment.

[0231] Optionally, a trivalent vaccine combines H5, M2 and neuraminidase embodiments of the invention.

[0232] Optionally, a trivalent vaccine of the invention combines an H5 embodiment, an M2 embodiment and a neuraminidase embodiment of the invention.

[0233] Optionally, a trivalent vaccine combines H1, M2 and neuraminidase embodiments of the invention.

[0234] Optionally, a trivalent vaccine of the invention combines an H1 embodiment, an M2 embodiment and a neuraminidase embodiment of the invention.

[0235] Optionally, the nucleic acid vector of the invention comprises: i) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or 29, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or 37, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 43 or 45 (examples of the embodiment of H5), and / or ii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0236] Optionally, the nucleic acid vector of the invention comprises: i) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 68 (example of an embodiment of H1), and / or ii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0237] Optionally, the nucleic acid vector of the invention comprises: i) a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or a complement thereof, and / or ii) a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement, and / or iii) a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or a complement thereof. Includes.

[0238] Optionally, the nucleic acid vector of the invention comprises: i) a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22 (amino acid sequence of FLU_T2_HA_3_I3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 22, or a complement thereof; and / or ii) a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 (amino acid sequence of FLU_T2_NA_3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 16, or a complement thereof; and / or iii) a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:14 (the amino acid sequence of FLU_T2_M2_1), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:14, or a complement thereof. Includes.

[0239] Optionally, the nucleic acid molecule of (i) comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 22 (the amino acid sequence of FLU_T2_HA_3_I3), or a complement thereof.

[0240] Optionally, the nucleic acid molecule of (ii) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 (the amino acid sequence of FLU_T2_NA_3), or a complement thereof.

[0241] Optionally, the nucleic acid molecule of (iii) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 (the amino acid sequence of FLU_T2_M2_1), or a complement thereof.

[0242] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO:22), or its complement, comprises the nucleotide sequence of SEQ ID NO:23, or its complement.

[0243] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), or its complement, comprises the nucleotide sequence of SEQ ID NO: 17, or its complement.

[0244] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14), or its complement, comprises the nucleotide sequence of SEQ ID NO: 15, or its complement.

[0245] Optionally, the vectors of the present invention further comprise a promoter operably linked to each nucleic acid molecule.

[0246] Optionally, the vector of the invention is a pEVAC-based vector.

[0247] The immune response can be a humoral and / or cellular immune response. A cellular immune response is the response of cells of the immune system (e.g., B cells, T cells, macrophages, or polymorphonucleocytes) to a stimulus such as an antigen or a vaccine. An immune response can include any cell of the body that participates in a host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate immune responses or inflammation.

[0248] Optionally, the polypeptide of the present invention induces a protective immune response.A protective immune response refers to an immune response that protects a subject from infection or disease (i.e., prevents infection or prevents the occurrence of disease associated with infection).Methods for measuring immune response are well known in the art and include, for example, measuring lymphocyte (e.g., B cell or T cell) proliferation and / or activity, cytokine or chemokine secretion, inflammation, or antibody production.

[0249] Optionally, the polypeptides of the present invention may induce the production of antibodies and / or T cell responses in a human or non-human animal to which the polypeptide has been administered (either as a polypeptide or, for example, expressed from an administered nucleic acid expression vector).

[0250] Similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between sequences (otherwise referred to as sequence identity). Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the greater the similarity between the two sequences. Homologs or variants of a given gene or protein have a relatively high degree of sequence identity when aligned using standard methods. Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids' Research 16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0251] Sequence identity between nucleic acid sequences or amino acid sequences can be determined by comparing the alignment of the sequences. If the equivalent position in the compared sequences is occupied by the same nucleotide or amino acid, the molecules are identical at that position. The score of the alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at the position shared by the compared sequences. When comparing sequences, optimal alignment may require that gaps are introduced in one or more of the sequences to take into account possible insertions and deletions in the sequence. The sequence comparison method may use gap penalties so that, when the number of identical nucleotides in the compared sequences is the same, sequence alignment with the fewest possible gaps, which indicates a higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. The calculation of maximum percent identity includes the generation of optimal alignment, taking into account gap penalties.

[0252] Suitable computer programs for performing sequence comparisons are widely available in the commercial and public sectors. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29; program available at http: / / bitincka.com / ledion / matgat), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410; program available at http: / / www.ebi.ac.uk / fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948; program available at http: / / www.ebi.ac.uk / tools / clustalw2) and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds.), pp 1-44, Addison Wesley; programs available at http: / / www.ebi.ac.uk / tools / emboss / align). All programs can be run using default parameters.

[0253] For example, sequence comparisons can be performed using the "Needle" method of the EMBOSS Pairwise Alignment Algorithms, which determines the optimal alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparison ("Protein Molecule" option) can be gap extension penalty: 0.5, gap opening penalty: 10.0, matrix: Blosum 62.

[0254] Sequence comparison may be performed over the entire length of the reference sequence.

[0255] The sequences described herein include references to amino acid sequences that contain amino acid residues "at positions corresponding to positions" in another amino acid sequence. Such corresponding positions can be identified from alignment of the sequences, for example, using the sequence alignment methods described herein or other sequence alignment methods known to those of skill in the art.

[0256] vector Vectors comprising the nucleic acid molecules of the invention are also provided according to the invention.

[0257] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:29.

[0258] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:35 or SEQ ID NO:37.

[0259] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:43 or 45.

[0260] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0261] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0262] Optionally, the vector of the invention further comprises a promoter operably linked to the nucleic acid.

[0263] Optionally, the promoter is for expression of a polypeptide encoded by the nucleic acid in a mammal.

[0264] Optionally, the promoter is for expressing a polypeptide encoded by the nucleic acid in yeast or insect cells.

[0265] Optionally, the vector is a vaccine vector.

[0266] Optionally, the vector is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, an mRNA vaccine vector, or a DNA vaccine vector.

[0267] Optionally, the vector is a DNA vector.

[0268] Optionally, the vector is an mRNA vector.

[0269] The polynucleotide of the present invention can comprise DNA or RNA molecules. For the embodiment in which the polynucleotide comprises an RNA molecule, it should be understood that the nucleic acid sequence of the polynucleotide is the same as or its complement as that set forth in each SEQ ID NO:, but each "T" nucleotide is replaced by "U".

[0270] As discussed in more detail below, the polynucleotides of the invention may contain one or more modified nucleosides.

[0271] A polynucleotide of the invention may contain one or more nucleotide analogs known to those of skill in the art.

[0272] The nucleic acid molecule of the present invention can include DNA or RNA molecules. For the embodiment in which the nucleic acid molecule comprises RNA, it is understood that the molecule can include an RNA sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 2, 4, or 6 (wherein each "T" nucleotide is replaced by "U"), or its complement.

[0273] For example, where an RNA vaccine vector is provided comprising a nucleic acid of the invention, it is understood that the nucleic acid sequence of the nucleic acid of the invention is an RNA sequence and therefore may comprise, for example, an RNA nucleic acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 2, 4, or 6 (wherein each "T" nucleotide is replaced by a "U"), or a complement thereof.

[0274] Viral vaccine vectors use a virus to deliver nucleic acid (e.g., DNA or RNA) into human or non-human animal cells. The nucleic acid contained in the virus encodes one or more antigens that, once expressed in the infected human or non-human animal cell, elicit an immune response. Both humoral and cell-mediated immune responses can be induced by viral vaccine vectors. Viral vaccine vectors combine many of the useful properties of nucleic acid vaccines with those of live attenuated vaccines. Like nucleic acid vaccines, viral vaccine vectors can elicit a range of immune responses (antibodies, T helper cells (CD4 + T cells), and cytotoxic T lymphocytes (CTL, CD8 +Viral vaccine vectors deliver nucleic acid to host cells for the production of antigenic proteins that can be tailored to stimulate immune responses (including T cell-mediated immunity). Unlike nucleic acid vaccines, viral vaccine vectors actively invade host cells and also have the potential to replicate similarly to live attenuated vaccines, further activating the immune system like adjuvants. Viral vaccine vectors therefore generally comprise live attenuated viruses genetically engineered to deliver nucleic acid (e.g., DNA or RNA) that encodes protein antigens from unrelated organisms. Although viral vaccine vectors can generally generate stronger immune responses than nucleic acid vaccines, for some diseases, viral vectors are used in combination with other vaccine technologies in a strategy called heterologous prime-boost. In this system, one vaccine is given as a prime step, followed by vaccination with an alternative vaccine as a booster. The heterologous prime-boost strategy aims to provide a stronger overall immune response. Viral vaccine vectors can be used as both prime and boost vaccines as part of this strategy. Viral vaccine vectors are reviewed by Ura et al., 2014 (Vaccines 2014, 2, 624-641) and Choi and Chang, 2013 (Clinical and Experimental Vaccine Research 2013;2:97-105).

[0275] Optionally, the viral vaccine vector is based on a viral delivery vector (e.g., a poxvirus (e.g., Modified Vaccinia Ankara (MVA), NYVAC, AVIPOX), herpesvirus (e.g., HSV of any host species, CMV, adenovirus), morbillivirus (e.g., measles), alphavirus (e.g., SFV, Sendai), flavivirus (e.g., yellow fever), or rhabdovirus (e.g., VSV) based viral delivery vector), a bacterial delivery vector (e.g., Salmonella, E. coli), an RNA expression vector, or a DNA expression vector.

[0276] Adenoviruses are currently the most widely used and advanced viral vectors developed for SARS2 vaccines. They are non-enveloped double-stranded DNA (dsDNA) viruses with the capacity to package up to 7.5 kb of foreign genes. Recombinant adenovirus vectors are widely used due to their high transduction efficiency, high transgene expression level, and wide viral tropism range. These vaccines are highly cell-specific, have high gene transfer efficiency, and are efficient in inducing immune responses. Adenovirus vaccines are effective in triggering and priming T cells, resulting in long-term and high-level antigenic protein expression and therefore long-lasting protection.

[0277] When a viral vector is used according to the present invention, it may be advantageous for each of the HA and / or M2 and / or neuraminidase embodiments of the present invention, the H5 and / or M2 and / or neuraminidase embodiments of the present invention, the H1 and / or M2 and / or neuraminidase embodiments of the present invention, or the FLU_T2_HA_3_I3 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the present invention, or the FLU_T2_HA_4 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the present invention to be encoded as part of the same viral vaccine vector. For example, it may be easier (and cheaper) to make a single vector encoding each of the H5, M2, and neuraminidase embodiments than to make several different vectors, each encoding a different H5, M2, or neuraminidase embodiment.

[0278] Optionally, the nucleic acid expression vector is a nucleic acid expression vector and a viral pseudotype vector.

[0279] Optionally, the nucleic acid expression vector is a vaccine vector.

[0280] Optionally, the nucleic acid expression vector comprises, in the 5' to 3' direction: a promoter; a splice donor site (SD); a splice acceptor site (SA); and a termination signal, wherein the multiple cloning site is located between the splice acceptor site and the termination signal sequence.

[0281] Optionally, the promoter comprises the CMV immediate early 1 enhancer / promoter (CMV-IE-E / P) and / or the termination signal comprises the termination signal of the bovine growth hormone gene (Tbgh) lacking a KpnI restriction endonuclease site.

[0282] Optionally, the nucleic acid expression vector further comprises an origin of replication and a nucleic acid encoding resistance to an antibiotic. Optionally, the origin of replication comprises a pUC-plasmid origin of replication and / or a nucleic acid encoding resistance to kanamycin.

[0283] Optionally, the vector is a pEVAC-based expression vector.

[0284] Optionally, the nucleic acid expression vector comprises the nucleic acid sequence of SEQ ID NO: 21 (pEVAC). The pEVAC vector has been found to be a highly versatile expression vector for generating viral pseudotypes and for direct DNA vaccination of animals and humans. The pEVAC expression vector is described in more detail in Example 11 below. Figure 8 shows the plasmid map of pEVAC.

[0285] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0286] Optionally, the or each vaccine vector is an RNA vaccine vector.

[0287] Optionally, the or each vaccine vector is an mRNA vaccine vector.

[0288] The polynucleotides of the present invention may comprise DNA molecules.

[0289] The or each polynucleotide of the pharmaceutical composition, combined preparation, or vector of the invention may comprise a DNA molecule.

[0290] The vector of the present invention may be a DNA vector.

[0291] The or each vector of the pharmaceutical composition or combined preparation of the invention may be a DNA vector.

[0292] A polynucleotide of the invention, or of a pharmaceutical composition, combined preparation, or vector of the invention, may be provided as part of a DNA vaccine.

[0293] Also provided according to the present invention is a DNA vaccine comprising a polynucleotide of the present invention, a vector of the present invention, or a pharmaceutical composition or combined preparation of the present invention comprising one or more polynucleotides, wherein the or each polynucleotide is a DNA molecule.

[0294] The polynucleotides of the present invention may comprise RNA molecules.

[0295] The or each polynucleotide of the pharmaceutical composition, combined preparation, or vector of the invention may comprise an RNA molecule.

[0296] The vector of the present invention may be an RNA vector.

[0297] The or each vector of the pharmaceutical composition or combined preparation of the invention may be an RNA vector.

[0298] A polynucleotide of the invention, or of a pharmaceutical composition, combined preparation, or vector of the invention, may be provided as part of an RNA vaccine.

[0299] Also provided according to the present invention is an RNA vaccine comprising a polynucleotide of the present invention, a vector of the present invention, or a pharmaceutical composition or combined preparation of the present invention comprising one or more polynucleotides, wherein the or each polynucleotide is an RNA molecule.

[0300] The polynucleotides of the present invention may comprise mRNA molecules.

[0301] The or each polynucleotide of the pharmaceutical composition, combined preparation, or vector of the present invention may comprise an mRNA molecule.

[0302] The vector of the present invention may be an mRNA vector.

[0303] The or each vector of the pharmaceutical composition or combined preparation of the invention may be an mRNA vector.

[0304] Messenger RNA (mRNA) vaccines A polynucleotide of the invention, or of a pharmaceutical composition, combined preparation, or vector of the invention, may be provided as part of an mRNA vaccine.

[0305] Also provided according to the present invention is an mRNA vaccine comprising a polynucleotide of the present invention, a vector of the present invention, or a pharmaceutical composition or combined preparation of the present invention comprising one or more polynucleotides, wherein the or each polynucleotide comprises an mRNA molecule.

[0306] Messenger RNA (mRNA) vaccines are a new form of vaccine (recently reviewed in Pardi et al., Nature Reviews Drug Discovery Volume 17, pages 261-279(2018); Wang et al., Molecular Cancer (2021) 20:33: mRNA vaccine: a potential therapeutic strategy). The first mRNA vaccines approved for use were BNT162b2 (BioNTech's vaccine produced by Pfizer) and mRNA-1273 (produced by Moderna) during the COVID-19 pandemic. mRNA vaccines have the unique property of promoting antigen expression transiently (typically for a few days). Expression of exogenous antigens is controlled by the lifespan of the coding mRNA, which is regulated by cellular degradation pathways. This transient nature of protein expression, which requires repeated administration for the treatment of genetic diseases and cancer, is extremely beneficial for vaccines, where prime or prime-boost vaccination is sufficient to generate highly specific adaptive immunity without any exposure to infectious disease.

[0307] mRNA-based vaccines trigger an immune response after synthetic mRNA encoding viral antigens is transfected into human cells. The cytosolic mRNA molecules are then translated by the host's own cellular machinery into specific viral antigens. These antigens are then presented on the cell surface where they can be recognized by immune cells and an immune response can be triggered.

[0308] The structural elements of the vaccine vector mRNA molecule are similar to those of natural mRNA, including a 5' cap, a 5' untranslated region (UTR), a coding region (e.g., containing an open reading frame encoding a polypeptide of the invention), a 3' UTR, and a poly(A) tail. The 5' UTR (also known as leader sequence, transcript leader, or leader RNA) is the region of the mRNA immediately upstream from the start codon. This region is important in regulating the translation of the transcript. In many organisms, the 5' UTR forms complex secondary structures to regulate translation. The 5' UTR begins at the transcription start site and ends one nucleotide (nt) before the start sequence of the coding region (usually AUG). In eukaryotes, the length of the 5' UTR tends to be between 100 and several thousand nucleotides in length. The difference in size is likely due to the complexity of eukaryotic regulation that the 5' UTR has, as well as the large preinitiation complex that must form to initiate translation. Eukaryotic 5' UTRs contain a Kozak consensus sequence (ACC) that includes the start codon AUG. AUG (start codon is underlined) (SEQ ID NO:36). The constructs described herein contain the extended Kozak sequence: GCCACC AUG (The start codon is underlined) (SEQ ID NO:37).

[0309] Two main types of RNA are currently being investigated as vaccines: non-replicating mRNA and self-amplifying RNA derived from viruses. Both types of vaccines share a common structure in the mRNA construct, but self-amplifying RNA vaccines contain an additional sequence in the coding region that contains an RNA-dependent RNA polymerase for RNA replication.

[0310] The BNT162b2 vaccine construct comprises an mRNA molecule encoding the trimerized full-length SARS2 S protein with a PP mutation (at residue positions 986-987) encapsulated in a lipid nanoparticle (LNP). The mRNA is encapsulated in 80 nm ionizable cationic lipid nanoparticles. The mRNA-1273 vaccine construct is also based on a LNP vector, but the synthetic mRNA encapsulated within the lipid construct encodes the full-length SARS2 S protein.

[0311] US Patent No. 10,702,600 B1 (ModernaTX) describes a betacoronavirus mRNA vaccine, including LNPs suitable for use in such a vaccine.

[0312] The nucleic acid vaccines (eg, mRNA) of the present invention may be formulated in lipid nanoparticles.

[0313] mRNA vaccines have several advantages over conventional vaccines that contain inactivated (or live attenuated), disease-causing organisms. First, mRNA-based vaccines can be developed quickly due to the flexibility of design and the ability of the constructs to mimic the structure and expression of antigens found in the course of natural infection. Whereas mRNA vaccines can be developed within days or months based on sequencing information obtained from the target virus, conventional vaccines often take years and require a deep understanding of the target virus to make the vaccine effective and safe. Second, these novel vaccines can be produced quickly. Due to the high yields from in vitro transcription reactions, mRNA production can be fast, inexpensive, and scalable (resulting from chemical synthesis rather than biological growth of cells or bacteria). Third, the vaccine is low risk. mRNA does not contain infectious viral elements or cellular debris (because it is synthetically produced) that pose the risk of infection and insertional mutagenesis. Anti-vector immunity is also avoided because mRNA is a minimally immunogenic gene vector and allows for repeated administration of the vaccine. The challenge of effective application of mRNA vaccines lies in cytosolic delivery. mRNA isolates are rapidly degraded by extracellular RNases and cannot penetrate the cell membrane to be transcribed in the cytosol. However, efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules to allow rapid uptake and expression in the cytoplasm. To date, numerous delivery methods have been developed, including lipid, polymer, or peptide-based delivery, virus-like replicon particles, cationic nanoemulsions, naked mRNA, and dendritic cell-based delivery (respectively discussed in Wang et al., supra). Decationic lipid nanoparticle (LNP) delivery is the most promising and commonly used mRNA vaccine delivery tool.

