Avian influenza vaccine and its manufacturing method

Abstract

The present disclosure relates to avian influenza vaccines produced through reverse genetics to protect against A / turkey / Indiana / 22-like viruses, methods of producing such vaccines, and methods of using such vaccines to protect poultry. The disclosure also relates to methods of producing influenza viruses that comprise a chimeric polynucleotide having an HA non-coding sequence from a non-pathogenic highly multiplying influenza virus and a polynucleotide encoding an HA protein from a highly pathogenic avian influenza virus or variants thereof, and / or have a chimeric polynucleotide having an NA non-coding sequence from a non-pathogenic highly multiplying influenza virus and a polynucleotide encoding the NA protein from a highly pathogenic avian influenza virus or variants thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 328,911, filed April 8, 2022, the contents of which are expressly incorporated by reference herein in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing XML as required by 37 CFR § 1.831 (a), which was submitted in XML file format via the USPTO Electronic Patent Filing System and is incorporated herein by reference in its entirety. The XML file was created on 03 / 22 / 2023, is named Sequence_Listing_CONVERSION-005622.xml, and is 9.74 KB.

[0003] FIELD OF THE PRESENT APPLICATION The present disclosure relates to avian influenza viruses produced through reverse genetics and used as vaccines to protect against highly pathogenic avian influenza (HPAI), methods of producing such viruses, and methods of using such vaccines to protect poultry. [Background technology]

[0004] Avian influenza, commonly known as "bird flu", has devastating effects on the poultry industry, farm livelihoods, international trade, and wild bird health. To stop the spread of avian influenza, the policy is often to cull all poultry in outbreak areas, whether infected or healthy. This means huge economic losses and long-lasting effects on farmer livelihoods. Avian influenza is also a major public health concern. Every time an avian influenza virus circulates in poultry, sporadic cases of avian influenza can be identified in humans.

[0005] As of March 23, 2022, the United States Animal and Plant Health Inspection Service (APHIS) has reported detection of HPAI-infected wild birds in South Dakota, Nebraska, Kansas, Iowa, Missouri, Illinois, Indiana, Kentucky, Tennessee, Alabama, Florida, Georgia, Ohio, South Carolina, North Carolina, Virginia, Maryland, Delaware, New Jersey, New York, Connecticut, Massachusetts, New Hampshire, and Maine. All confirmed infected birds tested positive for Eurasian H5N1 virus. Wild birds can transmit avian influenza to domestic poultry. Wild birds may be infected with the virus and not show signs of illness. Wild birds have spread the virus to domestic poultry, including numerous backyard poultry and commercial turkey, layer, and broiler operations in multiple states. To date, over 20 million poultry have died or been euthanized to curb the spread of the disease.

[0006] Influenza A viruses are genetically diverse pathogens that infect a variety of hosts, including birds, pigs, and humans. In the past 30 years, vaccines and vaccination have emerged as essential tools for influenza control. The use of vaccines in poultry can increase resistance to infection, prevent illness and mortality, reduce virus replication and shedding, and reduce virus transmission to birds and mammals, including humans.

[0007] Low pathogenicity avian influenza (LPAI) viruses usually cause little or no clinical symptoms in infected poultry. LPAI viruses are shed through feces and respiratory secretions of infected birds. It spreads mainly through direct contact between healthy and infected birds. It can also spread through indirect contact with contaminated equipment and materials. LPAI virus strains occur naturally in wild migratory waterfowl and shore birds without causing disease. Highly pathogenic avian influenza viruses (HPAI) are highly contagious and highly lethal, which is why they are a severe disease that requires rapid response. They infect poultry such as chickens, turkeys, pheasants, quail, ducks, and geese, and wild birds, especially waterfowl. HPAI spreads from bird to bird by direct contact or through contact with contaminated water, feces, equipment, vehicles, cages, and clothing and shoes of people who have come into contact with the virus.

[0008] DIVA stands for Differentiating Infected from Vaccinated Animals. In the case of avian influenza, this can be achieved by using a vaccine based on a strain (e.g. H5N2) different from the currently circulating field strain (e.g. H5N1) and by using a serological test that can distinguish between vaccine-induced antibodies (e.g. against N2) and antibodies against the field virus (N1).

[0009] Because new influenza virus strains can easily evolve and spread among various species, the continuous production of new vaccines is necessary. Due to the continuous emergence or re-emergence of different influenza strains, new influenza vaccines are continually desired. Such vaccines are typically made using antigenic portions of newly emerged virus strains, so polypeptides and polynucleotides (especially sequences of antigenic genes) of new, newly emerged, or newly re-emerged virus strains are highly desirable. Summary of the Invention

[0010] The present disclosure provides influenza vaccine viruses produced by reverse genetics that provide protection against current H5N1 field strains and / or provide DIVA (differentiating vaccinated from infected animals) capabilities.

[0011] The present disclosure relates to avian influenza viruses produced through reverse genetics and used as vaccines to protect against highly pathogenic avian influenza (HPAI), methods of producing such viruses, and methods of using such vaccines to protect poultry.

[0012] In one embodiment, the present disclosure relates to a chimeric polynucleotide comprising a hemagglutinin (HA) non-coding sequence of a non-pathogenic high growth influenza virus and a polynucleotide encoding an HA protein from a highly pathogenic avian influenza virus or a variant thereof, and / or a neuraminidase (NA) non-coding sequence of a non-pathogenic high growth influenza virus and a polynucleotide encoding an NA protein from a highly pathogenic avian influenza virus or a variant thereof. In some embodiments of the present disclosure, the polynucleotide encoding the variant of the HA protein has at least about 80% identity to the A / PuertoRico / 8 / 34 (PR8) HA NA protein. In some embodiments of the present disclosure, the NA protein is N1, N2, N3, N4, N5, N6, N7, N8, or N9 or a variant thereof. In some embodiments of the present disclosure, the polynucleotide encoding the variant of the NA protein has at least about 80% identity to the N1, N2, N3, N4, N5, N6, N7, N8, or N9 protein. In some embodiments of the present disclosure, the non-pathogenic high growth influenza virus is PR8.

[0013] In one embodiment, the disclosure relates to a rescued influenza virus comprising a polynucleotide encoding PR8 M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2, A / turkey / Indiana / 22-003707-003 / 2022 HA, and at least one of N1, N2, N3, N4, N5, N6, N7, N8, or N9 NA.

[0014] In one embodiment, the disclosure relates to a method of producing influenza virus comprising introducing into a population of host cells a plurality of vectors comprising polynucleotides corresponding to at least six internal segments of PR8, at least one polynucleotide encoding A / turkey / Indiana / 22-003707-003 / 2022 HA, and at least one polynucleotide encoding one of N1, N2, N3, N4, N5, N6, N7, N8, or N9, culturing the population of host cells at a temperature of 35° C. or less, and recovering virus. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a schematic diagram of the influenza A virus, showing the shape of the virus particle, the lipid bilayer, the location of the membrane-associated proteins HA, NA, M1 and M2, and the genome segments PB2, PB1, PA, HA, NP, NA, M and NS within the virus particle.

[0016] [Diagram 2] Figure 2 shows the eight gene segments present in influenza A virus particles: gene segment 1: PB2; gene segment 2: PB1; gene segment 3: PA; gene segment 4: HA; gene segment 5: NP; gene segment 6: NA; gene segment 7: MA; and gene segment 8: NS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Brief Description of the Sequence Listing The nucleotide sequences disclosed herein are listed in Table 1 below. [Table 1]

[0018] Detailed Description The present disclosure relates to avian influenza vaccines for protection against HPAI produced through reverse genetics, kits containing such vaccines, and methods of using such vaccines to protect poultry.

[0019] As used herein, the term "about" is defined as plus or minus 10% of the stated value. For example, about 1.0 g means between 0.9 g and 1.1 g.

[0020] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the term "or" is intended to include "and" unless the context clearly dictates otherwise.

[0021] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise.

[0022] The term "about" is intended to refer to a range substantially within the cited range, without departing from the scope of the disclosure. As used herein, "about" is understood by those skilled in the art and varies to some extent depending on the context in which it is used. If there are uses of the term that are not clear to those skilled in the art in light of the context in which the term is used, "about" means up to plus or minus 10% of the stated value.

[0023] The terms "individual," "subject," and "animal" are used interchangeably herein to refer to a vertebrate animal that supports negative stranded RNA virus infection, specifically influenza A virus infection.

[0024] Exemplary subjects may include vertebrates that have importance to humans because they are endangered, because they are economically important, such as those raised on farms for human consumption, and / or because they are socially important, such as animals kept as pets or in zoos. The methods and compositions of the present disclosure are particularly useful for warm-blooded vertebrates, including, but not limited to, birds, canines, cetaceans, felines, mustelids, rodents, equines, bovids, suidae, and primates. In an embodiment, the vertebrate is a bird, such as a waterfowl, chicken, or turkey. In an embodiment, the vertebrate is a mammal, such as a pig, horse, whale, dolphin, or human. In an embodiment, the vertebrate is a human. In an embodiment, the vertebrate is a chicken.

[0025] As used herein, the terms "isolated" and "purified" refer to material which is substantially or essentially free from components which normally accompany the referenced material in its natural state.

[0026] As used herein, "substantially free" means, with respect to a particular material, below the level of detection using standard detection methods for that material, be it unwanted nucleic acids, proteins, cells, viruses, infectious agents, etc.

[0027] As used herein, an "amino acid sequence" is a character string representing a polymer of amino acid residues, such as a protein, polypeptide, or polymer.

[0028] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to single- or double-stranded deoxyribonucleotide or ribonucleotide polymers, chimeras or analogs thereof.

[0029] As used herein, the terms "polynucleotide sequence" and "nucleic acid sequence" are used interchangeably and refer to a character string that represents a polynucleotide or a nucleic acid.Unless otherwise indicated, the specific nucleic acid sequences of the present disclosure include complementary sequences in addition to the sequence explicitly indicated, as appropriate.From any particular polynucleotide sequence, either the given nucleic acid sequence or the complementary polynucleotide sequence (e.g., complementary nucleic acid) can be determined.