[0314] Exogenous mRNA can be highly immunostimulatory. Single-stranded RNA (ssRNA) molecules are considered pathogen-associated molecular patterns (PAMPs) and are recognized by various Toll-like receptors (TLRs) that induce proinflammatory responses. Although strong cellular and humoral immune responses are desirable in response to vaccination, the innate immune responses induced by exogenous mRNAs can cause undesirable side effects in subjects. U-rich mRNA sequences are key elements for activating TLRs (Wang et al., supra). In addition, enzymatically synthesized mRNA preparations contain double-stranded RNA (dsRNA) contaminants as abnormal products of the in vitro transcription (IVT) process. dsRNA is a strong PAMP and induces downstream responses that lead to the inhibition of translation and the degradation of cellular mRNAs and ribosomal RNAs (Pardi et al., supra). Thus, mRNA can suppress antigen expression and thus reduce the effectiveness of vaccines.

[0315] Research over the past decade has shown that the immune stimulatory effects of mRNA can be shaped by purifying IVT mRNA, introducing modified nucleosides, complexing mRNA with various carrier molecules (Pardi et al., supra), adding poly(A) tails, or optimizing mRNA with GC-rich sequences (Wang et al., supra). Chemical modification of uridine is a common approach to minimize the immunogenicity of foreign mRNA. Incorporation of pseudouridine (ψ) and N1-methylpseudouridine (m1ψ) into IVT mRNA prevents TLR activation and other innate immune sensors, thus reducing proinflammatory signaling in response to exogenous mRNA. Such nucleoside modifications may also suppress recognition of dsRNA species (Pardi et al., supra) and reduce innate immune sensitization of exogenous mRNA translation (Hou et al. Nature Reviews Materials, 2021, https: / / doi.org / 10.1038 / s41578-021-00358-0).

[0316] Other chemical modifications of nucleosides include, but are not limited to, 5-methylcytidine (m5C), 5-methyluridine (m5U), N1-methyladenosine (m1A), N6-methyladenosine (m6A), 2-thiouridine (s2U), and 5-methoxyuridine (5moU) (Wang et al., supra).

[0317] The IVT mRNA molecules used in the mRNA-1273 and BNT162b2 COVID-19 vaccines were prepared by replacing uridines with m1ψ, and their sequences were optimized to encode a stabilized pre-fusion spike protein with two pivotal proline substitutions (Hou et al., supra). However, CureVac's mRNA vaccine candidate, CVnCoV, uses unmodified nucleosides and relies on a combination of mRNA sequence modifications to enable immune evasion without affecting the expressed protein. First, CVnCoV has a higher GC content (63%) than the competing vaccines (56% for BNT162b2) and the original SARS-CoV-2 virus itself (37%). Second, the vaccine contains a C-rich motif that binds to poly(C)-binding proteins, enhancing both mRNA stability and expression. Further modifications of CVnCoV are that it contains a histone stem-loop sequence, as well as a poly(A) tail to enhance mRNA longevity and translation (Hubert, B., 2021. The CureVac Vaccine, and a brief tour through some of the wonders of nature. URL https: / / berthub.eu / articles / posts / curevac-vaccine-and-wonders-of-biology / . (Accessed 15 September 2021). However, the vaccine performed disappointingly in Phase III clinical trials, which experts claim was due to the failure to incorporate chemically modified nucleosides into the mRNA sequence. Nevertheless, CureVac and Acuitas Therapeutics delivered mRNA encoding erythropoietin (EPO) with rich GC codons to pigs using lipid nanoparticles (LNPs). Their results showed that EPO-associated responses were elicited without immunogenicity (Wang et al., 2013). al., supra), suggesting that unmodified mRNA nucleoside-based vaccines still have potential.

[0318] The polynucleotides of the present invention may comprise mRNA molecules.

[0319] The or each polynucleotide of the pharmaceutical composition, combined preparation, or vector of the present invention may comprise an mRNA molecule.

[0320] The vector of the present invention may be an mRNA vector.

[0321] The or each vector of the pharmaceutical composition or combined preparation of the invention may be an mRNA vector.

[0322] A polynucleotide of the invention, or of a pharmaceutical composition, combined preparation, or vector of the invention, may be provided as part of an mRNA vaccine.

[0323] Also provided according to the present invention is an mRNA vaccine comprising a polynucleotide of the present invention, a vector of the present invention, or a pharmaceutical composition or combined preparation of the present invention comprising one or more polynucleotides, wherein the or each polynucleotide comprises an mRNA molecule.

[0324] RNA or mRNA of a polynucleotide of the invention, or of a pharmaceutical composition, combined preparation, vector or vaccine of the invention, may be produced by in vitro transcription (IVT).

[0325] A polynucleotide of the invention, or of a pharmaceutical composition, combined preparation, vector, or vaccine of the invention may comprise one or more modified nucleosides.

[0326] One or more modified nucleosides may be present in a polynucleotide of the invention, or in the DNA or RNA of the polynucleotide of a pharmaceutical composition, combined preparation, vector, or vaccine of the invention.

[0327] Optionally, at least one chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the chemical modification is at the 5-position of uracil. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, the chemical modification is N1-ethylpseudouridine.

[0328] For example, a polynucleotide of the invention, or an RNA or mRNA of a polynucleotide of a pharmaceutical composition, combined preparation, vector, or vaccine of the invention, may contain the following modified nucleosides: Pseudouridine (ψ); N1-methylpseudouridine (m1ψ) 5-Methylcytidine (m5C) 5-Methyluridine (m5U) N1-methyladenosine (m1A) N6-Methyladenosine (m6A) 2-Thiouridine (s2U) 5-Methoxyuridine (5moU) may include one or more of:

[0329] In some embodiments, 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is at the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, 100% of the uracils in the open reading frame have N1-methylpseudouridine at the 5-position of the uracil.

[0330] The polynucleotide may be present in an amount ranging from about 1% to about 100%, or any intervening percentage (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, 10%-95%, 10%-100%, 20%-25%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, The nucleic acid sequence may contain between 50% and 70%, 50% and 80%, 50% and 90%, 50% and 95%, 50% and 100%, 70% and 80%, 70% and 90%, 70% and 95%, 70% and 100%, 80% and 90%, 80% and 95%, 80% and 100%, 90% and 95%, 90% and 100%, and 95% and 100%, of modified nucleotides (or nucleosides) (either in relation to the overall nucleotide content or in relation to any one or more types of nucleotides, i.e., A, G, U, or C). Any remaining percentages are occupied by the presence of unmodified A, G, U, or C.

[0331] Optionally, a polynucleotide of the present invention, or a pharmaceutical composition, combined preparation, vector, or vaccine of the present invention, comprises an RNA molecule in which the nucleic acid sequence of the polynucleotide is the same as or a complement of that set forth in the respective SEQ ID NO:, but in which each "U" is replaced by m1ψ.

[0332] Optionally, the polynucleotides of the present invention, or the polynucleotides of the pharmaceutical compositions, combined preparations, vectors, or vaccines of the present invention, comprise an mRNA molecule in which the nucleic acid sequence of the polynucleotide is the same as or a complement of that set forth in the respective SEQ ID NO:, but in which each "U" is replaced by m1ψ.

[0333] Optionally, the polynucleotide of the invention, or the pharmaceutical composition, combination preparation, vector, or vaccine of the invention, comprises an RNA molecule in which the nucleic acid sequence of the polynucleotide is the same as or a complement of that set forth in the respective SEQ ID NO:, but in which at least 50% of the "U's" are replaced by m1ψ. The remaining "U's" may be all unmodified or may include both unmodified and one or more other modified nucleosides.

[0334] Optionally, the polynucleotide of the invention, or the polynucleotide of the pharmaceutical composition, combination preparation, vector, or vaccine of the invention, comprises an mRNA molecule in which the nucleic acid sequence of the polynucleotide is the same as or a complement of that set forth in the respective SEQ ID NO:, but in which at least 50% of the "U's" are replaced by m1ψ. The remaining "U's" may be all unmodified or may include both unmodified and one or more other modified nucleosides.

[0335] Optionally, a polynucleotide of the invention, or a pharmaceutical composition, combination preparation, vector, or vaccine of the invention, comprises an RNA molecule in which the nucleic acid sequence of the polynucleotide is the same as or a complement of that set forth in the respective SEQ ID NO:, but in which at least 90% of the "U's" are replaced by m1ψ. The remaining "U's" may be all unmodified or may include both unmodified and one or more other modified nucleosides.

[0336] Optionally, the polynucleotide of the invention, or the polynucleotide of the pharmaceutical composition, combination preparation, vector, or vaccine of the invention, comprises an mRNA molecule in which the nucleic acid sequence of the polynucleotide is the same as or a complement of that set forth in the respective SEQ ID NO:, but in which at least 90% of the "U's" are replaced by m1ψ. The remaining "U's" may be all unmodified or may include both unmodified and one or more other modified nucleosides.

[0337] The mRNA vaccines of the present invention may be co-administered with immunological adjuvants, such as MF59 (Novartis), TriMix, RNActive (CureVac AG), RNAdjuvant (also discussed in Wang et al., supra).

[0338] Thus, in a preferred embodiment, each vector of the pharmaceutical composition or combined preparation of the invention is an mRNA vaccine vector.

[0339] Also provided in accordance with the present invention is an isolated cell comprising or transfected with a vector of the present invention.

[0340] Fusion proteins comprising the polypeptides of the invention are also provided according to the invention.

[0341] Pseudotyped viruses comprising a polypeptide of the invention are also provided according to the invention.

[0342] Pharmaceutical Compositions Also provided in accordance with the present invention is a pharmaceutical composition comprising a polypeptide of the present invention and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0343] Pharmaceutical compositions of the invention may comprise polypeptides of the invention in any suitable combination (e.g., HA and / or M2 and / or neuraminidase embodiments of the invention, H5 and / or M2 and / or neuraminidase embodiments of the invention, H1 and / or M2 and / or neuraminidase embodiments of the invention, or FLU_T2_HA_3_I3 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention, or FLU_T2_HA_4 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention).

[0344] Optionally, one embodiment of each different category of embodiments is used in combination, for example, an HA embodiment (H5 or H1) and / or an M2 embodiment and / or an neuraminidase embodiment. Optionally, the pharmaceutical composition of the invention comprises: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3 (examples of the embodiment of H5), and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) A polypeptide comprising the amino acid sequence of SEQ ID NO: 16, a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0345] Optionally, the pharmaceutical composition of the present invention comprises: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or 29, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or 37, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 43 or 45 (examples of embodiments of H5), and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) A polypeptide comprising the amino acid sequence of SEQ ID NO: 16, a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0346] Optionally, the pharmaceutical composition of the present invention comprises: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 or a polypeptide comprising the amino acid sequence of SEQ ID NO: 68 (example of an embodiment of H1), and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0347] Optionally, the pharmaceutical composition of the present invention comprises: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 (amino acid sequence of FLU_T2_HA_3_I3), and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (amino acid sequence of FLU_T2_M2_1), and / or iii) A polypeptide comprising the amino acid sequence of SEQ ID NO: 16 (the amino acid sequence of FLU_T2_NA_3). Includes.

[0348] Optionally, the pharmaceutical composition of the present invention comprises: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 (amino acid sequence of FLU_T2_HA_3_I3) or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 22; and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (the amino acid sequence of FLU_T2_M2_1), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 14, and / or iii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 16 (the amino acid sequence of FLU_T2_NA_3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 16. Includes.

[0349] Optionally, the polypeptide of (i) comprises the amino acid sequence of SEQ ID NO: 22 (the amino acid sequence of FLU_T2_HA_3_I3).

[0350] Optionally, the polypeptide of (ii) comprises the amino acid sequence of SEQ ID NO: 14 (the amino acid sequence of FLU_T2_M2_1).

[0351] Optionally, the polypeptide of (iii) comprises the amino acid sequence of SEQ ID NO: 16 (the amino acid sequence of FLU_T2_NA_3).

[0352] Also provided in accordance with the invention are pharmaceutical compositions comprising a nucleic acid of the invention and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0353] Pharmaceutical compositions of the invention may comprise nucleic acid molecules of the invention in any suitable combination (e.g., HA and / or M2 and / or neuraminidase embodiments of the invention, H5 and / or M2 and / or neuraminidase embodiments of the invention, H1 and / or M2 and / or neuraminidase embodiments of the invention, or FLU_T2_HA_3_I3 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention, or FLU_T2_HA_4 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention).

[0354] Optionally, one embodiment of each different category of embodiments is used in combination, for example, an HA embodiment (H5 or H1) and / or an M2 embodiment and / or a neuraminidase embodiment.

[0355] Optionally, the pharmaceutical composition of the present invention comprises: i) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3 (examples of the embodiment of H5); and / or ii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2); and / or iii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase); Includes.

[0356] Optionally, the pharmaceutical composition of the present invention comprises: i) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or 29, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or 37, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 43 or 45 (examples of the embodiment of H5), and / or ii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0357] Optionally, the pharmaceutical composition of the present invention comprises: i) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 68 (example of an embodiment of H1), and / or ii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0358] Optionally, the pharmaceutical composition of the present invention comprises: i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or a complement thereof; and / or ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and / or iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Includes.

[0359] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement. Also provided is a pharmaceutical composition comprising:

[0360] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a pharmaceutical composition comprising:

[0361] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a pharmaceutical composition comprising:

[0362] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement. ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a pharmaceutical composition comprising:

[0363] Optionally, the pharmaceutical composition of the present invention comprises: i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22 (amino acid sequence of FLU_T2_HA_3_I3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 22, or a complement thereof; and / or ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 (amino acid sequence of FLU_T2_NA_3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 16, or a complement thereof; and / or iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:14 (the amino acid sequence of FLU_T2_M2_1), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:14, or a complement thereof. Includes.

[0364] Optionally, the polynucleotide of (i) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22 (the amino acid sequence of FLU_T2_HA_3_I3), or a complement thereof.

[0365] Optionally, the polynucleotide of (ii) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 (the amino acid sequence of FLU_T2_NA_3), or a complement thereof.

[0366] Optionally, the polynucleotide of (iii) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 (the amino acid sequence of FLU_T2_M2_1), or a complement thereof.

[0367] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO:22), or its complement, comprises the nucleotide sequence of SEQ ID NO:23, or its complement.

[0368] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), or its complement, comprises the nucleotide sequence of SEQ ID NO: 17, or its complement.

[0369] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14), or its complement, comprises the nucleotide sequence of SEQ ID NO: 15, or its complement.

[0370] Optionally, the pharmaceutical composition of the present invention comprises: i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_4 (SEQ ID NO:68) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement. Includes.

[0371] Optionally, the pharmaceutical composition of the present invention comprises: i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_4 (SEQ ID NO:68) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Includes.

[0372] Optionally, the pharmaceutical composition of the present invention comprises: i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Includes.

[0373] Optionally, the pharmaceutical composition of the present invention comprises: i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_4 (SEQ ID NO:68) or its complement; ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Includes.

[0374] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_4 (SEQ ID NO:68), or its complement, comprises the nucleotide sequence of SEQ ID NO:69, or its complement.

[0375] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), or its complement, comprises the nucleotide sequence of SEQ ID NO: 17, or its complement.

[0376] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14), or its complement, comprises the nucleotide sequence of SEQ ID NO: 15, or its complement.

[0377] Optionally, each polynucleotide comprises a DNA molecule.

[0378] Optionally, each polynucleotide comprises a messenger RNA (mRNA) molecule.

[0379] Also provided in accordance with the present invention is a pharmaceutical composition comprising a vector of the present invention and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0380] Optionally, the pharmaceutical composition of the present invention further comprises an adjuvant for enhancing the immune response in the subject to the composition, to the polypeptide, or to the polypeptide encoded by the nucleic acid.

[0381] Each different nucleic acid molecule of the pharmaceutical composition of the present invention can be provided as part of a separate vector.

[0382] Also provided in accordance with the present invention is a pharmaceutical composition comprising a vector of the present invention and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0383] Optionally, the pharmaceutical composition of the present invention further comprises an adjuvant for enhancing the immune response in the subject against the polypeptide of the composition or against a polypeptide encoded by the nucleic acid.

[0384] Also provided in accordance with the present invention is a pharmaceutical composition comprising a vector of the present invention and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0385] Combination Preparations The term "combination preparation" as used herein refers to a "kit of parts" in the sense that components (i) and (ii), or (i), (ii), and (iii) of the combination as defined herein can be administered independently or using different fixed combinations with different amounts of combination components (i) and (ii), or (i), (ii), and (iii). The components can be administered simultaneously or one after the other. When the components are administered one after the other, preferably the interval between administrations is selected so that the therapeutic effect of the combined use of the components is greater than the effect that would be obtained by the use of only any one of the combination components (i) and (ii), or (i), (ii), and (iii).

[0386] The components of the combination preparation may be present in one combination unit dosage form, or as a first unit dosage form of component (i) and a separate second unit dosage form of component (ii), or as a first unit dosage form of component (i), a separate second unit dosage form of component (ii), and a separate third unit dosage form of component (iii). The ratio of the total amount of the combination component (i) to the combination component (ii), or the combination component (i) to the combination component (ii) and the combination component (iii) to be administered in the combination preparation may vary, for example, to address the needs of the patient subpopulation to be treated or the needs of a single patient, which may be due to, for example, the specific disease, age, sex, or weight of the patient.

[0387] Preferably, there is at least one beneficial effect compared to the effective dosage of one or both of components (i) and (ii) or (i), (ii), and (iii) of the combination, such as an enhancement of the effect of component (i), or an enhancement of the effect of component (ii), or a mutual enhancement of the effects of components (i) and (ii) of the combination, an enhancement of the effect of component (i), or an enhancement of the effect of component (ii), or an enhancement of the effect of component (iii), or a mutual enhancement of the effects of components (i), (ii), and (iii) of the combination, such as a more than additive effect, an additional advantageous effect, fewer side effects, lower toxicity, or therapeutic effect of the combination, and very preferably a synergistic effect of components (i) and (ii), or (i), (ii), and (iii) of the combination.

[0388] The combination preparation of the present invention can be provided as a pharmaceutical combination preparation for administration to mammals, preferably humans.Component (i) can be provided with pharmaceutically acceptable carrier, excipient or diluent if necessary, and / or component (ii) can be provided with pharmaceutically acceptable carrier, excipient or diluent if necessary, or component (i) can be provided with pharmaceutically acceptable carrier, excipient or diluent if necessary, and / or component (ii) can be provided with pharmaceutically acceptable carrier, excipient or diluent if necessary, and / or component (iii) can be provided with pharmaceutically acceptable carrier, excipient or diluent if necessary.

[0389] Combined preparations of the invention may comprise polypeptides of the invention in any suitable combination (e.g. HA and / or M2 and / or neuraminidase embodiments of the invention, H5 and / or M2 and / or neuraminidase embodiments of the invention, H1 and / or M2 and / or neuraminidase embodiments of the invention, or FLU_T2_HA_3_I3 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention, or FLU_T2_HA_4 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention).

[0390] Optionally, one embodiment of each different category of embodiments is used in combination, for example, an HA embodiment (H5 or H1) and / or an M2 embodiment and / or a neuraminidase embodiment.

[0391] According to the present invention, i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3 (examples of the embodiment of H5), and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) A polypeptide comprising the amino acid sequence of SEQ ID NO: 16, a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) A combination preparation comprising:

[0392] According to the present invention, i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or 29, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or 37, or a polypeptide comprising the amino acid sequence of SEQ ID NO: 43 or 45 (examples of embodiments of H5), and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) A polypeptide comprising the amino acid sequence of SEQ ID NO: 16, a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) A combination preparation comprising:

[0393] According to the present invention, i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 or a polypeptide comprising the amino acid sequence of SEQ ID NO: 68 (example of an embodiment of H1), and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Also provided is a combination preparation comprising:

[0394] Optionally, the combination preparation of the present invention comprises i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 (amino acid sequence of FLU_T2_HA_3_I3), and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (amino acid sequence of FLU_T2_M2_1), and / or iii) A polypeptide comprising the amino acid sequence of SEQ ID NO: 16 (the amino acid sequence of FLU_T2_NA_3). Includes.