[0030] The term "gene" is used broadly herein to refer to any nucleic acid associated with a biological function. Thus, genes include coding sequences and / or regulatory sequences required for their expression. The term "gene" applies to a specific genomic sequence, as well as to the cDNA or mRNA encoded by that genomic sequence.

[0031] Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins. Non-expressed regulatory sequences include "promoters" and "enhancers," to which regulatory proteins, such as transcription factors, bind and result in transcription of adjacent or nearby sequences. A "tissue-specific" promoter or enhancer is one that regulates transcription in a particular tissue or cell type(s).

[0032] An "open reading frame" or "ORF" is a possible translational reading frame of DNA or RNA (e.g., that of a gene) that can be translated into a polypeptide. That is, the reading frame is not interrupted by a stop codon. It should be noted, however, that the term ORF does not necessarily indicate that the polynucleotide is actually translated into a polypeptide.

[0033] As used herein, "expression of a gene" or "expression of a nucleic acid" means, as indicated by the context, transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing), translation of RNA into a polypeptide (which may include subsequent modification of the polypeptide, e.g., post-translational modification), or both transcription and translation.

[0034] The term "vector" as used herein refers to a means by which nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, etc., which can replicate autonomously or be integrated into the chromosomes of host cells. Vectors can also be naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, polylysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, etc., which do not replicate autonomously. In many, if not all, common embodiments, the vectors of the present disclosure are plasmids.

[0035] An "expression vector" is a vector, such as a plasmid, that is capable of promoting the expression and replication of a nucleic acid incorporated therein. Typically, the nucleic acid to be expressed is "operably linked" to a promoter and / or enhancer and is under the transcriptional regulatory control of the promoter and / or enhancer.

[0036] The term "polypeptide" is used herein to refer to a polymer (e.g., a peptide or protein) comprising two or more amino acid residues. A polypeptide can optionally include modifications such as glycosylation. The amino acid residues of a polypeptide can be natural or non-natural and can be unsubstituted, unmodified, substituted, or modified.

[0037] As used herein, the term "DIVA" stands for Differentiating Infected from Vaccinated Animals. For avian influenza, this can be accomplished by using a vaccine based on a strain (e.g., H5N2) different from the current field strain (e.g., H5N1) and using a serological test that can distinguish vaccine-induced antibodies (e.g., to N2) from antibodies to the field virus (N1). Since unvaccinated animals die and do not develop a serological response, the DIVA vaccine of the present disclosure can be used to distinguish vaccinated and infected animals from vaccinated animals.

[0038] As used herein, a "recombinant virus" is one that has been manipulated in vitro, e.g., one in which changes have been introduced into the viral genome using recombinant nucleic acid techniques, or one that has been artificially produced.

[0039] As used herein, the terms "recombinant nucleic acid," "recombinant segment," and "recombinant polynucleotide" are used interchangeably and refer to a nucleic acid that has been modified in vitro. The sequence of a recombinant polynucleotide is not naturally occurring or does not correspond to a naturally occurring sequence or is not arranged as it is located in a natural genome.

[0040] "Pharmaceutical composition" refers to a composition containing a recombinant mutated influenza gene segment of the present disclosure or a recombinant influenza virus of the present disclosure and suitable for administration to a subject. The pharmaceutical composition is suitable for preventing, treating, reducing or ameliorating one or more influenza symptoms in a subject. For purposes of this disclosure, a pharmaceutical composition includes a vaccine.

[0041] As used herein, "diluent", "excipient", "carrier" and "adjuvant" are used interchangeably and refer to a diluent, excipient, carrier or adjuvant that is physiologically acceptable to a subject while retaining the therapeutic properties of the pharmaceutical composition administered therewith. Physiologically acceptable diluents, excipients, carriers or adjuvants and their formulations are known to those skilled in the art (see, for example, U.S. Pat. No. 9,017,691; Chaudhari SP, et al. 2012, Pharmaceutical Excipients: A Review," IJAPBC Vol 1(1)). Reed SG, et al. (2013, "Key Roles of Adjuvants in Modern Medicines," Nature Medicine 19(12): 1597-1608) provides a general overview of adjuvants used in vaccines.

[0042] As used herein, the term "attenuated" in relation to a virus refers to a virus that has reduced toxicity or pathogenicity compared to its non-attenuated counterpart, but is still viable or alive. Typically, attenuation renders an infectious agent, such as a virus, less harmful or pathogenic to an infected subject compared to a non-attenuated virus.

[0043] The terms "inoculated" and "vaccinated" are used interchangeably herein and refer to the act of introducing an influenza virus vaccine of the present disclosure into a bird.

[0044] Orthomyxoviruses are a family of RNA viruses that include seven genera: Influenza virus A, Influenza virus B, Influenza virus C, Influenza virus D, Isavir virus, Thogoto virus, and Cranja virus. Influenza A virus is one of the major uncontrolled pathogens worldwide, causing seasonal epidemics and global pandemics. Influenza A virus can infect various vertebrate hosts, including birds and mammals. The vertebrate hosts of influenza are birds, canines, cetaceans, felines, mustelidae, rodents, equines, bovidae, suidae, and primates. In some embodiments, the vertebrate is a bird, such as a waterfowl, chicken, or turkey. In some embodiments, the vertebrate is a mammal, such as a pig, horse, whale, dolphin, or human. In some embodiments, the vertebrate is a human. In some embodiments, the vertebrate is a chicken, turkey, dolphin, whale, pig, horse, or human.

[0045] The genome of influenza A viruses is a negative-sense, single-stranded, segmented RNA. Influenza A subtypes are named (HxNy) according to the type of hemagglutinin (H) and neuraminidase (N) present in the virus. To date, 18 different H antigens and 10 different N antigens are known.

[0046] Influenza virus particles (also called virions) consist of a viral envelope wrapped around a central core. The outer layer of the influenza virion is a lipid membrane taken from the host cell in which the virus grows. The lipid membrane is inserted with the hemagglutinin (HA), neuraminidase (NA), and matrix-2 (M2) proteins. Four M2 proteins form a proton-selective ion channel, the unit of which is a helix stabilized by two disulfide bonds. Under the lipid membrane, a layer of matrix protein (M1) forms the shell. Inside the virion are eight negative-stranded viral RNA segments consisting of RNA bound to the nucleoprotein (NP), and three polymerase subunits: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). Nonstructural protein 1 (NS1) and nonstructural protein 2 (NS2) are found in infected cells but are not packaged inside the virion.

[0047] The eight RNA segments of the influenza A virus genome are shown in Figure 1. Gene segment 1, also called the PB2 gene segment, encodes the cap-binding transcriptase PB2. Gene segment 2, also called the PB1 gene segment, encodes the elongation-associated proteins PB1, PB1-F2, and PB1N40 using alternative translation initiation sites. Ribosomal frameshift gene segment 3, also called the PA gene segment, encodes the polymerase acidic proteins PA and PA-X, and encodes PA-N155 and PA-N182 using alternative translation sites and N-terminal truncations. Gene segment 4, also called the HA gene segment, encodes the hemagglutinin protein HA. Gene segment 5, also called the NP gene segment, encodes the RNA-binding nucleoprotein NP. Gene segment 6, also called the NA gene segment, encodes the neuraminidase protein NA. Gene segment 7, also called the matrix gene, encodes two proteins M1 and M2. Gene segment 8, also called the nonstructural gene, encodes the nonstructural protein NS1, which, by alternative RNA splicing, encodes NS3 and the nuclear export protein NS2 / NEP.

[0048] Avian influenza is caused by influenza A viruses (influenza A). Influenza viruses of avian origin are broadly classified based on the combination of two protein groups on the surface of influenza A viruses: the hemagglutinin "H" protein, of which there are 16 types (H1-H16), and the neuraminidase "N" protein, of which there are 9 types (N1-N9). Many different combinations of the "H" and "N" proteins are possible. Each combination is considered a different subtype, and related viruses within a subtype can be called a lineage. Avian influenza viruses are classified as either "low pathogenic" or "highly pathogenic" based on their genetic characteristics and the severity of the disease they cause in chickens. Most viruses are low pathogenic, meaning that they cause no or only mild clinical signs of infection in poultry.

[0049] Influenza seed virus production by reverse genetics involves transfecting tissue culture cells with cDNA clones from the nonpathogenic, highly proliferating A / PuertoRico / 8 / 34 (PR8) backbone and donor antigen proteins.

[0050] The inventors have prepared recombinant influenza viruses expressing a modified hemagglutinin (HA) from A / Turkey / Indiana / 2022. The modified HA contains an alteration in its cleavage site, which allows the rescued virus to have a phenotype of low pathogenicity HA. The disclosed recombinant influenza viruses express either neuraminidase (NA) from PR8 (N1), NA from A / Turkey / Indiana / 2022 (N1), or NA from A / BlueWingedTeal / WY / AH0099021 / 2016 (N9). All three viruses were confirmed by sequencing to have the low pathogenicity cleavage site, the correct NA, and all grow at sufficiently high titers to be effective vaccine candidates. All of these vaccine variants are used as inactivated and adjuvanted vaccines.

[0051] Just as with human influenza vaccines, a close match of the influenza strain in the vaccine to the circulating virus is necessary for optimal protection. Although several RG vaccines against H5 influenza have been produced over the past 20 years, there is currently no commercially available vaccine that closely matches the HPAI viruses circulating in the United States. An advantage of the proposed vaccine is that it closely matches the field strain and therefore provides better protection compared to mismatched vaccines. Matching of the hemagglutinin protein is important for protection. Matching of the neuraminidase protein may provide some additional protection.

[0052] The present inventors have prepared several vaccine viruses containing attenuated hemagglutinin proteins that are homologously matched against circulating field strains of the 2022 North American H5N1 highly pathogenic avian influenza virus pandemic, belonging to antigenic clade 2.3.4.4b viruses, which provide improved protection compared to non-clade 2.3.4.4b vaccines, including those targeting antigenic clades 2.3.4.4a, 2.3.4.4c, 2.2, 2.3.2.1 and related H5 vaccine viruses.