[0395] Optionally, the combination preparation of the present invention comprises i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 (amino acid sequence of FLU_T2_HA_3_I3) or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 22; and / or ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (the amino acid sequence of FLU_T2_M2_1), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 14, and / or iii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 16 (the amino acid sequence of FLU_T2_NA_3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 16. Includes.

[0396] Optionally, the polypeptide of (i) comprises the amino acid sequence of SEQ ID NO: 22 (the amino acid sequence of FLU_T2_HA_3_I3).

[0397] Optionally, the polypeptide of (ii) comprises the amino acid sequence of SEQ ID NO: 14 (the amino acid sequence of FLU_T2_M2_1).

[0398] Optionally, the polypeptide of (iii) comprises the amino acid sequence of SEQ ID NO: 16 (the amino acid sequence of FLU_T2_NA_3).

[0399] Combination preparations of the invention may comprise nucleic acid molecules of the invention in any suitable combination (e.g. HA and / or M2 and / or neuraminidase embodiments of the invention, H5 and / or M2 and / or neuraminidase embodiments of the invention, H1 and / or M2 and / or neuraminidase embodiments of the invention, or FLU_T2_HA_3_I3 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention, or FLU_T2_HA_4 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention).

[0400] Optionally, one embodiment of each different category of embodiments is used in combination, for example, an HA embodiment (H5 or H1) and / or an M2 embodiment and / or a neuraminidase embodiment.

[0401] Optionally, the combination preparation of the present invention comprises i) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3 (examples of the embodiment of H5), and / or ii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0402] Optionally, the combination preparation of the present invention comprises i) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 or 29, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 or 37, or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 43 or 45 (examples of the embodiment of H5), and / or ii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0403] Optionally, the combination preparation of the present invention comprises i) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 68 (example of an embodiment of H1), and / or ii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (an example of an embodiment of M2), and / or iii) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 (example of an embodiment of neuraminidase) Includes.

[0404] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or a complement thereof; and / or ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and / or iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a combination preparation comprising:

[0405] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22 (amino acid sequence of FLU_T2_HA_3_I3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 22, or a complement thereof; and / or ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 (amino acid sequence of FLU_T2_NA_3), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 16, or a complement thereof; and / or iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:14 (the amino acid sequence of FLU_T2_M2_1), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:14, or a complement thereof. Also provided is a combination preparation comprising:

[0406] Optionally, the polynucleotide of (i) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22 (the amino acid sequence of FLU_T2_HA_3_I3), or a complement thereof.

[0407] Optionally, the polynucleotide of (ii) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 (the amino acid sequence of FLU_T2_NA_3), or a complement thereof.

[0408] Optionally, the polynucleotide of (iii) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 (the amino acid sequence of FLU_T2_M2_1), or a complement thereof.

[0409] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement. Also provided is a combination preparation comprising:

[0410] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a combination preparation comprising:

[0411] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a combination preparation comprising:

[0412] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a combination preparation comprising:

[0413] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO:22), or its complement, comprises the nucleotide sequence of SEQ ID NO:23, or its complement.

[0414] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), or its complement, comprises the nucleotide sequence of SEQ ID NO: 17, or its complement.

[0415] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14), or its complement, comprises the nucleotide sequence of SEQ ID NO: 15, or its complement.

[0416] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_4 (SEQ ID NO:68) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement. Also provided is a combination preparation comprising:

[0417] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_4 (SEQ ID NO:68) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a combination preparation comprising:

[0418] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a combination preparation comprising:

[0419] According to the present invention, i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_4 (SEQ ID NO:68) or its complement; ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. Also provided is a combination preparation comprising:

[0420] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_4 (SEQ ID NO:68), or its complement, comprises the nucleotide sequence of SEQ ID NO:69, or its complement.

[0421] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), or its complement, comprises the nucleotide sequence of SEQ ID NO: 17, or its complement.

[0422] Optionally, the nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14), or its complement, comprises the nucleotide sequence of SEQ ID NO: 15, or its complement.

[0423] Optionally, each polynucleotide comprises a DNA molecule.

[0424] Optionally, each polynucleotide comprises a messenger RNA (mRNA) molecule.

[0425] Each different nucleic acid molecule of the combinatorial preparation of the invention can be provided as part of a separate vector.

[0426] Optionally, the combined preparation of the invention further comprises an adjuvant to enhance the immune response in the subject against the polypeptide of the combined preparation or against the polypeptide encoded by the nucleic acid.

[0427] string Embodiments of the invention in which different polypeptides of the invention are encoded as part of the same polynucleotide (or nucleic acid) or provided in the same polypeptide (i.e. as "strings" of different subunits, e.g. HA and / or M2 and / or neuraminidase embodiments of the invention, H5 and / or M2 and / or neuraminidase embodiments of the invention, H1 and / or M2 and / or neuraminidase embodiments of the invention, or FLU_T2_HA_3_I3 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention, or FLU_T2_HA_4 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention) are particularly advantageous because the use of such "strings" as part of a vaccine requires testing only a single product comprising the "string" for safety and efficacy, rather than testing each different subunit individually. This dramatically reduces the time and cost of developing a vaccine compared to individual subunits. In some embodiments, a combination of different strings (polynucleotides and / or polypeptides), or a combination of one or more strings and one or more single subunits (polypeptides or encoded subunits) may be used.

[0428] Optionally, one embodiment of each different category of embodiments is used in combination, for example, an HA embodiment (H5 or H1) and / or an M2 embodiment and / or a neuraminidase embodiment.

[0429] Strategies for co-expression of multiple genes include the introduction of multiple vectors, the use of multiple promoters in a single vector, fusion proteins, intergenic proteolytic cleavage sites, internal ribosome entry sites (IRES), and "self-cleaving" 2A peptides. Multicistronic vectors based on IRES nucleotide sequences and self-cleaving 2A peptides are discussed in Shaimardanova et al. (Pharmaceutics 2019, 11, 580; doi:10.3390 / pharmaceutics11110580).

[0430] In one embodiment of the invention, the following subunits joined by the self-cleaving 2A peptide, known as panH1N1 (described below in Example 15 below):

[0431] FLU_T2_HA_3_I3 (amino acid sequence ID NO: 22), FLU_T2_NA_3 (amino acid sequence ID NO: 16), and FLU_T2_M2_1 (amino acid sequence ID NO: 14) A polypeptide is provided comprising the string:

[0432] The amino acid sequence of panH1N1 is provided as SEQ ID NO:63.

[0433] There is also provided in accordance with the present invention an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:63.

[0434] In accordance with the present invention, there is provided an isolated polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:63.

[0435] vaccine Vaccines of the invention may be provided together as separate polynucleotides each encoding a different subunit (such as the HA and / or M2 and / or neuraminidase embodiments of the invention, e.g. the H5 and / or M2 and / or neuraminidase embodiments of the invention, the H1 and / or M2 and / or neuraminidase embodiments of the invention, or the FLU_T2_HA_3_I3 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention, or the FLU_T2_HA_4 and / or Flu_T2_NA_3 and / or Flu_T2_M2_1 embodiments of the invention), e.g. as a nucleic acid vaccine (e.g. for administration together or separately), or may be combined together in one string as a single polynucleotide encoding all of the subunits. The separate polynucleotides can be administered together as a mixture (e.g., as a pharmaceutical composition comprising the separate polynucleotides), or can be co-administered or administered sequentially in any order (in which case the separate polynucleotides can be provided as a combined preparation for co-administration or sequential administration). Nucleic acid vaccines can be provided as DNA, RNA, or mRNA vaccines. The production and application of multicistronic constructs (e.g., where the subunits are provided in one string as a single polynucleotide) is discussed by Shaimardanova et al. (Pharmaceutics 2019, 11, 580; doi:10.3390 / pharmaceutics11110580).

[0436] Vaccine constructs of the invention may also be provided, for example, as separate polypeptides, each comprising a different subunit (e.g., the HA, M2, or neuraminidase embodiments of the invention, the H5, M2, or neuraminidase embodiments of the invention, the H1, M2, or neuraminidase embodiments of the invention, or the FLU_T2_HA_3_I3, or Flu_T2_NA_3, or Flu_T2_M2_1 embodiments of the invention, or the FLU_T2_HA_4, or Flu_T2_NA_3, or Flu_T2_M2_2 embodiments of the invention). _1 embodiment), or may be combined together in one string as a single polypeptide comprising all of the subunits (e.g., HA and M2 and neuraminidase embodiments of the invention, H5 and M2 and neuraminidase embodiments of the invention, H1 and M2 and neuraminidase embodiments of the invention, or FLU_T2_HA_3_I3 and Flu_T2_NA_3 and Flu_T2_M2_1 embodiments of the invention, or FLU_T2_HA_4 and Flu_T2_NA_3 and Flu_T2_M2_1 embodiments of the invention). The separate polypeptides may be administered together as a mixture (e.g., as a pharmaceutical composition comprising the separate polypeptides), or may be co-administered or administered sequentially in any order (in which case the separate polypeptides may be provided as a combined preparation for co-administration or sequential administration).

[0437] Optionally, one embodiment of each different category of embodiments is used in combination, for example, an HA embodiment (H5 or H1) and / or an M2 embodiment and / or a neuraminidase embodiment.

[0438] Strategies for co-expression of multiple genes include the introduction of multiple vectors, the use of multiple promoters in a single vector, fusion proteins, intergenic proteolytic cleavage sites, internal ribosome entry sites (IRES), and "self-cleaving" 2A peptides. Multicistronic vectors based on IRES nucleotide sequences and self-cleaving 2A peptides are discussed in Shaimardanova et al. (Pharmaceutics 2019, 11, 580; doi:10.3390 / pharmaceutics11110580).

[0439] In one embodiment of the present invention (described in Example 15 below), the following subunits joined by a self-cleaving 2A peptide:

[0440] FLU_T2_HA_3_I3 (amino acid sequence ID NO: 22), FLU_T2_NA_3 (amino acid sequence ID NO: 16), and FLU_T2_M2_1 (amino acid sequence ID NO: 14) A nucleic acid molecule having the nucleotide sequence of SEQ ID NO:25, which encodes a string of the following (known as panH1N1), is provided:

[0441] Also provided in accordance with the present invention is an isolated polynucleotide comprising a nucleotide sequence encoding FLU_T2_HA_3_I3 (amino acid sequence number 22), a nucleotide sequence encoding FLU_T2_NA_3 (amino acid sequence number 16), and a nucleotide sequence encoding FLU_T2_M2_1 (amino acid sequence number 14).

[0442] Also provided in accordance with the present invention are isolated polynucleotides comprising the nucleotide sequence of SEQ ID NO:23, the nucleotide sequence of SEQ ID NO:17, and the nucleotide sequence of SEQ ID NO:15.

[0443] In accordance with the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:63, or a complement thereof.

[0444] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:25, or its complement.

[0445] In accordance with the present invention, there is also provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:63, or a complement thereof.

[0446] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:25, or a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity along its entire length to the nucleotide sequence of SEQ ID NO:25, or a complement thereof, encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:63.

[0447] 1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:63, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:63, Also provided in accordance with the present invention is an isolated nucleic acid molecule, the nucleic acid comprising the nucleotide sequence of SEQ ID NO:25, or a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity along its entire length to the nucleotide sequence of SEQ ID NO:25, or a complement thereof.

[0448] Also provided in accordance with the present invention is an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:25, or a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity along its entire length to the nucleotide sequence of SEQ ID NO:25, or a complement thereof.

[0449] Optionally, an isolated nucleic acid molecule of the present invention comprises a DNA molecule, an RNA molecule, or an mRNA molecule.

[0450] When an mRNA vaccine is used in accordance with the present invention, it is preferred that each designed subunit of the string of the present invention is encoded as part of a separate mRNA vaccine vector.

[0451] Methods of Treatment and Use Also provided according to the present invention is a method of inducing an immune response to influenza virus in a subject, comprising administering to the subject an effective amount of a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, a pharmaceutical composition of the present invention, or a combined preparation.

[0452] Also provided according to the present invention is a method of immunizing a subject against influenza virus, comprising administering to the subject an effective amount of a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, a pharmaceutical composition of the present invention, or a combined preparation.

[0453] An effective amount is an amount that produces an antigen-specific immune response in a subject.

[0454] Further provided according to the invention is a polypeptide of the invention, a nucleic acid of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a combined preparation for use as a medicament.

[0455] Further provided according to the present invention is a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, a pharmaceutical composition of the present invention, or a combined preparation for use in the prevention, treatment, or amelioration of influenza virus infection.

[0456] Also provided according to the invention is the use of a polypeptide of the invention, a nucleic acid of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a combined preparation in the manufacture of a medicament for the prevention, treatment, or amelioration of influenza virus infection.

[0457] Administration Any suitable route of administration may be used. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal, intranasal, inhalation, or oral. Parenteral administration, for example, subcutaneous, intravenous, or intramuscular, is generally accomplished by injection. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectable solutions and suspensions may be prepared from the types of sterile powders, granules, and tablets previously described. Administration may be systemic or local. Routes for systemic administration generally include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal, and intraperitoneal injections, and / or intranasal routes of administration. Routes for local administration generally include, for example, topical routes of administration, but also include intradermal, transdermal, subcutaneous, or intramuscular injection, or intralesional, intracranial, intrapulmonary, intracardiac, and sublingual injection.

[0458] The composition may be administered in any suitable manner, for example, with a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition to be administered and the particular method used to administer the composition. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0459] Some of the compositions may potentially be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids (such as, for example, hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric, and phosphoric acids) and organic acids (such as, formic, acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, maleic, and fumaric acids), or by reaction with inorganic bases (such as, for example, sodium hydroxide, ammonium hydroxide, potassium hydroxide) and organic bases (such as, for example, mono-, di-, tri-, and aryl alkylamines, and substituted ethanolamines).

[0460] Administration can be achieved by single dose or multiple doses.The dose administered to a subject in the context of this disclosure should be sufficient to induce a beneficial therapeutic response in the subject over time, or to inhibit or prevent infection.The required dose will vary between subjects, depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the specific composition being used and its mode of administration.Appropriate doses can be determined by those skilled in the art using only routine experimentation.

[0461] The present disclosure includes a method that includes administering an RNA vaccine, an mRNA vaccine, or a DNA vaccine to a subject in need thereof.The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the specific composition, its mode of administration, its mode of activity, etc.

[0462] RNA or DNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage.However, it will be understood that the total daily use of RNA can be determined by the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level, prophylactically effective dose level, or suitable imaging dose level for any specific patient will depend on various factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound being used; the specific composition being used; the age, weight, general health, sex, and diet of the patient; the administration time, administration route, and excretion rate of the specific compound being used; the duration of treatment; the drugs used in combination with or simultaneously with the specific compound being used; and similar factors well known in the pharmaceutical field.

[0463] The effective amount of RNA or DNA provided herein can be as low as 20 pg, for example, administered as a single dose or as two 10 pg doses. In some embodiments, the effective amount is 20 μg to 300 μg or 25 μg to 300 μg total dose. For example, the effective amount can be 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250 μg, or 300 μg total dose. In some embodiments, the effective amount is a 20 μg total dose. In some embodiments, the effective amount is a 25 pg total dose. In some embodiments, the effective amount is a 50 μg total dose. In some embodiments, the effective amount is a 75 μg total dose. In some embodiments, the effective amount is a 100 μg total dose. In some embodiments, the effective amount is a 150 μg total dose. In some embodiments, the effective amount is a 200 μg total dose. In some embodiments, the effective amount is a 250 pg total dose. In some embodiments, the effective amount is a 300 μg total dose.

[0464] The RNA or DNA described herein can be formulated into dosage forms described herein, e.g., intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0465] Optionally, the RNA (eg, mRNA) or DNA vaccine is formulated in an amount effective to generate an antigen-specific immune response in a subject.

[0466] In some embodiments, the effective amount is a total dose of 25 μg to 1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a dose of 25 μg, administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 μg, administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 μg, administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 μg, administered to the subject a total of two times.

[0467] If necessary, a nucleic acid vaccine is administered to the subject at a dosage of 10 μg / kg to 400 μg / kg. In some embodiments, the dosage of the RNA or DNA polynucleotide (or nucleic acid) is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200μg, 100-200μg, 120-250μg, 150-250μg, 180-280μg, 200-300μg, 50-300μg, 80-300μg, 100-300μg, 40-300μg, 50-350μg, 100-350μg, 200-350μg, 300-350μg, 320-400μg, 40-380μg, 40-100μg, 100-400μg, 200-400μg, or 300-400μg. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day 0. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day 21.

[0468] Pharmaceutically Acceptable Carriers Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carriers and compositions can be sterile, and the formulation is adapted to the mode of administration. The compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions can be liquid solutions, suspensions, emulsions, tablets, pills, capsules, sustained release formulations, or powders. The compositions can be formulated as suppositories with traditional binders and carriers, such as triglycerides. Oral formulations can contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Any of the common pharmaceutical carriers, such as sterile saline or sesame oil, can be used. The vehicle can also contain conventional pharmaceutical auxiliary substances, such as pharma- ceutically acceptable salts to adjust osmotic pressure, buffers, preservatives, and the like. Other vehicles that can be used with the compositions and methods provided herein are normal saline and sesame oil.

[0469] In some embodiments, the composition includes a pharma- ceutically acceptable carrier and / or an adjuvant. For example, the adjuvant can be alum, Freund's complete adjuvant, a biological adjuvant, or an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide).

[0470] Pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, 15th Edition (1975), by E. W. Martin, Mack Publishing Co., Easton, PA, describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions (e.g., one or more influenza vaccines) and additional agents.

[0471] Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually contain injectable fluids that contain pharma- ceutically and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc., as vehicles). For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can contain, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances (e.g., wetting or emulsifying agents), preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0472] Optionally, the compositions of the invention are administered intramuscularly.

[0473] Optionally, the composition is administered intramuscularly, intradermally, subcutaneously, by needle or by gene gun or electroporation.