[0053] The use of heterologous neuraminidase protein is being considered for DIVA purposes. DIVA is an acronym that stands for differentiating vaccinated from infected animals, but for trade purposes it is generally understood to mean differentiating vaccinated, unexposed poultry from vaccinated and infected poultry. Currently, avian influenza vaccination has significant trade implications, as countries do not import poultry or poultry products from countries that vaccinate against avian influenza. Reliable identification of vaccinated and infected animals with a simple serological test could overcome the negative trade impacts associated with avian influenza. The use of a vaccine with the N9 neuraminidase protein would allow serological monitoring of the N1 neuraminidase protein of the field virus. Previous studies have shown only a small loss of protection when heterologous neuraminidase proteins are used.

[0054] The construction of the three viruses reported in this application takes advantage of one unique feature not previously reported. All influenza gene segments have protein coding regions flanked on both ends by non-coding sequences. Non-coding sequences are known to be important in regulating gene expression and packaging of genes into viral particles. The non-coding regions (NCRs) have small regions of highly conserved sequence, but also have regions of variability. As part of the plasmid construction for RG rescue, a different non-coding region was used for the N9 neuraminidase gene. This included sequences found in the original virus and the same coding sequence with non-coding regions from the PR8 virus. The inventors hypothesized that a virus with a PR8 non-coding region that matches the rest of the virus would replicate better. This change resulted in an almost 2 log increase in titer with the PR8 NCR from the native NCR. This effectively makes a virus that did not grow well enough for commercial use as a vaccine seed strain into one that makes an excellent seed strain. The use of the PR8 NCR has not previously been identified as a significant factor in viral titer.

[0055] Preparation of vaccine viruses included chimeric constructs of an attenuated HA coding sequence from a circulating field strain and a non-coding sequence from PR8 virus, or an NA coding sequence from a circulating field strain and a non-coding sequence from PR8 virus. The use of chimeric constructs improved expression of hemagglutinin and neuraminidase proteins, resulting in higher titers of rescued virus. Improved growth in embryonic chicken eggs allows for more economical vaccine production, thereby reducing vaccine production costs.

[0056] Although the principle of DIVA has been discussed for many years, it has not been routinely used against avian influenza, in part due to the lack of a convenient companion diagnostic. Published studies support the heterologous NA DIVA strategy as a viable option (Avellaneda, G., et al., 2010, "A Heterologous Neuraminidase Subtype Strategy for the Differentiation of Vaccinated and Infected Animals (DIVA) Strategy for Avian Influenza Virus using a more flexible Neuraminidase Inhibition Test," Avian Diseases : 54: 278-286; Jadhao, S J., et al., 2009, "Comparative efficacy of North American and antigenically matched reverse genetics derived H5N9 DIVA marker vaccines against highly pathogenic Asian H5N1 avian influenza in chickens," Vaccine: 27: 6247-6260). Vaccine viruses were prepared that contain a neuraminidase gene that is heterologous to the field strain. The inclusion of a neuraminidase gene with a different subtype than the field virus provides the ability to distinguish vaccinated and uninfected birds from those that are infected after vaccination by serological testing. The use of a simple and cost-effective companion diagnostic is necessary to complete serological surveillance.

[0057] In one embodiment, the present disclosure relates to a polynucleotide comprising a hemagglutinin (HA) non-coding sequence from a non-pathogenic highly proliferating influenza virus and a polynucleotide encoding an attenuated HA protein from a highly pathogenic avian influenza virus or variant thereof, or a polynucleotide comprising a neuraminidase (NA) non-coding sequence from a non-pathogenic highly proliferating influenza virus and a polynucleotide encoding an NA protein from a highly pathogenic avian influenza virus or variant thereof. In some embodiments of the present disclosure, the non-coding region in the polynucleotide is from A / PuertoRico / 8 / 34 (PR8). In certain embodiments of the present disclosure, the HA protein or variant thereof encoded by the polynucleotide of the present disclosure is from a highly pathogenic avian influenza virus. In certain embodiments of the present disclosure, the NA protein or variant thereof encoded by the polynucleotide of the present disclosure is from a highly pathogenic avian influenza virus. In certain embodiments of the present disclosure, the HA protein or variant thereof encoded by the polynucleotide of the present disclosure is mutated to include an attenuated cleavage site. In some embodiments of the disclosure, the HA protein or variant thereof is from A / turkey / Indiana / 22-003707-003 / 2022. In some embodiments of the disclosure, the NA protein or variant thereof encoded by the polynucleotide is N1, N2, N3, N4, N5, N6, N7, N8 or N9. In some embodiments of the disclosure, the NA protein or variant thereof encoded by the polynucleotide is from A / turkey / Indiana / 22-003707-003 / 2022. In some embodiments of the disclosure, the NA protein or variant thereof encoded by the polynucleotide is from A / Blue Winged Teal / Wyoming / AH0099021 / 2016.

[0058] In certain embodiments, the present disclosure relates to polynucleotides encoding variant attenuated HA proteins from A / turkey / Indiana / 22-003707-003 / 2022. Influenza virus hemagglutinin or hemagglutinin (HA) glycoprotein is a homotrimeric membrane integral protein found on the surface of influenza viruses. HA proteins have multifunctional activities as adhesins and membrane fusion proteins. Polynucleotides encoding HA protein variants may have less than 100% identity to polynucleotides encoding attenuated A / turkey / Indiana / 22-003707-003 / 2022 HA protein but may retain the multifunctional activity of the HA protein. For example, an attenuated HA protein can be encoded by a polynucleotide having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:3, while retaining multifunctional activity as an attachment factor and membrane fusion protein.

[0059] In some embodiments, the present disclosure relates to a polynucleotide encoding an NA protein from a highly pathogenic avian influenza virus or a variant thereof. Influenza neuraminidase (NA) is found on the virion surface and plays an essential role in the release and spread of progeny virions from infected cells. A polynucleotide encoding an NA protein variant may have less than 100% identity to a polynucleotide encoding an A / turkey / Indiana / 22-003707-003 / 2022 NA protein or an A / Blue Winged Teal / Wyoming / AH0099021 / 2016 NA protein while still retaining its virus release and spread activity. For example, the NA protein can be encoded by a polynucleotide having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, while retaining its viral release and spreading activity.

[0060] In one embodiment, the disclosure relates to a rescued influenza virus comprising a polynucleotide encoding influenza A M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2 from a non-pathogenic, highly proliferating influenza virus, a polynucleotide encoding HA from A / turkey / Indiana / 22-003707-003 / 2022 modified to contain a low pathogenicity cleavage site, and a polynucleotide encoding an N1, N2, N3, N4, N5, N6, N7, N8, or N9 NA. In some embodiments of the disclosure, the polynucleotide encoding NA in the rescued influenza virus is from A / turkey / Indiana / 22-003707-003 / 2022 or A / Blue Winged Teal / Wyoming / AH0099021 / 2016. In some embodiments of the disclosure, the rescued influenza virus polynucleotides encoding influenza A M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2 are derived from PR8.

[0061] In one embodiment, the disclosure relates to a vector comprising the polynucleotide of the disclosure. In some embodiments of the disclosure, the vector is a plasmid, cosmid, phage, virus, or a fragment of a virus. In one embodiment, the disclosure relates to a rescued influenza virus comprising a polynucleotide encoding influenza A M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2 from a non-pathogenic high growth influenza virus, a polynucleotide encoding HA from A / turkey / Indiana / 22-003707-003 / 2022 modified to contain a low pathogenicity cleavage site, and a polynucleotide encoding N1, N2, N3, N4, N5, N6, N7, N8, or N9 NA. In some embodiments of the disclosure, the polynucleotide encoding NA in the rescued influenza virus is from A / turkey / Indiana / 22-003707-003 / 2022 or A / Blue Winged Teal / Wyoming / AH0099021 / 2016. In some embodiments of the disclosure, the polynucleotide encoding influenza A M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2 in the rescued influenza virus is from PR8. In one embodiment, the disclosure relates to an immunogenic composition comprising an immunologically effective amount of a rescued influenza virus of the disclosure.

[0062] In one embodiment, the disclosure relates to a method of producing influenza virus comprising introducing a plurality of vectors comprising polynucleotides corresponding to at least six internal segments of a non-pathogenic high growth influenza virus, a chimeric polynucleotide comprising a non-coding sequence from a non-pathogenic high growth influenza virus and a polynucleotide encoding a HA protein from a highly pathogenic avian influenza virus or variant thereof, and a chimeric polynucleotide comprising a non-coding sequence from a non-pathogenic high growth influenza virus and a polynucleotide encoding a NA protein from a highly pathogenic avian influenza virus or variant thereof into a population of host cells, culturing the population of host cells at a temperature of 35° C. or less, and recovering the virus. In some embodiments of the disclosure, the influenza virus is produced with PR8, HA from A / turkey / Indiana / 22-003707-003 / 2022 or a variant thereof as the non-pathogenic virus, and NA of N1, N2, N3, N4, N5, N6, N7, N8, or N9.

[0063] In one embodiment, the present disclosure relates to a method of producing influenza virus, comprising introducing a plurality of vectors comprising polynucleotides corresponding to at least six internal segments of a non-pathogenic, highly multiplying influenza virus, a polynucleotide encoding A / turkey / Indiana / 22-003707-003 / 2022 HA or a variant thereof, and an N1, N2, N3, N4, N5, N6, N7, N8, or N9 NA polynucleotide into a population of host cells, culturing the population of host cells at a temperature of 35° C. or less, and recovering the virus. In some embodiments of the present disclosure, the non-pathogenic virus in the method of producing influenza virus is PR8. In some embodiments of the present disclosure, the NA polynucleotide in the method of producing influenza virus is from A / turkey / Indiana / 22-003707-003 / 2022 or A / Blue Winged Teal / Wyoming / AH0099021 / 2016.

[0064] Embodiments of the present disclosure are shown and described herein. It will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the disclosure. Various alternatives to the embodiments of the disclosure described herein may be used in carrying out the present disclosure. It is intended that the included claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents are covered thereby. All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. EXAMPLES

[0065] Having generally described this disclosure, the same will be better understood with reference to specific examples, which are included herein solely to further illustrate the disclosure and are not intended to limit the scope of the disclosure, which is defined by the claims.

[0066] Example 1 Virus and vaccine preparation Reverse genetics was used to assemble viruses containing polynucleotides encoding A / turkey / Indiana / 22-003707-003 / 2022 viral HA and / or NA, or NA from PR8, N3, N4, or N9 viruses.