[0474] Aspects of the present invention are defined in the following numbered paragraphs: 1. A globular head domain of hemagglutinin subtype 5 (H5), and optionally a stem domain of hemagglutinin, comprising the following amino acid residues at positions 156, 157, 171, 172, and 205 of the head domain: ·156: R; · 157: P or S, preferably P; 171: D or N; 172: T or A, preferably T; and 205: K or R, preferably K An isolated polypeptide having the formula: 2. The following amino acid residues at positions 156, 157, 171, 172, and 205 in the head domain: ·156: R; ·157: P; ·171: D; 172: T; and 205: K 2. The isolated polypeptide of paragraph 1, having the following structure: 3. An amino acid sequence of SEQ ID NO: 7 or 8, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO: 7 or 8 and comprising the following amino acid residues at positions corresponding to positions 156, 157, 171, 172, and 205 of SEQ ID NO: 7 or 8: ·156: R; ·157: P; ·171: D; 172: T; and 205: K 3. The isolated polypeptide of paragraph 1 or 2, comprising an amino acid sequence having the following structure: 4. The following amino acid residues at positions 156, 157, 171, 172, and 205 in the head domain: ·156: R; ·157: P; ·171: N; 172: T; and 205: K 2. The isolated polypeptide of paragraph 1, having the following structure: 5. An amino acid sequence of SEQ ID NO: 10 or 11, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO: 10 or 11 and comprising the following amino acid residues at positions corresponding to positions 156, 157, 171, 172, and 205 of SEQ ID NO: 10 or 11: ·156: R; ·157: P; ·171: N; 172: T; and 205: K 5. The isolated polypeptide of paragraph 1 or 4, comprising an amino acid sequence having the following structure: 6. The following amino acid residues at positions 156, 157, 171, 172, and 205 in the head domain: ·156: R; ·157: S; ·171: N; 172: A; and 205: R 2. The isolated polypeptide of paragraph 1, having the following structure: 7. An amino acid sequence of SEQ ID NO:1 or 3, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of SEQ ID NO:1 or 3 and comprising the following amino acid residues at positions corresponding to positions 156, 157, 171, 172, and 205 of SEQ ID NO:1 or 3: ·156: R; ·157: S; ·171: N; 172: A; and 205: R 7. The isolated polypeptide of paragraph 1 or 6, comprising an amino acid sequence having the following structure: 8. The following amino acid residues at positions 416 and 434 of the stem domain: 416: F; and 434: F 2. The isolated polypeptide of any of the preceding paragraphs, having the following structure: 9. The amino acid sequence: R(P / S)SFFRNVVWLIKKN(D / N)(T / A)YPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQT(K / R) (SEQ ID NO:13), or having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along the entire length of SEQ ID NO:13 and the following amino acid residues at positions corresponding to positions 1, 2, 16, 17, and 50 of SEQ ID NO:13: ·1: R; 2: P or S, preferably P; 16: D or N; 17: T or A, preferably T; and 50: K or R, preferably K The amino acid sequence 1. An isolated polypeptide comprising: 10. The following amino acid residues at positions 1, 2, 16, 17, and 50 of the amino acid sequence, or at positions corresponding to positions 1, 2, 16, 17, and 50 of SEQ ID NO:13: ·1: R; ·2: P; ·16: D; 17: T; and 50: K 10. The isolated polypeptide of paragraph 9, having the following structure: 11. The following amino acid residues at positions 1, 2, 16, 17, and 50 of the amino acid sequence, or at positions corresponding to positions 1, 2, 16, 17, and 50 of SEQ ID NO:13: ·1: R; ·2: P; ·16: N; 17: T; and 50: K 10. The isolated polypeptide of paragraph 9, having the following structure: 12. The following amino acid residues at positions 1, 2, 16, 17, and 50 of the amino acid sequence, or at positions corresponding to positions 1, 2, 16, 17, and 50 of SEQ ID NO:13: ·1: R; ·2: S; ·16: N; 17: A; and 50: R 10. The isolated polypeptide of paragraph 9, having the following structure: 13. An amino acid sequence of any of SEQ ID NOs: 5, 9, or 12, or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to any of SEQ ID NOs: 5, 9, or 12, and comprising the following amino acid residues at positions corresponding to positions 148 and 166 of SEQ ID NOs: 5, 9, or 12: 148: F; and 166: F 2. An isolated polypeptide comprising an amino acid sequence having the following structure: 14. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:14, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:14. 15. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:16, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:16. 16. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:18, or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:18. 17. An isolated nucleic acid molecule encoding a polypeptide according to any of paragraphs 1 to 16, or an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleic acid molecule, or a complement thereof. 18. The isolated nucleic acid molecule of paragraph 17, comprising the nucleotide sequence of SEQ ID NO:2, 4, or 6, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to SEQ ID NO:2, 4, or 6, or a complement thereof. 19. The isolated nucleic acid molecule of paragraph 17, comprising the nucleotide sequence of SEQ ID NO:15, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15 over its entire length, or a complement thereof. 20. The isolated nucleic acid molecule of paragraph 17, comprising the nucleotide sequence of SEQ ID NO:17, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to SEQ ID NO:17, or a complement thereof. 21. The isolated nucleic acid molecule of paragraph 17, comprising the nucleotide sequence of SEQ ID NO:19, or a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:19 over its entire length, or a complement thereof. 22. A vector comprising a nucleic acid molecule according to any of paragraphs 17 to 21. 23. A vector according to paragraph 22, comprising a nucleic acid molecule encoding a polypeptide according to any of paragraphs 1 to 12. 24. A vector according to paragraph 22 or 23, comprising a nucleic acid molecule encoding a polypeptide according to paragraph 14. 25. A vector according to any of paragraphs 22 to 24, comprising a nucleic acid molecule encoding a polypeptide according to paragraph 15 or 16. 26. A vector according to any of paragraphs 22 to 25, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8. 27. A vector according to any of paragraphs 22 to 26, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11. 28. A vector according to any of paragraphs 22 to 27, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3. 29. The vector according to any of paragraphs 22 to 28, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:14. 30. A vector according to any of paragraphs 22 to 29, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:16. 31. A vector according to any of paragraphs 22 to 30, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:18. 32. A vector according to any of paragraphs 22 to 31, further comprising a promoter operably linked to the or each nucleic acid molecule. 33. A vector according to paragraph 32, wherein the or each promoter is for expression of a polypeptide encoded by the nucleic acid in a mammalian cell. 34. A vector according to paragraph 33, wherein the or each promoter is for expression of a polypeptide encoded by the nucleic acid in a yeast or insect cell. 35. The vector according to any of paragraphs 22 to 34, which is a vaccine vector. 36. The vector according to paragraph 35, which is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector. 37. An isolated cell comprising a vector according to any of paragraphs 22 to 36. 38. A fusion protein comprising a polypeptide according to any of paragraphs 1 to 16. 39. A pharmaceutical composition comprising a polypeptide according to any of paragraphs 1 to 16, and a pharma- ceutically acceptable carrier, excipient, or diluent. 40. A pharmaceutical composition according to paragraph 39, comprising a polypeptide according to any of paragraphs 1 to 12. 41. A pharmaceutical composition according to paragraph 39 or 40, comprising a polypeptide according to paragraph 14. 42. A pharmaceutical composition according to any of paragraphs 39 to 41, comprising a polypeptide according to paragraph 15 or 16. 43. A pharmaceutical composition according to any of paragraphs 39 to 42, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8. 44. A pharmaceutical composition according to any of paragraphs 39 to 43, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11. 45. A pharmaceutical composition according to any of paragraphs 39 to 44, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3. 46. ​​A pharmaceutical composition according to any of paragraphs 39 to 45, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:14. 47. A pharmaceutical composition according to any of paragraphs 39 to 46, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 16. 48. A pharmaceutical composition according to any of paragraphs 39 to 47, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:18. 49. A pharmaceutical composition comprising a nucleic acid according to any of paragraphs 17 to 21, and a pharma- ceutically acceptable carrier, excipient, or diluent. 50. A pharmaceutical composition according to paragraph 49, comprising a nucleic acid molecule encoding a polypeptide according to any of paragraphs 1 to 12. 51. A pharmaceutical composition according to paragraph 49 or 50, comprising a nucleic acid molecule encoding a polypeptide according to paragraph 14. 52. A pharmaceutical composition according to any of paragraphs 49 to 51, comprising a nucleic acid molecule encoding a polypeptide according to paragraph 15 or 16. 53. A pharmaceutical composition according to any of paragraphs 49 to 52, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8. 54. A pharmaceutical composition according to any of paragraphs 49 to 53, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11. 55. A pharmaceutical composition according to any of paragraphs 49 to 54, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3. 56. A pharmaceutical composition according to any of paragraphs 49 to 55, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:14. 57. A pharmaceutical composition according to any of paragraphs 49 to 56, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:16. 58. A pharmaceutical composition according to any of paragraphs 49 to 57, comprising a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:18. 59. A pharmaceutical composition comprising a vector according to any of paragraphs 22 to 36, and a pharma- ceutically acceptable carrier, excipient, or diluent. 60. A pharmaceutical composition according to any of paragraphs 39 to 59, further comprising an adjuvant for enhancing an immune response in a subject against a polypeptide of the composition or against a polypeptide encoded by the nucleic acid. 61. A method for inducing an immune response to influenza virus in a subject, comprising administering to the subject an effective amount of a polypeptide described in any of paragraphs 1 to 16, a nucleic acid described in any of paragraphs 17 to 21, a vector described in any of paragraphs 22 to 36, or a pharmaceutical composition described in any of paragraphs 39 to 60. 62. A method for immunizing a subject against influenza virus, comprising administering to the subject an effective amount of a polypeptide described in any of paragraphs 1 to 16, a nucleic acid described in any of paragraphs 17 to 21, a vector described in any of paragraphs 22 to 36, or a pharmaceutical composition described in any of paragraphs 39 to 60. 63. A polypeptide according to any of paragraphs 1 to 16, a nucleic acid according to any of paragraphs 17 to 21, a vector according to any of paragraphs 22 to 36, or a pharmaceutical composition according to any of paragraphs 39 to 60, for use as a medicament. 64. A polypeptide according to any of paragraphs 1 to 16, a nucleic acid according to any of paragraphs 17 to 21, a vector according to any of paragraphs 22 to 36, or a pharmaceutical composition according to any of paragraphs 39 to 60, for use in the prevention, treatment, or amelioration of influenza virus infection. 65. Use of a polypeptide according to any of paragraphs 1 to 16, a nucleic acid according to any of paragraphs 17 to 21, a vector according to any of paragraphs 22 to 36, or a pharmaceutical composition according to any of paragraphs 39 to 60 in the manufacture of a medicament for the prevention, treatment, or amelioration of influenza virus infection. 66. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 22 (FLU_T2_HA_3_I3). 67. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 16 (FLU_T2_NA_3). 68. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (FLU_T2_M2_1). 69. An isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22 (FLU_T2_HA_3_I3) or its complement. 70. The polynucleotide according to paragraph 69, wherein the nucleotide sequence comprises the sequence of SEQ ID NO: 23 or its complement. 71. An isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 (FLU_T2_NA_3) or its complement. 72. The polynucleotide according to paragraph 71, wherein the nucleotide sequence comprises the sequence of SEQ ID NO: 17 or its complement. 73. An isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 (FLU_T2_M2_1), or its complement. 74. The polynucleotide according to paragraph 73, wherein the nucleotide sequence comprises the sequence of SEQ ID NO: 15 or its complement. 75. An isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) and the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), or a complement thereof. 76. An isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) and the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14), or a complement thereof. 77. An isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) and the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14), or a complement thereof. 78. An isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22), the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), and the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14), or a complement thereof. 79. A polynucleotide according to any of paragraphs 69 to 78, comprising a DNA molecule. 80. The polynucleotide of any of paragraphs 69 to 78, comprising a messenger RNA (mRNA) molecule. 81. i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement. 23. A pharmaceutical composition comprising: 82. i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. 23. A pharmaceutical composition comprising: 83. i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. 23. A pharmaceutical composition comprising: 84. i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. 23. A pharmaceutical composition comprising: 85. The pharmaceutical composition of any of paragraphs 81, 82, or 84, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement comprises the nucleotide sequence of SEQ ID NO: 23 or its complement. 86. The pharmaceutical composition of any of paragraphs 81, 83, or 84, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), or its complement, comprises the nucleotide sequence of SEQ ID NO: 17, or its complement. 87. The pharmaceutical composition of any of paragraphs 82, 83, or 84, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement comprises the nucleotide sequence of SEQ ID NO: 15 or its complement. 88. A pharmaceutical composition according to any of paragraphs 81 to 87, wherein each polynucleotide comprises a DNA molecule. 89. A pharmaceutical composition according to any of paragraphs 81 to 87, wherein each polynucleotide comprises a messenger RNA (mRNA) molecule. 90. i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement. A combination preparation comprising: 91. i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. A combination preparation comprising: 92. i) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. A combination preparation comprising: 93. i) An isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; ii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and iii) an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement. A combination preparation comprising: 94. The combined preparation of any of paragraphs 90, 91, or 93, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement comprises the nucleotide sequence of SEQ ID NO: 23 or its complement. 95. The combined preparation of any of paragraphs 90, 92, or 93, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), or its complement, comprises the nucleotide sequence of SEQ ID NO: 17, or its complement. 96. The combined preparation of any of paragraphs 91, 92, or 93, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement comprises the nucleotide sequence of SEQ ID NO: 15 or its complement. 97. A combined preparation according to any of paragraphs 90 to 96, wherein each polynucleotide comprises a DNA molecule. 98. The combined preparation described in any of paragraphs 90 to 96, wherein each polynucleotide comprises a messenger RNA (mRNA) molecule. 99. A vector comprising a polynucleotide according to any of paragraphs 69 to 80. 100. The vector of paragraph 99, further comprising a promoter operably linked to the nucleotide sequence. 101. The vector according to paragraph 99, further comprising, for each nucleotide sequence of the vector encoding a separate polypeptide, a separate promoter operably linked to that nucleotide sequence. 102. The vector according to paragraph 99, which is a DNA vector. 103. The vector according to paragraph 99, which is a messenger (mRNA) vector. 104. i) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement 23. A pharmaceutical composition comprising: 105. i) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement 23. A pharmaceutical composition comprising: 106. i) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and ii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement 23. A pharmaceutical composition comprising: 107. i) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; ii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement, and iii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement 23. A pharmaceutical composition comprising: 108. The pharmaceutical composition of any of paragraphs 104, 105, or 107, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22), or its complement, comprises the nucleotide sequence of SEQ ID NO: 23, or its complement. 109. The pharmaceutical composition of any of paragraphs 104, 106, or 107, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), or its complement, comprises the nucleotide sequence of SEQ ID NO: 17, or its complement. 110. The pharmaceutical composition of any of paragraphs 105, 106, or 107, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14), or its complement, comprises the nucleotide sequence of SEQ ID NO: 15, or its complement. 111. A pharmaceutical composition according to any of paragraphs 104 to 110, wherein each vector comprises a promoter operably linked to the coding nucleotide sequence. 112. The pharmaceutical composition according to any of paragraphs 104 to 110, wherein each vector is a DNA vector. 113. The pharmaceutical composition according to any of paragraphs 104 to 110, wherein each vector is a messenger (mRNA) vector. 114. i) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement A combination preparation comprising: 115. i) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; and ii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement A combination preparation comprising: 116. i) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement; and ii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement A combination preparation comprising: 117. i) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement; ii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement, and iii) a vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement A combination preparation comprising: 118. The combined preparation described in any of paragraphs 114, 115, or 117, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) or its complement comprises the nucleotide sequence of SEQ ID NO: 23 or its complement. 119. The combined preparation of any of paragraphs 114, 116, or 117, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) or its complement comprises the nucleotide sequence of SEQ ID NO: 17 or its complement. 120. The combined preparation of any of paragraphs 115, 116, or 117, wherein the nucleotide sequence encoding the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) or its complement comprises the nucleotide sequence of SEQ ID NO: 15 or its complement. 121. A combined preparation according to any of paragraphs 114 to 120, wherein each vector comprises a promoter operably linked to the coding nucleotide sequence. 122. The combined preparation according to any of paragraphs 114 to 120, wherein each vector is a DNA vector. 123. The combined preparation according to any of paragraphs 114 to 120, wherein each vector is a messenger (mRNA) vector. 124. A vector according to paragraph 100 or 101, a pharmaceutical composition according to paragraph 111, or a combined preparation according to paragraph 121, wherein the or each promoter is for expression of a polypeptide encoded by the polynucleotide in a mammalian cell. 125. A vector according to paragraph 100 or 101, a pharmaceutical composition according to paragraph 111, or a combined preparation according to paragraph 121, wherein the or each promoter is for expression of a polypeptide encoded by the polynucleotide in yeast or insect cells. 126. A vector according to any of paragraphs 99 to 103, a pharmaceutical composition according to any of paragraphs 104 to 113, or a combined preparation according to any of paragraphs 114 to 123, wherein the or each vector is a vaccine vector. 127. The vector, pharmaceutical composition, or combined preparation according to paragraph 126, wherein the or each vaccine vector is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, an mRNA vaccine vector, or a DNA vaccine vector. 128. A vector described in any of paragraphs 99 to 102, 124, 126, or 127, a pharmaceutical composition described in any of paragraphs 104, 105, 107 to 112, 124, 126, or 127, or a combined preparation described in any of paragraphs 114, 115, 117 to 122, 124, 126, or 127, wherein the vector comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22) comprises the nucleotide sequence of SEQ ID NO: 24 or its complement. 129. An isolated cell comprising a vector according to any of paragraphs 99 to 103, 124, 126 or 127. 130. An isolated polypeptide comprising the amino acid sequence of FLU_T2_HA_3_I3 (sequence number 22), the amino acid sequence of FLU_T2_NA_3 (sequence number 16), and the amino acid sequence of FLU_T2_M2_1 (sequence number 14). 131. An isolated polypeptide comprising the amino acid sequence of FLU_T2_HA_3_I3 (sequence number 22) and the amino acid sequence of FLU_T2_NA_3 (sequence number 16). 132. An isolated polypeptide comprising the amino acid sequence of FLU_T2_HA_3_I3 (sequence number 22) and the amino acid sequence of FLU_T2_M2_1 (sequence number 14). 133. An isolated polypeptide comprising the amino acid sequence of FLU_T2_NA_3 (sequence number 16) and the amino acid sequence of FLU_T2_M2_1 (sequence number 14). 134. i) a polypeptide comprising the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22); and ii) a polypeptide comprising the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) 23. A pharmaceutical composition comprising: 135. i) a polypeptide comprising the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22); and ii) a polypeptide comprising the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) 23. A pharmaceutical composition comprising: 136. i) a polypeptide comprising the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16); and ii) a polypeptide comprising the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) 23. A pharmaceutical composition comprising: 137. i) a polypeptide comprising the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22); ii) a polypeptide comprising the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), and iii) A polypeptide comprising the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14). 23. A pharmaceutical composition comprising: 138. i) a polypeptide comprising the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22); and ii) a polypeptide comprising the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16) A combination preparation comprising: 139. i) a polypeptide comprising the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22); and ii) a polypeptide comprising the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) A combination preparation comprising: 140. i) a polypeptide comprising the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16); and ii) a polypeptide comprising the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14) A combination preparation comprising: 141. i) a polypeptide comprising the amino acid sequence of FLU_T2_HA_3_I3 (SEQ ID NO: 22); ii) a polypeptide comprising the amino acid sequence of FLU_T2_NA_3 (SEQ ID NO: 16), and iii) A polypeptide comprising the amino acid sequence of FLU_T2_M2_1 (SEQ ID NO: 14). A combination preparation comprising: 142. A pharmaceutical composition comprising an isolated polynucleotide according to any of paragraphs 69 to 80, and a pharma- ceutically acceptable carrier, excipient, or diluent. 143 A pharmaceutical composition comprising a vector according to any of paragraphs 99 to 103, and a pharma- ceutically acceptable carrier, excipient, or diluent. 144. A pharmaceutical composition comprising an isolated polypeptide according to any of paragraphs 66-68 or 130-133, and a pharma- ceutically acceptable carrier, excipient, or diluent. 145. The pharmaceutical composition of any of paragraphs 81 to 89, 104 to 113, 124 to 128, 134 to 137, or 142 to 144, further comprising an adjuvant for enhancing an immune response in a subject to a polypeptide of the composition or to a polypeptide encoded by the nucleotide. 146. The polynucleotide of any of paragraphs 182 to 188, comprising one or more modified nucleosides. 147. The vector according to any of paragraphs 99 to 103 or 124 to 128, wherein the polynucleotide of the vector comprises one or more modified nucleosides. 148. A pharmaceutical composition according to any of paragraphs 81 to 89, 104 to 113, 124 to 128, 134 to 137, or 142 to 145, wherein the or each polynucleotide of the composition comprises one or more modified nucleosides. 149. A combined preparation according to any of paragraphs 90 to 98, 114 to 128, or 138 to 141, wherein each nucleic acid of the combined preparation comprises one or more modified nucleosides. 150. The polynucleotide according to paragraph 146, the vector according to paragraph 147, the pharmaceutical composition according to paragraph 148, or the combined preparation according to paragraph 149, wherein the or each polynucleotide comprises messenger RNA (mRNA). 151. The polynucleotide according to paragraph 146 or 150, the vector according to paragraph 147 or 150, the pharmaceutical composition according to paragraph 148 or 150, or the combined preparation according to paragraph 149 or 150, wherein one or more modified nucleosides comprises a 1-methylpseudouridine modification. 152. The polynucleotide according to paragraph 146 or 150 or 151, the vector according to paragraph 147 or 150 or 151, the pharmaceutical composition according to paragraph 148 or 150 or 151, or the combined preparation according to paragraph 149 or 150 or 151, wherein one or more modified nucleosides comprises a 1-methylpseudouridine modification. 153. A polynucleotide according to any one of paragraphs 146 or 150 to 152, a vector according to any one of paragraphs 147 or 150 to 152, a pharmaceutical composition according to any one of paragraphs 148 or 150 to 152, or a combined preparation according to any one of paragraphs 149 to 152, wherein at least 80% of the uridines in the open reading frame are modified. 154. A fusion protein comprising a polypeptide according to any of paragraphs 66 to 68 or 130 to 133. 155. A pseudotyped viral particle comprising a polypeptide according to any of paragraphs 66 to 68 or 130 to 133. 156. A method of inducing an immune response to influenza virus in a subject, comprising administering to the subject an effective amount of a polypeptide according to any of paragraphs 66 to 68 or 130 to 133, a polynucleotide according to any of paragraphs 69 to 80, 146 or 150 to 153, a vector according to any of paragraphs 99 to 103, 124 to 128, 147 or 150 to 153, a pharmaceutical composition according to any of paragraphs 81 to 89, 104 to 113, 124 to 128, 134 to 137, 142 to 145, 148 or 150 to 153, or a combined preparation according to any of paragraphs 90 to 98, 114 to 128, 138 to 141 or 149 to 153. 157. A method of immunizing a subject against influenza virus, comprising administering to the subject an effective amount of a polypeptide according to any of paragraphs 66 to 68 or 130 to 133, a polynucleotide according to any of paragraphs 69 to 80, 146 or 150 to 153, a vector according to any of paragraphs 99 to 103, 124 to 128, 147 or 150 to 153, a pharmaceutical composition according to any of paragraphs 81 to 89, 104 to 113, 124 to 128, 134 to 137, 142 to 145, 148 or 150 to 153, or a combined preparation according to any of paragraphs 90 to 98, 114 to 128, 138 to 141 or 149 to 153. 158. A polypeptide according to any of paragraphs 66 to 68 or 130 to 133, a polynucleotide according to any of paragraphs 69 to 80, 146 or 150 to 153, a vector according to any of paragraphs 99 to 103, 124 to 128, 147 or 150 to 153, a pharmaceutical composition according to any of paragraphs 81 to 89, 104 to 113, 124 to 128, 134 to 137, 142 to 145, 148 or 150 to 153, or a combined preparation according to any of paragraphs 90 to 98, 114 to 128, 138 to 141 or 149 to 153 for use as a medicament. 159. A polypeptide according to any of paragraphs 66 to 68 or 130 to 133, a polynucleotide according to any of paragraphs 69 to 80, 146 or 150 to 153, a vector according to any of paragraphs 99 to 103, 124 to 128, 147 or 150 to 153, a pharmaceutical composition according to any of paragraphs 81 to 89, 104 to 113, 124 to 128, 134 to 137, 142 to 145, 148 or 150 to 153, or a combined preparation according to any of paragraphs 90 to 98, 114 to 128, 138 to 141 or 149 to 153 for use in the prevention, treatment or amelioration of influenza virus infection. 160. Use of a polypeptide according to any of paragraphs 66 to 68 or 130 to 133, a polynucleotide according to any of paragraphs 69 to 80, 146 or 150 to 153, a vector according to any of paragraphs 99 to 103, 124 to 128, 147 or 150 to 153, a pharmaceutical composition according to any of paragraphs 81 to 89, 104 to 113, 124 to 128, 134 to 137, 142 to 145, 148 or 150 to 153, or a combined preparation according to any of paragraphs 90 to 98, 114 to 128, 138 to 141 or 149 to 153 in the manufacture of a medicament for the prevention, treatment or amelioration of influenza virus infection. 161. i) A polypeptide according to any one of paragraphs 1 to 12; ii) a polypeptide according to paragraph 14, and iii) a polypeptide according to paragraph 15 or 16. A combination preparation comprising: 162. i) a polypeptide according to any one of paragraphs 1 to 12, and ii) a polypeptide according to paragraph 14. A combination preparation comprising: 163. i) a polypeptide according to any one of paragraphs 1 to 12, and ii) a polypeptide according to paragraph 15 or 16 A combination preparation comprising: 164. i) a polypeptide according to paragraph 14, and ii) a polypeptide according to paragraph 15 or 16 A combination preparation comprising: 165. i) A polynucleotide encoding a polypeptide according to any one of paragraphs 1 to 12; ii) a polynucleotide encoding a polypeptide according to paragraph 14, and iii) a polynucleotide encoding a polypeptide according to paragraph 15 or 16. A combination preparation comprising: 166. i) a polynucleotide encoding a polypeptide according to any one of paragraphs 1 to 12; and ii) a polynucleotide encoding a polypeptide according to paragraph 14. A combination preparation comprising: 167. i) a polynucleotide encoding a polypeptide according to any one of paragraphs 1 to 12; and ii) a polynucleotide encoding a polypeptide according to paragraph 15 or 16. A combination preparation comprising: 168. i) a polynucleotide encoding a polypeptide according to paragraph 14; and ii) a polynucleotide encoding a polypeptide according to paragraph 15 or 16. A combination preparation comprising: 169. A combined preparation according to any of paragraphs 161, 162, or 163, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8. 170. A combined preparation according to any of paragraphs 161, 162, or 163, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11. 171. A combined preparation according to any of paragraphs 161, 162, or 163, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3. 172. A combined preparation according to any of paragraphs 161, 162, or 164, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:14. 173. A combined preparation according to any of paragraphs 161, 163, or 164, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:16. 174. A combined preparation according to any of paragraphs 161, 163, or 164, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:18. 175. A combined preparation according to any of paragraphs 165, 166, or 167, comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or 8. 176. A combined preparation according to any of paragraphs 165, 166, or 167, comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 11. 177. A combined preparation according to any of paragraphs 165, 166, or 167, comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3. 178. A combined preparation according to any of paragraphs 165, 166, or 168, comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:14. 179. A combined preparation according to any of paragraphs 165, 167, or 168, comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:16. 180. A combined preparation according to any of paragraphs 165, 167, or 168, comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:18. 181. The combined preparation of any of paragraphs 165 to 168 or 175 to 180, wherein each polynucleotide comprises a DNA molecule. 182. The combined preparation of any of paragraphs 165 to 168 or 175 to 180, wherein each polynucleotide comprises a messenger RNA (mRNA) molecule. 183. The combined preparation according to any of paragraphs 165 to 168 or 175 to 180, wherein each polynucleotide is provided by a vector. 184. The combined preparation according to paragraph 183, wherein each vector comprises a promoter operably linked to the coding nucleotide sequence. 185. The combined preparation according to paragraph 183 or 184, wherein each vector is a DNA vector. 186. The combined preparation according to paragraph 183 or 184, wherein each vector is a messenger (mRNA) vector. 187. The combined preparation according to paragraph 183, wherein each promoter is for expression of a polypeptide encoded by the polynucleotide in a mammalian cell. 188. The combined preparation according to paragraph 183, wherein each promoter is for expression of a polypeptide encoded by a polynucleotide in yeast or insect cells. 189. The combined preparation according to any of paragraphs 183 to 188, wherein each vector is a vaccine vector. 190. The combined preparation according to paragraph 189, wherein each vaccine vector is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, an mRNA vaccine vector, or a DNA vaccine vector. 191. A combined preparation according to any of paragraphs 165 to 168, 175 to 190, wherein each nucleic acid of the combined preparation comprises one or more modified nucleosides. 192. The combined preparation of paragraph 191, wherein each polynucleotide comprises messenger RNA (mRNA). 193. The combination preparation of paragraph 191 or 192, wherein one or more modified nucleosides comprises a 1-methylpseudouridine modification. 194. The combination preparation of paragraphs 191 or 192 or 193, wherein one or more modified nucleosides comprises a 1-methylpseudouridine modification. 195. A combined preparation according to any of paragraphs 191 to 194, wherein at least 80% of the uridines in the open reading frame are modified.