[0067] Coding sequence from the A / turkey / Indiana / 22-003707-003 / 2022 (TK / IN / 22) virus was obtained from GISAID (Global Initiative on Sharing All Influenza Data). The hemagglutinin (HA) gene sequence was modified to change the high pathogenicity cleavage site to a low pathogenicity cleavage site. The HA coding sequence was modified to change the cleavage site from REKRRKR / GLF (set forth in SEQ ID NO:1) to RETR / GLF (set forth in SEQ ID NO:2), which included the deletion of three basic amino acids and the substitution of a lysine at position -2 from the cleavage site with a threonine. Additionally, the gene sequence was modified to include non-coding gene sequences that were not included in the published sequence. This sequence was de novo synthesized by IDT DNA to include non-coding and coding sequences with the addition of non-influenza sequences (DNA assembly sites) to facilitate insertion into a plasmid. The N1 neuraminidase sequence gene from A / turkey / Indiana / 22-003707-003 / 2022 was also synthesized de novo with coding, non-coding and non-influenza sequences. The N9 neuraminidase gene from A / Blue Winged Teal / Wyoming / AH0099021 / 2016 was also synthesized de novo with coding, non-coding and non-influenza sequences. The sequence of A / turkey / Indiana / 22-003707-003 / 2022 HA is shown in SEQ ID NO:3, the sequence of A / turkey / Indiana / 22-003707-003 / 2022 NA is shown in SEQ ID NO:4 and the sequence of A / Blue Winged Teal / Wyoming / AH0099021 / 2016 NA is shown in SEQ ID NO:5.

[0068] Using DNA assembly enzymes, the sequences of the individual genes were inserted into a plasmid containing an RNA polymerase promoter and a transcription promoter. The influenza HA and NA coding fragments were inserted between the PR8 HA and NA noncoding regions, and the internal PR8 genes were found to be normal. This reverse genetics plasmid is used to transcribe mRNAs that express viral proteins required for viral packaging along with the viral RNA. In addition to the hemagglutinin (HA) gene, the neuraminidase (NA) gene and six other viral gene segments are required for rescue. The six internal genes are from the PR8 vaccine virus. The neuraminidase gene was obtained from either the A / turkey / Indiana / 22-003707-003 / 2022, PR8 virus, or N3, N4, or N9 influenza viruses.

[0069] Polynucleotides encoding the other six required genes, PB2, PB1, PA, NP, M1 / M2, and NS, from the PR8 vaccine strain, together with a plasmid carrying the HA coding sequence and a neuraminidase (NA) gene plasmid, were introduced and transfected into cell culture to rescue the live vaccine virus. A total of eight genes are required to rescue the virus.

[0070] The eight gene segments were transfected into 293T cells using commercially available TransIT-LT1 transfection reagent, and two days later the cell supernatants were inoculated into embryonated chicken eggs, and five days later the allantoic fluid was examined for hemagglutination activity, evidence of rescued virus.

[0071] Rescued viruses were sequenced to confirm the presence of the hypopathogenic cleavage site. To confirm that the rescued viruses were hypopathogenic, they were grown in cell culture with and without the addition of trypsin. Hypopathogenic viruses require trypsin to grow in cell culture medium.

[0072] The low pathogenic viruses were titered and three viruses were selected for further study: A / turkey / Indiana / 22-003707-003 / 2022 (HA-LPAI), PR / 8 (N1, MA, NS, PA, PB1, PB2, and NP) 10^9.2 EID50 / mL; A / turkey / Indiana / 22-003707-003 / 2022 (HA-LPAI and N1), PR / 8 (MA, NS, PA, PB1, PB2, and NP) 10^9.4 EID50 / mL. With the original non-coding region, A / turkey / Indiana / 22-003707-003 / 2022 (HA-LPAI), A / Blue Winged Teal / Wyoming / AH0099021 / 2016 (N9), and PR / 8 (MA, NS, PA, PB1, PB2, and NP) showed 10^7.6 EID50 / mL. With the PR8 non-coding region, A / turkey / Indiana / 22-003707-003 / 2022 (HA-LPAI), A / Blue Winged Teal / Wyoming / AH0099021 / 2016 (N9), and PR / 8 (MA, NS, PA, PB1, PB2, and NP) showed 10^9.5 EID50 / mL.

[0073] Two candidate viruses were selected: an H5N1 virus containing polynucleotides encoding the A / turkey / Indiana / 22-003707-003 / 2022 NA and an attenuated HA, and an H5N9 virus containing polynucleotides encoding the A / turkey / Indiana / 22-003707-003 / 2022 attenuated HA and the A / Blue Winged Teal / Wyoming / AH0099021 / 2016 NA (N9).

[0074] This example shows the rescue of candidate viruses containing attenuated A / turkey / Indiana / 22-003707-003 / 2022 HA and NA, and candidate viruses containing attenuated A / turkey / Indiana / 22-003707-003 / 2022 HA and A / Blue Winged Teal / Wyoming / AH0099021 / 2016 NA with PR3 non-coding regions or A / Blue Winged Teal / Wyoming / AH0099021 / 2016 non-coding regions.