[0475] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings. [Brief description of the drawings]

[0476] [Figure 1] FIG. 1 shows the results of neutralization assays illustrating the strength of neutralizing antibody responses to various pseudotyped viruses with H5 from different clades and subclades. [Diagram 2] FIG. 2 shows an amino acid sequence comparison of different embodiments of the polypeptide of the present invention. [Diagram 3] FIG. 3 shows an amino acid sequence comparison of different embodiments of the polypeptide of the present invention and the prior art COBRA sequence. [Figure 4]FIG. 4 shows the results of a flow cytometry-based immunofluorescence assay testing the ability of mouse sera obtained after immunizing mice with an embodiment of the invention to target M2 molecules from various influenza A isolates. [Diagram 5] FIG. 5 shows the results of a Pseudotype-based Enzyme-Linked Lectin Assay (pELLA) using FLU_T2_NA_3. [Figure 6] FIG. 6 shows the results of pELLA using FLU_T2_NA_4. [Figure 7] FIG. 7 shows pELLA results with N9 mAb. [Figure 8] FIG. 8 shows the plasmid map of the pEVAC vector. [Figure 9] FIG. 9 shows the logeIC50 plot of pEVAC_Flu_T2_HA_3_I-3 and other controls. [Figure 10] FIG. 10 shows inhibition of A / Brisbane / 02 / 2018 neuraminidase enzymatic activity by sera from mice vaccinated with (A) PBS, (B) primary strain-A / Brisbane / 02 / 2018, (C) N1_Final_1, (D) N1_Final_2 (Flu_T2_NA_3). [Figure 11] 11a and 11b show the panH1N1 vaccination protocol in pigs. [Figure 12] Figure 12 shows nasal shedding of viral RNA in pigs following infection in the four different vaccinated groups, monitored daily by RT-qPCR. Figure 12b shows viral titration measurements from bronchoalveolar lavage (BAL) fluid, nasal turbinate, and tracheal samples from pigs in each group. [Figure 13] Figure 13a shows the results of the HAI assay across the four vaccination groups against SW / EN / 09 at different time points, and Figure 13b shows the results of the NP competitive ELISA (Idvet). [Figure 14]FIG. 14 shows serum neutralization titers against SW / EN / 09 on different days after vaccination / infection. [Figure 15] Figure 15a shows the results of a T cell peptide stimulation assay in which splenocytes were stimulated with peptides spanning the A / swine / England / 1353 / 2009 and a / Victoria / 2454 / HAs. Figure 15b shows an HAI assay. The top panel shows the distribution of hemagglutinin inhibition titers at 0, 28, 42, and 63 days after vaccination and 8 days after infection. Titers were checked against the A / swine / England / 1353 / 2009 and a / Victoria / 2454 / 2019 strains. The bottom panel shows the average value for each group. [Figure 16] FIG. 16 shows a 3D model of the DIOS panH1N1 designed vaccine, including HA, NA, and M2 polypeptides. [Figure 17] Figure 17a shows the results of a serum neutralization assay in mice against a panel of H1 pseudoviruses using FLU_T2_HA_3_I3, and Figure 17b shows the results of an HAI assay in mice against a panel of H1 wild-type viruses. [Figure 18] FIG. 18 shows viral RNA shedding in panH1N1 and control vaccinated pigs at several time points following A / swine / EN / 1353 / 09 infection 10 weeks after prime. [Figure 19] Figures 19a and 19b show the results of serum neutralization assays in pigs using panH1N1 against the H1 clade at various time points, and Figure 19c shows the results of neutralization assays against a panel of H1 pseudoviruses using panH1N1 in pigs. [Figure 20]Figures 20a and 20b show ELLAs (enzyme-linked lectin assays) to assess the inhibitory activity of the NA component of panH1N1 against A / swine / England / 1353 / 2009 (Figure 20a) and A / England / 195 / 2009 (Figure 20b) at a range of time points post-vaccination / infection. Figure 20c shows an ELLA against a panel of NA-expressing pseudoviruses at 42 days post-vaccination. [Figure 21] FIG. 21 summarizes the influenza hemagglutinin H5 amino acid sequence differences for different embodiments of the invention, including embodiment differences at positions A-E of H5. [Figure 22-1] Figure 22 shows a multiple sequence alignment comparing the amino acid sequence of an embodiment of the invention with two influenza isolates, where the amino acid residue differences are underlined and the amino acid differences across the designed sequences FLU_T2_HA_1 and FLU_T3_HA_1 / 2 / 3 / 4 / 5 are highlighted. [Figure 22-2] Figure 22 shows a multiple sequence alignment comparing the amino acid sequence of an embodiment of the invention with two influenza isolates, where the amino acid residue differences are underlined and the amino acid differences across the designed sequences FLU_T2_HA_1 and FLU_T3_HA_1 / 2 / 3 / 4 / 5 are highlighted. [Figure 22-3] Figure 22 shows a multiple sequence alignment comparing the amino acid sequence of an embodiment of the invention with two influenza isolates, where the amino acid residue differences are underlined and the amino acid differences across the designed sequences FLU_T2_HA_1 and FLU_T3_HA_1 / 2 / 3 / 4 / 5 are highlighted. [Diagram 23] FIG. 23 shows the seroneutralization data of the T3 H5 vaccine design against a panel of nine antigenically distinct H5Nx. [Figure 24] Figure 24 shows an update of Figure 17a. Two additional seasonal H1 wild type strains are used as a challenge to the designed panH1N1 vaccine. [Diagram 25] Figure 25 summarizes the novel amino acid residue changes in the designed sequences of FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3. These novel amino acid residue changes are shown in bold and underlined. [Figure 26] Figure 26 shows the important amino acid residue positions of influenza H5. Residues shown in bold and underlined format are novel amino acid residues in the H5 Tier 4 design. [Figure 27] FIG. 27 summarizes the amino acid residues of H5 FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3 at additional key residue positions of H5. [Figure 28-1] Figure 28 shows a multiple sequence alignment of the H5 amino acid sequences of FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3, known wild-type influenza H5 strains, and previously designed H5 sequences. The amino acid residue positions in the figure correspond to those of A / Sichuan / 2014. [Figure 28-2] Figure 28 shows a multiple sequence alignment of the H5 amino acid sequences of FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3, known wild-type influenza H5 strains, and previously designed H5 sequences. The amino acid residue positions in the figure correspond to those of A / Sichuan / 2014. [Figure 28-3] Figure 28 shows a multiple sequence alignment of the H5 amino acid sequences of FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3, known wild-type influenza H5 strains, and previously designed H5 sequences. The amino acid residue positions in the figure correspond to those of A / Sichuan / 2014. [Figure 28-4]Figure 28 shows a multiple sequence alignment of the H5 amino acid sequences of FLU_T4_HA_1, FLU_T4_HA_2, and FLU_T4_HA_3, known wild-type influenza H5 strains, and previously designed H5 sequences. The amino acid residue positions in the figure correspond to those of A / Sichuan / 2014. [Figure 29-1] Figures 29A-I show the neutralizing activity of candidate H5 vaccine antigens, previously designed sequences, and wild-type sequences against a panel of clade 2.3.4.4 H5 viruses. [Figure 29-2] Figures 29A-I show the neutralizing activity of candidate H5 vaccine antigens, previously designed sequences, and wild-type sequences against a panel of clade 2.3.4.4 H5 viruses. [Figure 30-1] Figures 30A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / gyrfalcon / Washington / 41088-6 / 2014) clade 2.3.4.4c. challenge strain. [Figure 30-2] Figures 30A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / gyrfalcon / Washington / 41088-6 / 2014) clade 2.3.4.4c. challenge strain. [Figure 30-3] Figures 30A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / gyrfalcon / Washington / 41088-6 / 2014) clade 2.3.4.4c. challenge strain. [Figure 31-1] Figures 31A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / Sichuan / 26221 / 2014 clade 2.3.4.4a challenge strain. [Figure 31-2] Figures 31A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / Sichuan / 26221 / 2014 clade 2.3.4.4a challenge strain. [Figure 31-3]Figures 31A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / Sichuan / 26221 / 2014 clade 2.3.4.4a challenge strain. [Figure 32-1] Figures 32A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / Anhui / 2021-00011 / 2020 clade 2.3.4.4h challenge strain. [Figure 32-2] Figures 32A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / Anhui / 2021-00011 / 2020 clade 2.3.4.4h challenge strain. [Figure 32-3] Figures 32A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / Anhui / 2021-00011 / 2020 clade 2.3.4.4h challenge strain. [Figure 33-1] Figures 33A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / mute swan / England / 053054 / 2021 clade 2.3.4.4b challenge strain. [Figure 33-2] Figures 33A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / mute swan / England / 053054 / 2021 clade 2.3.4.4b challenge strain. [Figure 33-3] Figures 33A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / mute swan / England / 053054 / 2021 clade 2.3.4.4b challenge strain. [Figure 34-1] Figures 34A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / Hangzhou / 01 / 2021 clade 2.3.4.4b challenge strain. [Figure 34-2]Figures 34A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / Hangzhou / 01 / 2021 clade 2.3.4.4b challenge strain. [Figure 34-3] Figures 34A-I show individual neutralization curves of mice immunized with designed or wild-type sequences against the A / Hangzhou / 01 / 2021 clade 2.3.4.4b challenge strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0477] Table of sequence numbers SEQ ID NO: Description 1 FLU_T2_HA_1: Amino acid sequence of HA0 2 FLU_T2_HA_1: Nucleic acid sequence of HA0 3 FLU_T2_HA_1: Amino acid sequence of the head region 4 FLU_T2_HA_1: Nucleic acid sequence of the head region 5 FLU_T2_HA_1: Amino acid sequence of the stem region 6 FLU_T2_HA_1: Nucleic acid sequence of the stem region 7 FLU_T3_HA_1: Amino acid sequence of HA0 8 FLU_T3_HA_1: Amino acid sequence of the head region 9 FLU_T3_HA_1: Amino acid sequence of the stem region 10 FLU_T3_HA_2: Amino acid sequence of HA0 11 FLU_T3_HA_2: Amino acid sequence of the head region 12 FLU_T3_HA_2: Amino acid sequence of the stem region 13. Fragment of the Globular Head Domain of H5 14 FLU_T2_M2_1: Amino acid sequence 15 FLU_T2_M2_1: Nucleic acid sequence 16 FLU_T2_NA_3 (N1_FINAL_2): Amino acid sequence 17 FLU_T2_NA_3 (N1_FINAL_2): Nucleic acid sequence 18 FLU_T2_NA_4 (N1_FINAL_3): Amino acid sequence 19 FLU_T2_NA_4 (N1_FINAL_3): Nucleic acid sequence 20 pEVAC multiple cloning site sequence 21 pEVAC complete sequence 22 FLU_T2_HA_3_I3: Amino acid sequence 23 FLU_T2_HA_3_I3: Nucleic acid sequence 24 pEVAC- FLU_T2_HA_3_I3: Nucleic acid sequence 25 panH1N1: Nucleic acid sequence 26 pEVAC_panH1N1: Nucleic acid sequence 27 FLU_T3_HA_3: Amino acid sequence of HA0 28 FLU_T3_HA_3: Nucleic acid sequence of HA0 29 FLU_T3_HA_3: Amino acid sequence of the head region 30 FLU_T3_HA_3: Nucleic acid sequence of the head region 31 FLU_T3_HA_3: Amino acid sequence of the first stem region 32 FLU_T3_HA_3: Nucleic acid sequence of the first stem region 33 FLU_T3_HA_3: Amino acid sequence of the second stem region 34 FLU_T3_HA_3: Nucleic acid sequence of the second stem region 35 FLU_T3_HA_4: Amino acid sequence of HA0 36 FLU_T3_HA_4: Nucleic acid sequence of HA0 37 FLU_T3_HA_4: Amino acid sequence of the head region 38 FLU_T3_HA_4: Nucleic acid sequence of the head region 39 FLU_T3_HA_4: Amino acid sequence of the first stem region 40 FLU_T3_HA_4: Nucleic acid sequence of the first stem region 41 FLU_T3_HA_4: Amino acid sequence of the second stem region 42 FLU_T3_HA_4: Nucleic acid sequence of the second stem region 43 FLU_T3_HA_5: Amino acid sequence of HA0 44 FLU_T3_HA_5: Nucleic acid sequence of HA0 45 FLU_T3_HA_5: Amino acid sequence of the head region 46 FLU_T3_HA_5: Nucleic acid sequence of the head region 47 FLU_T3_HA_5: Amino acid sequence of the first stem region 48 FLU_T3_HA_5: Nucleic acid sequence of the first stem region 49 FLU_T3_HA_5: Amino acid sequence of the second stem region 50 FLU_T3_HA_5: Nucleic acid sequence of the second stem region 51 FLU_T3_HA_1: Amino acid sequence of the first stem region 52 FLU_T3_HA_1: Nucleic acid sequence of the first stem region 53 FLU_T3_HA_1: Amino acid sequence of the second stem region 54 FLU_T3_HA_1: Nucleic acid sequence of the second stem region 55 FLU_T3_HA_1: Nucleic acid sequence of HA0 56 FLU_T3_HA_1: Nucleic acid sequence of the head region 57 FLU_T3_HA_2: Amino acid sequence of the first stem region 58 FLU_T3_HA_2: Nucleic acid sequence of the first stem region 59 FLU_T3_HA_2: Amino acid sequence of the second stem region 60 FLU_T3_HA_2: Nucleic acid sequence of the second stem region 61 FLU_T3_HA_2: Nucleic acid sequence of HA0 62 FLU_T3_HA_2: Nucleic acid sequence of the head region 63 panH1N1: Amino acid sequence 64 A / whooper swan / Mongolia / 244 / 2005 H5 (H5_WSN) 65 A / gyrfalcon / Washington / 41088-6 / 2014 (H5_GYR) 66 First 2A self-cleaving peptide sequence: GSGEGRGSLLTCGDVEENPGP 67 Second 2A self-cleaving peptide sequence: GSGATNFSLLKQAGDVEENPGP 68 FLU_T2_HA_4 - Amino acid sequence 69 FLU_T2_HA_4 - Nucleic acid sequence 70 pEVAC-FLU_T2_HA_4 nucleic acid sequence 71 FLU_T4_HA_1: Amino acid sequence of HA0 72 FLU_T4_HA_1: Nucleic acid sequence of HA0 73 FLU_T4_HA_1: Amino acid sequence of the head region 74 FLU_T4_HA_1: Nucleic acid sequence of the head region 75 FLU_T4_HA_1: Amino acid sequence of the first stem region 76 FLU_T4_HA_1: Nucleic acid sequence of the first stem region 77 FLU_T4_HA_1: Amino acid sequence of the second stem region 78 FLU_T4_HA_1: Nucleic acid sequence of the second stem region 79 pEVAC-FLU_T4_HA_1 - Nucleic acid sequence 80 FLU_T4_HA_2: Amino acid sequence of HA0 81 FLU_T4_HA_2: Nucleic acid sequence of HA0 82 FLU_T4_HA_2: Amino acid sequence of the head region 83 FLU_T4_HA_2: Nucleic acid sequence of the head region 84 FLU_T4_HA_2: Amino acid sequence of the first stem region 85 FLU_T4_HA_2: Nucleic acid sequence of the first stem region 86 FLU_T4_HA_2: Amino acid sequence of the second stem region 87 FLU_T4_HA_2: Nucleic acid sequence of the second stem region 88 pEVAC-FLU_T4_HA_2 - Nucleic acid sequence 89 FLU_T4_HA_3: Amino acid sequence of HA0 90 FLU_T4_HA_3: Nucleic acid sequence of HA0 91 FLU_T4_HA_3: Amino acid sequence of the head region 92 FLU_T4_HA_3: Nucleic acid sequence of the head region 93 FLU_T4_HA_3: Amino acid sequence of the first stem region 94 FLU_T4_HA_3: Nucleic acid sequence of the first stem region 95 FLU_T4_HA_3: Amino acid sequence of the second stem region 96 FLU_T4_HA_3: Nucleic acid sequence of the second stem region 97 pEVAC-FLU_T4_HA_3 - Nucleic acid sequence 98 FLU_T3_NA_3 Amino acid sequence 99 FLU_T3_NA_3 Nucleic acid sequence 100 A / Sichuan / 2014 H5 amino acid sequence EXAMPLES