Claims

1. a polynucleotide encoding a hemagglutinin (HA) non-coding sequence from a non-pathogenic highly multiplying influenza virus and an HA protein from a highly pathogenic avian influenza virus or a variant thereof; or Polynucleotides encoding neuraminidase (NA) non-coding sequences from non-pathogenic, highly multiplying influenza viruses and NA proteins from highly pathogenic avian influenza viruses or variants thereof A chimeric polynucleotide comprising

2. 2. The chimeric polynucleotide of claim 1, wherein the non-pathogenic, highly proliferative influenza virus is A / PuertoRico / 8 / 34 (PR8).

3. 2. The chimeric polynucleotide of Claim 1, wherein the polynucleotide encoding an HA protein encodes an attenuated HA protein.

4. 4. The chimeric polynucleotide of claim 3, wherein the polynucleotide encoding an HA protein encodes an attenuated A / turkey / Indiana / 22-003707-003 / 2022 HA protein or a variant thereof.

5. The chimeric polynucleotide of claim 1, wherein the polynucleotide encoding the NA protein is N1, N2, N3, N4, N5, N6, N7, N8 or N9.

6. 2. The chimeric polynucleotide of Claim 1, wherein the NA protein is derived from A / turkey / Indiana / 22-003707-003 / 2022, A / Blue Winged Teal / Wyoming / AH0099021 / 2016, or a variant thereof.

7. A vector comprising the chimeric polynucleotide of claim 1.

8. 8. The vector of claim 7 which is a plasmid, cosmid, phage, virus, or a fragment of a virus.

9. A cell comprising the vector of claim 7.

10. A rescued influenza virus comprising a polynucleotide encoding M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2 derived from a non-pathogenic highly multiplying influenza A virus, a polynucleotide encoding an attenuated HA derived from a highly pathogenic avian influenza virus, and a polynucleotide encoding an N1, N2, N3, N4, N5, N6, N7, N8, or N9 NA.

11. The rescued influenza virus of claim 10, wherein the polynucleotide encoding the HA encodes an attenuated A / turkey / Indiana / 22-003707-003 / 2022 HA or a variant having 90% identity thereto.

12. 11. The rescued influenza virus of claim 10, wherein the polynucleotide encoding the NA is derived from A / turkey / Indiana / 22-003707-003 / 2022, A / Blue Winged Teal / Wyoming / AH0099021 / 2016, or a variant thereof.

13. 13. The rescued influenza virus of claim 12, wherein the polynucleotide encoding the NA is derived from A / turkey / Indiana / 22-003707-003 / 2022 or a variant thereof.

14. 13. The rescued influenza virus of claim 12, wherein the polynucleotide encoding the NA is derived from A / Blue Winged Teal / Wyoming / AH0099021 / 2016 or a variant thereof.

15. 11. The rescued influenza virus of claim 10, wherein the polynucleotides encoding M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2 influenza A are derived from PR8.

16. A vector comprising any one of the polynucleotides according to claim 10.

17. An immunogenic composition comprising an immunologically effective amount of the rescued influenza virus of claim 10.

18. 1. A method for producing influenza virus, comprising: introducing into a population of host cells a plurality of vectors comprising a polynucleotide corresponding to at least six internal segments of a non-pathogenic highly proliferating influenza virus, a chimeric polynucleotide comprising an HA non-coding sequence from a non-pathogenic highly proliferating influenza virus and a polynucleotide encoding an attenuated HA protein from a highly pathogenic avian influenza virus or variant thereof, and a chimeric polynucleotide comprising an NA non-coding sequence from a non-pathogenic highly proliferating influenza virus and a polynucleotide encoding an NA protein from a highly pathogenic avian influenza virus or variant thereof; Cultivating the population of host cells at a temperature of about 35° C. or less; and Harvesting the virus A method comprising:

19. The method of claim 18, wherein the non-pathogenic virus is PR8, the HA is from A / turkey / Indiana / 22-003707-003 / 2022 or a variant thereof, and the NA is N1, N2, N3, N4, N5, N6, N7, N8 or N9.

20. 19. The method of claim 18, wherein the NA is derived from A / turkey / Indiana / 22-003707-003 / 2022, A / Blue Winged Teal / Wyoming / AH0099021 / 2016, or a variant thereof.

21. 1. A method for producing influenza virus, comprising: introducing into a population of host cells a plurality of vectors, the plurality of vectors including polynucleotides corresponding to at least six internal segments of a non-pathogenic, highly multiplying influenza virus, a polynucleotide encoding an attenuated A / turkey / Indiana / 22-003707-003 / 2022 HA or variant thereof, and an N1, N2, N3, N4, N5, N6, N7, N8 or N9 NA polynucleotide; Cultivating the population of host cells at a temperature of 35° C. or less; and Harvesting the virus A method comprising:

22. 22. The method of claim 21, wherein the non-pathogenic virus is PR8.

23. 22. The method of claim 21 , wherein the NA polynucleotide is derived from A / turkey / Indiana / 22-003707-003 / 2022, A / Blue Winged Teal / Wyoming / AH0099021 / 2016, or a variant thereof.

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