[0478] Example 1 - FLU_T2_HA_1 This example provides the influenza hemagglutinin H5 head and stem region amino acid sequence for an embodiment of the invention known as FLU_T2_HA_1. In SEQ ID NO:1 below, the amino acid residues in the stem region are shown underlined. The amino acid residues in the head region are the remaining residues. FLU_T2_HA_1 - HA0 amino acid sequence (SEQ ID NO:1): [ka] FLU_T2_HA_1 - HA0 nucleic acid sequence (SEQ ID NO:2): [ka] [ka] FLU_T2_HA_1 - Head region amino acid sequence (SEQ ID NO:3): [ka]

[0479] The amino acid residues at positions 156, 157, 171, 172, and 205 are shown underlined in the above sequence (and are R, S, N, A, and R, respectively). FLU_T2_HA_1 - Head region nucleic acid sequence (SEQ ID NO:4): [ka] FLU_T2_HA_1 - stem region amino acid sequence (SEQ ID NO:5): [ka]

[0480] The amino acid residues at positions 416 and 434 (or positions 148 and 166 if counted from the beginning of the stem region) are shown underlined in the above sequence (and are F and F, respectively). FLU_T2_HA_1 - stem region nucleic acid sequence (SEQ ID NO:6): [ka]

[0481] Example 2 FLU_T2_HA_1 was tested for its ability to elicit broadly neutralizing antibody responses against viruses pseudotyped with H5 from different clades and subclades.

[0482] Immunization of mice with DNA vaccines: Female BALB / c mice (8-10 weeks old) were immunized 4 times (weeks 0, 2, 4, and 6) and bled 6-7 times (weeks 0, 2, 4, 6, 8, 10, and 12) with the following: · 50 μg FLU_T2_HA_1 DNA in pEVAC vector (see “H5N1 Anc.” in Figure 1); 50 μg A / whooper swan / Mongolia / 244 / 2005 (H5) DNA (see “WSN” in Figure 1) in pEVAC vector (this is the primary isolate sequenced from a whooper swan in 2005 (i.e., the H5 control)); or 50μl PBS.

[0483] DNA was injected subcutaneously into the dorsal flank of mice. DNA and PBS were endotoxin-free.

[0484] Ability of mouse sera to neutralize viruses pseudotyped with H5 from different clades and subclades: Mouse sera collected after immunization were tested against the following pseudotyped viruses (with H5 from different clades and subclades): · A / gyrfalcon / Washington / 41088-6 / 2014 (H5, clade 2.3.4.4); · A / turkey / Turkey / 1 / 2005 (H5, clade 2.2.1); · A / whooper Swan / Mongolia / 244 / 2005 (H5, clade 2.2) - homologous to H5 controls; · A / Indonesia / 5 / 2005 (H5, clade 2.1.3.2); · A / Vietnam / 1194 / 2004 (H5, clade 1); · A / goose / Guiyang / 337 / 2006 (H5, clade 4); A / chicken / Vietnam / NCVD-016 / 2008 (H5, clade 7.1)

[0485] Figure 1 shows the results of a neutralization assay illustrating the strength of neutralizing antibody responses to various pseudotyped viruses. The results illustrate the ability of each vaccine to elicit broadly neutralizing antibody responses to a diverse panel of H5-pseudotyped viruses from different clades and subclades.

[0486] The results show that administration of the FLU_T2_HA_1 DNA vaccine to mice conferred a significantly greater cross-clade immune response than immunization with the A / whooper swan / Mongolia / 244 / 2005 H5 control vaccine and naive mouse sera.

[0487] Example 3 - FLU_T3_HA_1 and FLU_T3_HA_2 This example describes the design of the amino acid sequences of two further embodiments of the invention, FLU_T3_HA_1 and FLU_T3_HA_2.

[0488] As described above in Example 2, mouse sera obtained after immunization with the FLU_T2_HA_1 DNA vaccine neutralized many clades of H5, but were less effective against clades 2.3.4 and 7.1. These two clades are currently circulating in birds and are among the most prevalent co-circulating H5N1 viruses in poultry in Asia, with sporadic cases occurring periodically in humans and other animals.

[0489] Epitope regions in the H5 head region important for neutralization of clade 2.3.4 and clade 7.1 were identified using available protein structure data, and the amino acid sequences of these epitopes were compared to FLU_T2_HA_1 to identify amino acid positions that may abrogate neutralization of these two clades by mouse sera.

[0490] Amino acid positions within FLU_T2_HA_1 were identified that, when altered to specific amino acid residues, could induce antibody responses capable of neutralizing clades 2.3.4 and 7.1 without abrogating neutralization of other clades. These positions are at amino acid residues 157, 171, 172, and 205 of the H5 protein (see positions A, B, and C in FIG. 2). The effect of these mutations on the stability of the HA protein and its interaction with known antibodies against clades 2.3.4 and 7 was checked by energy calculations. Mutations that stabilized the protein and its interaction with such antibodies were selective, although they minimally altered the neutralization of other clades. The resulting new HA sequences were designated FLU_T3_HA_1 and FLU_T3_HA_2.

[0491] 2 shows an amino acid sequence comparison of FLU_T2_HA_1 with FLU_T3_HA_1 and FLU_T3_HA_2. FLU_T3_HA_1 is described in more detail in Example 4, and FLU_T3_HA_2 is described in more detail in Example 5 below.

[0492] Example 4 - FLU_T3_HA_1 This example provides the amino acid sequence of the influenza hemagglutinin H5 head and stem regions for one embodiment of the invention known as FLU_T3_HA_1. In SEQ ID NO: 7 below, the amino acid residues in the stem region are shown underlined. The amino acid residues in the head region are the remaining residues. FLU_T3_HA_1 - HA0 amino acid sequence (SEQ ID NO:7): [ka] FLU_T3_HA_1 - Head region amino acid sequence (SEQ ID NO:8): [ka]

[0493] The amino acid residues at positions 156, 157, 171, 172, and 205 are shown underlined in the above sequence (R, P, D, T, and K, respectively). FLU_T3_HA_1 - stem region amino acid sequence (SEQ ID NO:9): [ka] [ka]

[0494] The amino acid residues at positions 416 and 434 are shown underlined in the above sequence (F and F, respectively).

[0495] Example 5 – Influenza H5 T3_HA_2 This example provides the amino acid sequence of the influenza H5 head and stem regions for one embodiment of the invention known as FLU_T3_HA_2. In SEQ ID NO: 4 below, the amino acid residues in the stem region are shown underlined. The amino acid residues in the head region are the remaining residues. FLU_T3_HA_2 - HA0 amino acid sequence (SEQ ID NO: 10): [ka] FLU_T3_HA_2 - Head region amino acid sequence (SEQ ID NO: 11): [ka]

[0496] The amino acid residues at positions 156, 157, 171, 172, and 205 are shown underlined in the above sequence (R, P, N, T, and K, respectively). FLU_T3_HA_2 - stem region amino acid sequence (SEQ ID NO: 12): [ka]

[0497] The amino acid residues at positions 416 and 434 are shown underlined in the above sequence (F and F, respectively).

[0498] Example 6 – Comparison of FLU_T3_HA_1 and FLU_T3_HA_2 with prior art COBRA H5 Tier 2 designs FIG. 3 shows an amino acid comparison of FLU_T3_HA_1 and FLU_T3_HA_2 with the prior art COBRA H5 Tier 2 design. There are amino acid differences at three positions (A, B, and C) in the head region that were introduced into FLU_T3_HA_1 and FLU_T3_HA_2 to increase the affinity of the antigen for antibodies of important clades. The amino acid differences are at residue numbers 156, 157, 171, 172, and 205 in the head region. There are additional amino acid differences at two positions (C and D) in the stem region that were introduced into FLU_T3_HA_1 and FLU_T3_HA_2 to stabilize the stem region in both pre-fusion and post-fusion states. The amino acid differences are at residue numbers 416 and 434 in the stem region.

[0499] Example 7 - FLU_T2_M2_1 This example provides the amino acid and nucleic acid sequences of the influenza M2 region for one embodiment of the present invention known as FLU_T2_M2_1. FLU_T2_M2_1 - amino acid sequence (SEQ ID NO: 14): [ka] FLU_T2_M2_1 - nucleic acid sequence (SEQ ID NO: 15): [ka]

[0500] Example 8 – Immune responses induced by FLU_T2_M2_1 This example describes a flow cytometry-based immunofluorescence assay to test the ability of mouse sera obtained after immunization of mice with FLU_T2_M2_1 DNA vaccine to target M2 molecules derived from influenza A isolates of different subtypes.

[0501] Immunization of mice with DNA vaccines: Four groups of six Balb / c mice (8-10 weeks old) were immunized four times (weeks 0, 2, 4, and 6) and bled six times (weeks 0, 2, 4, 6, 8, and 10) with: · 50 μg FLU_T2_M2_1 DNA in pEVAC vector (see “M2 ancestor” in Figure 5); · 50 μg FLU_T1_M2_1 DNA in pEVAC vector (M2 derived from H1N1pdm, see "M2 H1N1" in Figure 5); 50 μg FLU_T1_M2_2 DNA in pEVAC vector (M2 derived from H3N2, see "M2 H3N2" in Figure 5); or 50μl PBS.

[0502] DNA was injected subcutaneously into the dorsal flank of mice. DNA and PBS were endotoxin-free.

[0503] Ability of mouse sera to target M2 from influenza isolates of different subtypes: HEK293T cells were transfected with pEVAC vectors expressing M2 DNA from the following isolates: · A / Brisbane / 2 / 2018(H1N1); A / Kansas / 14 / 2017(H3N2); A / England / 195 / 2009(H1N1); A / Anhui / 1 / 2013 (H7N9); and ·A / Japan / WRAIR1059P / 2008(H3N2)

[0504] Serum was pooled for each group (6 mice / group), serially diluted, and incubated with cells for 30 min at room temperature. Mouse IgG isotype antibody was used as a negative control staining. After incubation, cells were washed twice with PBS and then incubated with goat anti-mouse AF647 secondary antibody for 30 min at room temperature protected from light. Cells were washed two more times with PBS before FACS separation. Analysis was performed using an Attune NxT FACS (Thermo Fisher).

[0505] Figure 4 shows the results of a flow cytometry-based immunofluorescence assay illustrating the ability of mouse serum antibodies to target M2 from different influenza isolates. The results illustrate the ability of each vaccine to target M2 from influenza isolates of different subtypes.

[0506] The results show that administration of the FLU_T2_M2_1 DNA vaccine (M2 ancestor) to mice elicited significantly greater immune responses against M2 across different influenza subtypes than immunization with M2 derived from H1N1 or H3N2 isolates as well as naive mouse sera.

[0507] Example 9 - FLU_T2_NA_3 and FLU_T2_NA_4 This example provides the amino acid and nucleic acid sequences of influenza neuraminidase regions for embodiments of the invention known as FLU_T2_NA_3 and FLU_T2_NA_4. FLU_T2_NA_3 (N1_FINAL_2) - Amino acid sequence (SEQ ID NO: 16): [ka] FLU_T2_NA_3 (N1_FINAL_2) - nucleic acid sequence (SEQ ID NO: 17): [ka] FLU_T2_NA_4 (N1_FINAL_3) - Amino acid sequence (SEQ ID NO: 18): [ka] FLU_T2_NA_4(N1_FINAL_3) - nucleic acid sequence (SEQ ID NO: 19): [ka]

[0508] Example 10 – Antibody inhibition of neuraminidase activity of FLU_T2_NA_3 and FLU_T2_NA_4 This example describes the screening of neuraminidase polypeptides according to an embodiment of the invention (FLU_T2_NA_3 and FLU_T2_NA_4) against a panel of monoclonal antibodies recognizing different neuraminidase epitopes.

[0509] Neuraminidase vaccines induce binding antibodies that inhibit the activity of the neuraminidase enzyme. This has been shown to correlate with reduced disease severity but not necessarily protect against infection. They also reduce transmission from infected vaccinated people because the virus requires NA activity to exit infected cells.

[0510] Pseudotype-based enzyme-linked lectin assay (pELLA) A lentivirus pseudotype carrying the neuraminidase of a selected influenza virus strain (e.g., N9 from A / Shanghai / 02 / 2013 (H7N9)) or a neuraminidase of a polypeptide according to one embodiment of the present invention (e.g., T2_NA_3) is generated.

[0511] These pseudotypes bearing NA are used to digest the carbohydrate, fetuin, from pre-coated ELISA plates in a dilution series, and the resulting products from digested fetuin contain terminal galactose residues that can be recognized by peanut lectin (conjugated to horseradish peroxidase).

[0512] The more NA digests fetuin, the more galactose residues are exposed, and therefore the more peanut lectin (HRPO) will bind to the galactose, giving an ELISA-based readout proportional to the enzymatic activity of NA (Couzens et al., J Virol Methods. 2014 Dec 15;210:7-14).

[0513] The NA-pseudotypes are first titrated, and then inhibition assays are performed with antibodies or serum to "knock down" the activity of the enzyme with antibodies. Since this is a functional assay, only antibodies that interfere with the enzyme activity of NA are detected.

[0514] Figure 5: Panel of monoclonal antibodies tested against FLU_T2_NA_3 (N1_FINAL_2): Strong inhibition of NA activity by: 2D4, Z2B3, 3H4, 1H8, 2D9, 3H10, 4E9, 4G2, 1H5, 2G6, A67C Weak inhibition by: 3C2 Not inhibited by: AF9C, 4C4, 2B5, 1C7, 3A2 FLU_T2_NA_3 (in Figure 5 = N1_FINAL_2 = na2 = na2p1)

[0515] Figure 6: Panel of monoclonal antibodies tested against FLU_T2_NA_4 (N1_FINAL_3): Strong inhibition of NA activity by: Z2B3, 2D4, 1H8, 3H4, 2D9, 3H10, 4E9, 1H5, 2G6, 4G2, A67C Weak inhibition by: 4C4, 3C2 Not inhibited by: AF9C, 2B5, 1C7, 3A2 FLU_T2_NA_4 (In Figure 6, =N1_FINAL_3=p1na3=na3)

[0516] Figure 7: Panel of monoclonal antibodies tested against FLU_T2_NA_18 (N9_FINAL_1), FLU_T2_NA_19 (N9_FINAL_2), FLU_T2_NA_20 (N9_FINAL_3): Strong inhibition of NA activity by: 1E8, 7F8, 5H11, 7A4, 7F12, 2F6, Z2B3, 1E8 Weak inhibition by: I2H3 Not hindered by: N / A

[0517] For wild type N9 (A / Shanghai / 02 / 2013): Strong inhibition by: 1E8, 5H11, 7A4, 2F6, 7F12, Z2B3 No inhibition by: 7F8 and I2H3

[0518] From the results described above and shown in Figures 5-7, it is concluded that the neuraminidase polypeptides according to embodiments of the invention (FLU_T2_NA_3 and FLU_T2_NA_4) contain epitopes conserved between seasonal H1N1, N1 from pandemic H1N1 and N1 from avian H5N1, as well as epitopes conserved between N1 and N9 (Z2B3 mAb).

[0519] Monoclonal antibody panel: Hongquan Wan, FDA mAb: [Table 3]

[0520] mAb from Alain Townsend, Oxford: mAb_AF9C N1 derived from seasonal and pandemic H1N1 Rijal et al., Journal of Virology, February 2020 Volume 94 Issue 4, 1-17; mAb_Z2B3 N1 and N9 Rijal et al., Journal of Virology, February 2020 Volume 94 Issue 4, 1-17

[0521] FACS binding assay: NA is expressed on the cell surface in HEK293T / 17 cells and serum / mAb is allowed to bind to it. Binding is detected with a secondary antibody against mouse or human serum antibodies. The cells are passed through a fluorescence activated cell sampler (FACS cytometer) to measure the amount of binding present in the sample. This binding is independent of whether the antibody interferes with enzyme activity. These may be antibodies that act via an ADCC mechanism via immune cells.

[0522] Example 11 –pEVAC expression vector Figure 8 shows a map of the pEVAC expression vector. The sequence of the multiple cloning site of the vector is given below, followed by its entire nucleotide sequence. The sequence of pEVAC multiple cloning site (MCS) (SEQ ID NO:20): [ka] The full sequence of pEVAC (SEQ ID NO:21): CMV-IE-E / P:248-989 CMV immediate early 1 enhancer / promoter KanR: 3445-4098 Kanamycin resistance SD: 990-1220 Splice Donor SA: 1221-1343 splice acceptor Tbgh: 1392-1942 termination signal derived from bovine growth hormone pUC-ori: 2096-2769 pUC-plasmid replication origin [ka] [ka] [ka]

[0523] Example 12 FLU_T2_HA_3_I3 This example provides the amino acid and nucleic acid sequences of the influenza H1 region for one embodiment of the present invention known as FLU_T2_HA_3_I3. FLU_T2_HA_3_I3 - amino acid sequence (SEQ ID NO: 22): [ka] FLU_T2_HA_3_I3 - nucleic acid sequence (SEQ ID NO: 23) [ka] [ka]

[0524] Example 13 pEVAC-FLU_T2_HA-3-I-3 This example provides the nucleic acid sequence of pEVAC-FLU_T2_HA-3-I-3. pEVAC-FLU_T2_HA-3-I-3 - nucleic acid sequence (SEQ ID NO: 24): Locus 17ADKK4C_I-3_pVRC8400EVAC_Ar 6083bp DNA circular Feature Location / Identifier Promoter complement (5925..5953) / label="AmpR_promoter" Promoter 868..987 / label="CMV2_promoter" CDS complement (4963..5778) / label="Kana(R)" Replication origin complement(3818..4437) / label="pBR322_origin" Primer Complement(29..51) / label="pGEX_3_primer" Primer 855..875 / label="CMV_fwd_primer" Primer 899..918 / label="pCEP_fwd_primer" Primer 901..925 / label="LNCX_primer" PolyA site 3071..3295 / label="BGH\pA" Promoter 394..904 / label="CMV_Promoter" CDS 1343..3063 / label="I-3" [ka] [ka] [ka]

[0525] Example 14 Broad-coverage H1N1 vaccine candidate This example provides a broad coverage H1N1 string-based vaccine construct (panH1N1 vaccine candidate). The panH1N1 comprises an isolated polynucleotide comprising nucleotide sequences encoding the covalently linked designed subunits of FLU_T2_HA_3_I3 (SEQ ID NO:23), FLU_T2_NA_3 (SEQ ID NO:17), and FLU_T2_M2_1 (SEQ ID NO:15).

[0526] Figure 9 shows the log e I C 50 The plot is shown.

[0527] The induction of neutralizing antibodies by our vaccine candidate - Flu_T2_HA_3_I-3 against A / Brisbane / 02 / 2018, A / California / 07 / 2009, A / swine / Guangxi / 2013 and A / swine / Henan / SN10 / 2018 was confirmed using pMN assay. The different controls used were: primary strains viz. A / Brisbane / 02 / 2018, A / Michigan / 45 / 2015, cobra design: H1N1 cobra, our seasonal H1N1 vaccine candidate: Flu_T2_HA_2, and monoclonal antibodies - mAb 4F8 and mAb FI6.

[0528] FIG. 10 shows inhibition of A / Brisbane / 02 / 2018 neuraminidase enzymatic activity by sera from mice vaccinated with (A) PBS, (B) primary strain-A / Brisbane / 02 / 2018, (C) N1_Final_1, (D) N1_Final_2 (Flu_T2_NA_3).

[0529] The data show superior neutralization breadth for some isolates, or comparable breadth for others, compared to the Cobra candidate.

[0530] Example 15 panH1N1 vaccine candidate This example provides the nucleic acid sequence of the broad coverage H1N1 vaccine candidate of the present invention, known as panH1N1. panH1N1 comprises an isolated polynucleotide comprising a nucleotide sequence encoding the covalently linked designed subunits of FLU_T2_HA_3_I3 (SEQ ID NO:23), FLU_T2_NA_3 (SEQ ID NO:17), and FLU_T2_M2_1 (SEQ ID NO:15). The amino acid sequence of panH1N1 (SEQ ID NO:63) is also provided. panH1N1 - nucleic acid sequence (SEQ ID NO:25) [ka] [ka] pEVAC_panH1N1 - nucleic acid sequence (SEQ ID NO:26) [ka] [ka] [ka] [ka] panH1N1 - amino acid sequence (SEQ ID NO:63) [ka]

[0531] The panH1N1 amino acid sequence shown above (SEQ ID NO:63) contains a first 2A self-cleaving peptide sequence (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO:66) highlighted in bold between the amino acid sequences of the FLU_T2_HA_3_I3 and FLU_T2_NA_3 subunits, and a second 2A self-cleaving peptide sequence (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO:67) highlighted in bold between the amino acid sequences of the FLU_T2_NA_3 and FLU_T2_M2_1 subunits.

[0532] Strategies for co-expression of multiple genes include the introduction of multiple vectors, the use of multiple promoters in a single vector, fusion proteins, intergenic proteolytic cleavage sites, internal ribosome entry sites (IRES), and "self-cleaving" 2A peptides. Multicistronic vectors based on IRES nucleotide sequences and self-cleaving 2A peptides are discussed in Shaimardanova et al. (Pharmaceutics 2019, 11, 580; doi:10.3390 / pharmaceutics11110580).

[0533] 2A self-cleaving peptides are viral oligopeptides, 18-22 amino acids long, that mediate the "cleavage" of polypeptides during translation in eukaryotic cells (Liu et al., Scientific Reports 7, Article number: 2193 (2017)). The designation "2A" refers to a specific region of the viral genome, and various viral 2As are generally named after the viruses from which they originate. The first 2A discovered was F2A (foot-and-mouth disease virus), after which E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (thosea asigna virus 2A) were also identified. The mechanism of 2A-mediated "self-cleavage" is that the ribosome skips the formation of a glycyl-prolyl peptide bond at the C-terminus of 2A. The highly conserved sequence GDVEXNPGP is shared by different 2As at the C-terminus and is essential for the formation of steric hindrance and ribosome skipping. There are three possibilities for 2A-mediated skipping events: (1) successful translation skipping and restart resulting in two "truncated" proteins: the protein upstream of 2A binds to the complete 2A peptide minus the C-terminal proline, and the protein downstream of 2A binds to a single N-terminal proline; (2) successful skipping, but the ribosome is dropped and translation is aborted, resulting in only the protein upstream of 2A; or (3) failed skipping and continued translation resulting in a fusion protein. Overall, 2A peptides result in relatively high levels of downstream protein expression compared to other strategies for co-expression of multiple genes, and they are small in size and therefore have a low risk of disrupting the function of the co-expressed genes.

[0534] (Example 16) Immunogenicity and efficacy of a broadly reactive H1N1 influenza vaccine in pigs background: Long-term, ongoing antigenic change (drift) of influenza A virus strains in the human population requires twice-yearly updates of human seasonal vaccine compositions. The development of a broadly cross-reactive "universal" vaccine that does not require such frequent updates would be a significant advantage. The objective of this study was to evaluate a novel broadly cross-reactive vaccine technology in a swine model of influenza.

[0535] method: The test vaccine in this study was a structure-based computationally synthetic multi-gene antigen of the human-origin H1N1 influenza A virus, panH1N1 (also called DIOSynVax-H1N1), which was administered intradermally (ID) without a needle as DNA to five pigs using the PharmaJet® Tropis® system. Two whole inactivated virus (WIV) vaccines of the same pandemic lineage, A / swine / England / 1353 / 2009 (WIV) served as controls. 1353 ) and A / Victoria / 2454 / 2019 (WIV Vic ) in oil-in-water adjuvant intramuscularly to five pigs each at the same 4-week interval. Six weeks after the second immunization, all groups were challenged with the porcine-origin pH1N1 strain A / swine / England / 1353 / 2009 (intranasally at 1.7 × 10 per pig). 6 TCID 50 Pigs were monitored at days post inoculation (dpi) 8 or 9 dpi until the end of the experiment. The immunization and blood sampling protocol for the tested pigs is shown in Figures 11a and b.

[0536] result: Nasal shedding of viral RNA was monitored daily by RT-qPCR (Figure 12). All challenged animals shed viral RNA, peaking at 2-5 dpi by RT-qPCR (Figure 12a). Area under the curve analysis showed broadly reactive panH1N1 (P=0.0012) or WIV. 1353(P=0.0003) in animals vaccinated with the vaccine, naive controls or WIV Vic Compared to the group vaccinated with , the nasal shedding of viral RNA was significantly reduced (Figure 12b). All pigs had resolved infection by 8-9 dpi, as shown in the bottom graph of Figure 12b, which shows viral titration measurements from bronchoalveolar lavage (BAL) fluid, nasal turbinate, and tracheal samples from pigs in each group.

[0537] Influenza virus-specific serum antibody levels were monitored longitudinally by hemagglutinin inhibition assay (HAI, FIG. 13a) and NP ELISA (FIG. 13b). Both assays revealed significant antibody levels in the WIV-vaccinated group even after a single vaccination. Pigs immunized with panH1N1 generated HA antibody responses after boost vaccination (i.e., after day 28) comparable to the WIV vaccine. The antibodies were found to be neutralizing, as shown by serum neutralization assay in FIG. 14. The panH1N1 vaccine was found to be neutralizing, as shown by serum neutralization assay in FIG. 15. 1353 Further evidence that panH1N1 vaccinated groups provide similar protection to panH1N1 vaccinated groups is shown in ELISopt and HAI assays in Figure 15. Specifically, Figure 15a shows a T cell peptide stimulation assay where splenocytes were stimulated with peptides spanning the A / Swine / england / 1353 / 2009 HA and the A / Victoria / 2545 / 2019 HA. Higher values ​​on the y-axis indicate higher T cell responses. Each point represents one pig. The panH1N1 vaccinated group has better T cell responses after infection than the control. Figure 15b shows an HAI assay. The top panel shows the distribution of hemagglutinin inhibition titers at 0, 28, 42, and 63 days after vaccination and 8 days after infection. Titers were checked against the A / swine / England / 1353 / 2009 and a / Victoria / 2454 / 2019 strains. The bottom panel shows the average value for each group.

[0538] Conclusion: Importantly, this study demonstrated that pigs immunized with a broadly neutralizing panH1N1 vaccine were able to tolerate the challenge strain and the homologous whole virion inactivated adjuvant vaccine (WIV 1353 ), demonstrated proof of concept that the vaccine provided protection. In contrast, a WIV vaccine made from a strain of human origin derived from the same pH1N1 1A.3.3.2 lineage (WIVvic) failed to provide any protection in the presence of significant antibody levels.

[0539] (Example 17) Optimized vaccine generates neutralizing immune responses and protects against human and swine H1N1 influenza in mice and pigs background The zoonotic transmission of influenza A (IAV) and its continuous evolution in multiple species, especially birds and pigs, increases the possibility of new strains emerging at the human-animal interface. Strain-specific vaccination remains the cornerstone of influenza prevention and control, but the associated drawbacks reduce vaccine effectiveness. To address the threat of seasonal zoonotic pandemics, we present a superior digitally designed immune-optimized synthetic (DIOS) vaccine that induces broad H1, N1, and M2 subtype-specific immunity and protection against diverse strains in mouse and pig models.

[0540] method For mouse immunogenicity studies, individual immunogen FLU_T2_HA_3_I3 was injected subcutaneously four times at 2-week intervals with a terminal bleed 10 weeks after the first immunization. For pig challenge, a prime-boost regimen (4-week intervals) was utilized and the panH1N1 vaccine candidate was administered intradermally by PharmaJet® Tropis. Intramuscular delivery to controls included whole inactivated virus (WIV) representing swine and human influenza. Pigs were challenged with A / swine / EN / 1353 / 09 10 weeks after prime. Efficacy was measured as a reduction in virus shedding. Serum neutralization titers were monitored using pseudotype neutralization (pMN), enzyme-linked lectin assay (ELLA), and hemagglutinin inhibition (HAI).

[0541] result Figure 16 shows a diagram of the hemagglutinin (HA), neuraminidase (NA), and M2 surfaces of the A / swine / EN / 1353 / 09, A / Victoria / 2454 / 2019 H1N1 strains, and our DIOS vaccine candidate panH1N1. Colored residues indicate defined antigenic sites, with non-conserved residues between swine / EN / 09 and panH1N1 highlighted in red and those between swine / EN / 09 and Victoria / 19 in magenta.

[0542] As shown in Figure 17a and Figure 24, we observed excellent immune responses in all mice (n=6) vaccinated with FLU_T2_HA_3_I3 (referred to as DIOS(HA) in Figure 17 and H1N1dpm in Figure 24) against all H1N1 strains tested, comparable to or better than the control A / Michigan / 45 / 15 (H1) (*p<0.05). Values ​​are calculated as the dilution of serum that resulted in 50% neutralization of the virus by pMN. Figure 17b shows that administration of FLU_T2_HA_3_I3 in mice induces effective antibody binding responses against the six H1 wild-type influenza viruses tested.

[0543] In pigs, reduction in viral shedding in nasal swabs (expressed as mean log relative equivalent units (REU) of viral RNA) was observed in panH1N1 (n=5) and WIV 1353 Serum neutralizing antibodies against the HA and NA of A / swine / England / 1353 / 2009 and other related H1N1 viruses were also observed after challenge in the panH1N1 and WIV (n=5) groups (Figure 18). 1353 Figures 19a and 19b show serum neutralization titers against VI / 2570 / 19 and EN / 195 / 09 monitored at specific time points, and Figure 19c shows serum neutralization upon panH1N1 vaccination against a panel of H1-expressing pseudoviruses 42 days after vaccination.

[0544] Figures 20a and 20b show ELLAs (enzyme-linked lectin assays) to assess the inhibitory activity of the NA component of panH1N1 against A / swine / England / 1353 / 2009 (Figure 20a) and A / England / 195 / 2009 (Figure 20b) at a range of time points post-vaccination / infection. Figure 20c shows an ELLA against a panel of NA-expressing pseudoviruses at 42 days post-vaccination.

[0545] panH1N1 is referred to as DIOS in Figures 16 and 18, 19, and 20.

[0546] conclusion We have demonstrated immunogenicity and efficacy of DIOS (panH1N1 and individual FLU_T2_HA3_I3) vaccines against related IAV H1N1 strains in vitro and in vivo in mice and pigs. This approach may target different aspects of influenza and provide broader protection within the same subtype. This could aid in pandemic preparedness while protecting against spreading human influenza. This platform could be diverted to other subtypes with the goal of producing a universal influenza vaccine.

[0547] (Example 18) FLU_T3_HA_3 This example provides the amino acid sequences of the influenza hemagglutinin H5 head and stem regions for one embodiment of the invention known as FLU_T3_HA_3. In SEQ ID NO:27 below, the amino acid residues of the stem region are shown underlined. The amino acid residues of the head region are the remaining residues. Similarly, in SEQ ID NO:28 below, the nucleic acid residues of the stem region are shown underlined. The nucleic acid residues of the head region are the remaining residues. FLU_T3_HA_3 - amino acid sequence of HA0 (SEQ ID NO:27): [ka] [ka] FLU_T3_HA_3 - nucleic acid sequence of HA0 (SEQ ID NO: 28) [ka] FLU_T3_HA_3 - Amino acid sequence of the head region (SEQ ID NO:29) [ka] [ka]

[0548] The amino acid residues at positions 156, 157, 171, 172, and 205 are shown underlined and highlighted in grey in the above sequence (and are R, S, N, A, and R, respectively). The deleted amino acid residues at residues 144 or 145 are shown as " / " and highlighted in greyscale. FLU_T3_HA_3 - nucleic acid sequence of the head region (SEQ ID NO: 30) [ka] FLU_T3_HA_3 - Amino acid sequence of the first stem region (SEQ ID NO:31) [ka] FLU_T3_HA_3 - nucleic acid sequence of the first stem region (SEQ ID NO: 32) [ka] FLU_T3_HA_3 - amino acid sequence of the second stem region (SEQ ID NO:33) [ka]

[0549] The amino acid residues at positions 416 and 434 (or positions 148 and 166 if counted from the beginning of the stem region) are shown underlined in the above sequence (and are F and F, respectively). FLU_T3_HA_3 - nucleic acid sequence of the second stem region (SEQ ID NO: 34) [ka]

[0550] (Example 19) FLU_T3_HA_4 This example provides the amino acid sequences of the influenza hemagglutinin H5 head and stem regions for one embodiment of the invention known as FLU_T3_HA_4. In SEQ ID NO:35 below, the amino acid residues of the stem region are shown underlined. The amino acid residues of the head region are the remaining residues. Similarly, in SEQ ID NO:36 below, the nucleic acid residues of the stem region are shown underlined. The nucleic acid residues of the head region are the remaining residues. FLU_T3_HA_4 - amino acid sequence of HA0 (SEQ ID NO:35): [ka] FLU_T3_HA_4 - nucleic acid sequence of HA0 (SEQ ID NO: 36) [ka] FLU_T3_HA_4 - Amino acid sequence of the head region (SEQ ID NO:37) [ka]

[0551] The amino acid residues at positions 156, 157, 171, 172, and 205 are shown highlighted in the above sequence (and are R, S, N, A, and R, respectively). The amino acid residues at positions 148, 149, and 238 are also shown highlighted and are V, P, and E, respectively. FLU_T3_HA_4 - nucleic acid sequence of the head region (SEQ ID NO: 38) [ka] FLU_T3_HA_4 - Amino acid sequence of the first stem region (SEQ ID NO:39) [ka] FLU_T3_HA_4 - nucleic acid sequence of the first stem region (SEQ ID NO: 40) [ka] FLU_T3_HA_4 - Amino acid sequence of the second stem region (SEQ ID NO: 41) [ka]

[0552] The amino acid residues at positions 416 and 434 (or positions 148 and 166 if counted from the beginning of the stem region) are shown underlined in the above sequence (and are F and F, respectively). FLU_T3_HA_4 - nucleic acid sequence of the second stem region (SEQ ID NO: 42) [ka] [ka]

[0553] (Example 20) FLU_T3_HA_5 This example provides the amino acid sequences of the influenza hemagglutinin H5 head and stem regions for one embodiment of the invention known as FLU_T3_HA_5. In SEQ ID NO:43 below, the amino acid residues of the stem region are shown underlined. The amino acid residues of the head region are the remaining residues. Similarly, in SEQ ID NO:44 below, the nucleic acid residues of the stem region are shown underlined. The nucleic acid residues of the head region are the remaining residues. FLU_T3_HA_5 - amino acid sequence of HA0 (SEQ ID NO: 43): [ka] FLU_T3_HA_5 - nucleic acid sequence of HA0 (SEQ ID NO: 44) [ka] [ka] FLU_T3_HA_5 - Amino acid sequence of the head region (SEQ ID NO:45) [ka]

[0554] The amino acid residues at positions 156, 157, 171, 172, and 205 are shown highlighted in the above sequence and are R, S, N, A, and R, respectively. The amino acid residues at positions 148, 149, and 238 are also shown highlighted and are S, S, and E, respectively. FLU_T3_HA_5 - nucleic acid sequence of the head region (SEQ ID NO: 46) [ka] [ka] FLU_T3_HA_5 - Amino acid sequence of the first stem region (SEQ ID NO: 47) [ka] FLU_T3_HA_5 - nucleic acid sequence of the first stem region (SEQ ID NO: 48) [ka] FLU_T3_HA_5 - Amino acid sequence of the second stem region (SEQ ID NO: 49) [ka]

[0555] The amino acid residues at positions 416 and 434 (or positions 148 and 166 if counted from the beginning of the stem region) are shown underlined in the above sequence (and are F and F, respectively). FLU_T3_HA_5 - nucleic acid sequence of the second stem region (SEQ ID NO: 50) [ka]

[0556] Example 21 FLU_T3_HA_1 This example provides the amino acid and nucleic acid sequences of the stem region of influenza hemagglutinin H5 for one embodiment of the present invention known as FLU_T3_HA_1. Example 4 above provides the amino acid and nucleic acid sequences of the composite stem region of FLU_T3_HA_1, however the stem region is separated by the head region. This example also provides the nucleic acid sequences of the H5 head and stem regions, with the stem region underlined. FLU_T3_HA_1 - Amino acid sequence of the first stem region (SEQ ID NO:51) [ka] FLU_T3_HA_1 - nucleic acid sequence of the first stem region (SEQ ID NO:52) [ka] FLU_T3_HA_1 - Amino acid sequence of the second stem region (SEQ ID NO:53) [ka] FLU_T3_HA_1 - nucleic acid sequence of the second stem region (SEQ ID NO:54) [ka] FLU_T3_HA_1 - nucleic acid sequence of HA0 (SEQ ID NO:55) [ka] [ka] FLU_T3_HA_1 - nucleic acid sequence of the head region (SEQ ID NO:56) [ka]

[0557] Example 22 FLU_T3_HA_2 This example provides the amino acid and nucleic acid sequences of the stem region of influenza hemagglutinin H5 for one embodiment of the invention known as FLU_T3_HA_2. Example 5 above provides the amino acid and nucleic acid sequences of the composite stem region of FLU_T3_HA_2, however the stem region is separated by the head region. This example also provides the nucleic acid sequences of the H5 head and stem regions, with the stem region underlined. FLU_T3_HA_2 - Amino acid sequence of the first stem region (SEQ ID NO:57) [ka] FLU_T3_HA_2 - nucleic acid sequence of the first stem region (SEQ ID NO:58) [ka] FLU_T3_HA_2 - amino acid sequence of the second stem region (SEQ ID NO:59) [ka] FLU_T3_HA_2 - nucleic acid sequence of the second stem region (SEQ ID NO: 60) [ka] FLU_T3_HA_2 - nucleic acid sequence of HA0 (SEQ ID NO: 61) [ka] FLU_T3_HA_2 - nucleic acid sequence of the head region (SEQ ID NO: 62) [ka] [ka]

[0558] Example 23 Residue differences in the amino acid sequences of influenza Tier 3 H5 vaccine candidates FLU_T3_HA_1 to FLU_T3_HA_5 and influenza Tier 2 H5 design FLU_T2_HA_1 FIG. 21 summarizes the differences in the amino acid sequence of influenza hemagglutinin H5, including differences in positions A-E of H5 of the embodiments for different embodiments of the present invention. Positions A, B, and C of H5 are in the epitope region in the head region, and mutations at these positions shown in the figure increase the affinity of H5 for binding antibodies. Positions D and E are in the stem region of H5, and mutations at these positions increase the stability of the stem region in both pre-fusion and post-fusion states. Mutations of amino acid residues at positions 148, 149, and 238 of FLU_T3_HA_4 and at position 238 of FLU_T3_HA_5 are in the receptor binding site. Mutations of these residues reduce the affinity of HA to its receptor (sialic acid) on the surface of target cells, thus increasing the bioavailability of HA for antigen presentation.

[0559] Figure 22 shows a multiple sequence alignment of the HA amino acid sequences of FLU_T2_HA_1, FLU_T3_HA_1 through FLU_T3_HA_5, and two influenza isolates H5_WSN (SEQ ID NO: 64) and H5 GYR (SEQ ID NO: 65). In the figure, the amino acid residue differences are underlined and the amino acid differences across the designed sequences FLU_T2_HA_1 and FLU_T3_HA_1 / 2 / 3 / 4 / 5 are highlighted. The amino acid residues at positions A, B, and C in the head region and D and E in the stem region are boxed. These amino acid residues are at residue positions 156, 157, 171, 172, and 205 in the head region and residue positions 416 and 434 in the stem region. >A / WSN / Mongolia / 244 / 2005 (sequence number 64) Amino acid sequence [ka] >A / gyrfalcon / Washington / 41088-6 / 2014_H5N8 (FLU_T1_HA_9, SEQ ID NO: 65) Amino acid sequence [ka]

[0560] (Example 24) Iteratively designed H5Nx antigens generate broad immune responses in mice background Annual outbreaks of avian influenza (H5Nx) have a large socio-economic impact worldwide. In addition, there is a constant risk that it will spill over into naive human populations and cause a pandemic. The constant antigenic drift in the surface glycoproteins of influenza-hemagglutinin and reassortment with different neuraminidase subtypes adds a complex dimension to the design of universal H5 influenza vaccine antigens that can provide broad protection against H5Nx. Herein, we discuss H5Nx antigen designs that have been iteratively optimized to increase coverage of H5Nx.

[0561] method The H5Nx sequences available from the NCBI virus database were downloaded, cleaned, and trimmed to obtain a non-redundant dataset of H5 sequences. Phylogenetic relationships between these sequences were inferred, and a phylogenetically optimized sequence was designed as our first vaccine candidate FLU_T2_HA_1 (referred to as DIOS-T2_HA_9 in FIG. 23). The immunogenicity of the vaccine design was confirmed in Balb / c mice. Mouse sera were tested for neutralization using a pseudotype neutralization assay against multiple H5 viruses. Based on these results, FLU_T2_HA_1 was further optimized using epitope optimization to achieve broad neutralization, generating a panel of the next tier vaccine designs FLU_T3_HA_1 / 2 / 3 / 4 / 5 (...

Claims

1. An isolated polypeptide comprising: a) the amino acid sequence of SEQ ID NO: 71 (amino acid sequence of FLU_T4_HA_1:HA0); The amino acid sequence of SEQ ID NO: 73 (FLU_T4_HA_1: amino acid sequence of the head region); or the amino acid sequence of SEQ ID NO: 75 (FLU_T4_HA_1: the amino acid sequence of the first stem region); or b) an amino acid sequence of SEQ ID NO: 80 (amino acid sequence of FLU_T4_HA_2:HA0), or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to a sequence of SEQ ID NO: 80 along its entire length, and including an amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), and / or an amino acid sequence of SEQ ID NO: 100 (A / Sichuan / 2014 H5). and / or an amino acid sequence comprising an amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); optionally, the polypeptide further comprises an amino acid residue E at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue A at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue A at a position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue T at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue R at a position corresponding to amino acid residue 344 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or an amino acid residue K at a position corresponding to amino acid residue 345 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid sequence of SEQ ID NO: 82 (FLU_T4_HA_2: amino acid sequence of the head region), or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to a sequence of SEQ ID NO: 82, and including an amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), and / or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto; an amino acid sequence comprising an amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); optionally, the polypeptide further comprises an amino acid residue E at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue A at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue A at a position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or an amino acid residue T at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); the amino acid sequence of SEQ ID NO: 84 (FLU_T4_HA_2: amino acid sequence of the first stem region); or the amino acid sequence of SEQ ID NO: 86 (FLU_T4_HA_2: the amino acid sequence of the second stem region); or c) the amino acid sequence of SEQ ID NO: 89 (amino acid sequence of FLU_T4_HA_3:HA0), or having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the sequence of SEQ ID NO: 89 along its entire length, and including amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or an amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); optionally, the polypeptide further comprises an amino acid sequence comprising an amino acid residue T at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue Q at a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); an amino acid residue R at a position corresponding to amino acid residue 344 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or an amino acid residue K at a position corresponding to amino acid residue 345 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); 91 (FLU_T4_HA_3: amino acid sequence of the head region), or having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:91, and including an amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5), an amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO:100 (A / Sichuan / 2014 H5), an amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO:100 (A / Sichuan / 2014 H5), an amino acid residue H ... or amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); optionally, the polypeptide further comprises an amino acid residue T at a position corresponding to amino acid residue 172 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); or an amino acid residue Q at a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); the amino acid sequence of SEQ ID NO: 93 (FLU_T4_HA_3: amino acid sequence of the first stem region); or Amino acid sequence of SEQ ID NO: 95 (FLU_T4_HA_3: amino acid sequence of the second stem region) 1. An isolated polypeptide comprising:

2. 10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of claim 1, or its complement.

3. An isolated nucleic acid molecule, a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 71 (amino acid sequence of FLU_T4_HA_1:HA0) or its complement, wherein optionally the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 71 (amino acid sequence of FLU_T4_HA_1:HA0) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 72 (nucleic acid sequence of FLU_T4_HA_1:HA0); The nucleotide sequence of SEQ ID NO: 72 (nucleic acid sequence of FLU_T4_HA_1:HA0) or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:73 (FLU_T4_HA_1:head region amino acid sequence) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO:73 (FLU_T4_HA_1:head region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO:74 (FLU_T4_HA_1:head region nucleic acid sequence); The nucleotide sequence of SEQ ID NO: 74 (FLU_T4_HA_1: nucleic acid sequence of the head region) or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:75 (FLU_T4_HA_1:first stem region amino acid sequence) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO:75 (FLU_T4_HA_1:first stem region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO:76 (FLU_T4_HA_1:first stem region nucleic acid sequence); The nucleotide sequence of SEQ ID NO: 76 (FLU_T4_HA_1: nucleic acid sequence of the first stem region) or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:77 (FLU_T4_HA_1: second stem region amino acid sequence) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO:77 (FLU_T4_HA_1: second stem region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO:78 (FLU_T4_HA_1: second stem region nucleic acid sequence); The nucleotide sequence of SEQ ID NO: 78 (FLU_T4_HA_1: nucleic acid sequence of the second stem region) or its complement; or The nucleotide sequence of SEQ ID NO:79 (pEVAC-FLU_T4_HA_1) or its complement; or b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 80 (amino acid sequence of FLU_T4_HA_2:HA0) or its complement, wherein optionally the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 80 (amino acid sequence of FLU_T4_HA_2:HA0) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 81 (nucleic acid sequence of FLU_T4_HA_2:HA0); The nucleotide sequence of SEQ ID NO: 81 (nucleic acid sequence of FLU_T4_HA_2:HA0) or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 80 (amino acid sequence of FLU_T4_HA_2:HA0), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to SEQ ID NO: 80 along its entire length and comprising amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5) and / or amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 82 (FLU_T4_HA_2:head region amino acid sequence) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 82 (FLU_T4_HA_2:head region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 83 (FLU_T4_HA_2:head region nucleic acid sequence); The nucleotide sequence of SEQ ID NO: 83 (FLU_T4_HA_2: nucleic acid sequence of the head region) or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 82 (FLU_T4_HA_2: amino acid sequence of the head region), or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to SEQ ID NO: 82 along its entire length and comprising amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5) and / or amino acid residue E at a position corresponding to amino acid residue 238 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 84 (FLU_T4_HA_2:first stem region amino acid sequence) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 84 (FLU_T4_HA_2:first stem region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 85 (FLU_T4_HA_2:first stem region nucleic acid sequence); The nucleotide sequence of SEQ ID NO: 85 (FLU_T4_HA_2: nucleic acid sequence of the first stem region) or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 86 (FLU_T4_HA_2: second stem region amino acid sequence) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 86 (FLU_T4_HA_2: second stem region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 87 (FLU_T4_HA_2: second stem region nucleic acid sequence); The nucleotide sequence of SEQ ID NO: 87 (FLU_T4_HA_2: nucleic acid sequence of the second stem region) or its complement; or The nucleotide sequence of SEQ ID NO: 88 (pEVAC-FLU_T4_HA_2) or its complement; or c) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 89 (amino acid sequence of FLU_T4_HA_3:HA0) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 89 (amino acid sequence of FLU_T4_HA_3:HA0) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 90 (nucleic acid sequence of FLU_T4_HA_3:HA0); An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 90 (nucleic acid sequence of FLU_T4_HA_3:HA0) or its complement; 89 (amino acid sequence of FLU_T4_HA_3:HA0), or having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO: 89, and including an amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue H ... H at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue L at a position corresponding to amino acid residue 142 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), an amino acid residue L at a position corresponding to amino acid residue 1 100 (A / Sichuan / 2014 H5), or an amino acid sequence comprising an amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), or an amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5), or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:91 (FLU_T4_HA_3:head region amino acid sequence) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO:91 (FLU_T4_HA_3:head region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO:92 (FLU_T4_HA_3:head region nucleic acid sequence); The nucleotide sequence of SEQ ID NO: 92 (FLU_T4_HA_3: nucleic acid sequence of the head region) or its complement; an amino acid sequence of SEQ ID NO:91 (FLU_T4_HA_3: amino acid sequence of the head region), or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the sequence of SEQ ID NO:91, and including an amino acid residue F at a position corresponding to amino acid residue 107 of SEQ ID NO:100 (A / Sichuan / 2014 H5), an amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO:100 (A / Sichuan / 2014 H5), an amino acid residue N at a position corresponding to amino acid residue 142 of SEQ ID NO:100 (A / Sichuan / 2014 H5), an amino acid residue F at a position corresponding to amino acid residue 142 ... nucleotide sequence, or its complement, comprising an amino acid sequence comprising amino acid residue T at a position corresponding to amino acid residue 200 of SEQ ID NO: 100 (A / Sichuan / 2014 H5) or amino acid residue N at a position corresponding to amino acid residue 231 of SEQ ID NO: 100 (A / Sichuan / 2014 H5); a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:93 (FLU_T4_HA_3:first stem region amino acid sequence) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO:93 (FLU_T4_HA_3:first stem region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO:94 (FLU_T4_HA_3:first stem region nucleic acid sequence); The nucleotide sequence of SEQ ID NO: 94 (FLU_T4_HA_3: nucleic acid sequence of the first stem region) or its complement; a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:95 (FLU_T4_HA_3: second stem region amino acid sequence) or its complement, wherein optionally said nucleotide sequence encoding the amino acid sequence of SEQ ID NO:95 (FLU_T4_HA_3: second stem region amino acid sequence) is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO:96 (FLU_T4_HA_3: second stem region nucleic acid sequence); The nucleotide sequence of SEQ ID NO: 96 (FLU_T4_HA_3: nucleic acid sequence of the second stem region) or its complement; or Nucleotide sequence of SEQ ID NO:97 (pEVAC-FLU_T4_HA_3) or its complement An isolated nucleic acid molecule comprising:

4. 4. The isolated nucleic acid molecule of claim 2 or 3, comprising a messenger RNA (mRNA) molecule.

5. A vector comprising the nucleic acid molecule of claim 2 or 3.

6. 6. The vector of claim 5, further comprising a promoter operably linked to the or each nucleic acid molecule.

7. 7. A vector according to claim 6, wherein the or each promoter is for expression of a polypeptide encoded by the nucleic acid in a mammalian cell.

8. 6. The vector of claim 5, which is a vaccine vector, optionally a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, a messenger RNA (mRNA) vector, or a DNA vaccine vector.

9. An isolated cell comprising the vector of claim 5.

10. A fusion protein comprising the polypeptide of claim 1.

11. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.

12. A pharmaceutical composition comprising a nucleic acid according to claim 2 or 3, or a vector comprising a nucleic acid molecule according to claim 2 or 3, and a pharmaceutically acceptable carrier, excipient, or diluent.

13. 13. The pharmaceutical composition of claim 12, wherein the nucleic acid comprises a messenger RNA (mRNA) molecule and / or the vector is a messenger (mRNA) vector.

14. A pharmaceutical composition comprising a polypeptide of claim 1, a nucleic acid of claim 2 or 3, or a vector comprising the nucleic acid molecule of claim 2 or 3, and a pharmaceutically acceptable carrier, excipient, or diluent, further comprising an adjuvant for enhancing an immune response in a subject to the polypeptide of the composition or to a polypeptide encoded by the nucleic acid.

15. A composition comprising a polypeptide described in claim 1, a nucleic acid described in claim 2 or 3, or a vector comprising a nucleic acid molecule described in claim 2 or 3, for use as a pharmaceutical, or a pharmaceutical composition comprising a polypeptide described in claim 1, a nucleic acid described in claim 2 or 3, or a vector comprising a nucleic acid molecule described in claim 2 or 3, and a pharmaceutically acceptable carrier, excipient, or diluent.

16. A composition comprising a polypeptide described in claim 1, a nucleic acid described in claim 2 or 3, or a vector comprising a nucleic acid molecule described in claim 2 or 3, or a pharmaceutical composition comprising a polypeptide described in claim 1, a nucleic acid described in claim 2 or 3, or a vector comprising a nucleic acid molecule described in claim 2 or 3, and a pharmaceutically acceptable carrier, excipient, or diluent, for use in preventing, treating, or ameliorating influenza virus infection.

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