Avian influenza vaccine and manufacturing method thereof

Abstract

The present disclosure relates to avian influenza vaccines produced by reverse genetics to prevent A / Turkey / Indiana / 22-like virus, methods of producing such vaccines, and methods of using such vaccines to protect poultry. The present disclosure also relates to methods of producing an influenza virus comprising a chimeric polynucleotide having an HA non-coding sequence from 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 chimeric polynucleotide comprising a polynucleotide having an NA non-coding sequence from a non-pathogenic high growth influenza virus and encoding an NA protein from a highly pathogenic avian influenza virus or a variant thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 328,911, filed on April 8, 2022. The entire contents of this provisional patent application are expressly incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing XML file as required by 37 CFR §1.831(a), which has been submitted in XML format through the US Patent and Trademark Office's patent electronic filing system, the entire contents of which are incorporated herein by reference. This XML file was created at March 22, 2023 , named Sequence_Listing_CONVERSION-005622.xml , the size is 9.74KB . Technical Field

[0005] The present disclosure relates to avian influenza viruses produced by reverse genetics and used as vaccines against highly pathogenic avian influenza (HPAI), methods of producing such viruses, and methods of using such vaccines to protect poultry. Background Art

[0006] Avian influenza, commonly known as "bird flu," has devastating consequences for the poultry industry, farmers' livelihoods, international trade, and the health of wild birds. To curb the spread of avian influenza, outbreaks often involve culling all poultry, both infected and healthy. This translates into severe economic losses and long-term impacts on farmers' livelihoods. Bird flu is also a major public health concern. Whenever the avian influenza virus circulates among poultry, sporadic cases of avian influenza are sometimes detected in humans.

[0007] As of March 23, 2022, the U.S. Animal and Plant Health Inspection Service (APHIS) reported 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 the Eurasian H5N1 virus. Wild birds can transmit avian influenza to domestic poultry. Wild birds can also become infected but may not show any symptoms. Wild birds have spread the virus to domestic poultry, including numerous free-range poultry flocks and commercial turkey, laying, and broiler chicken operations in multiple states. To date, more than 20 million poultry have died or been euthanized to control the spread of the disease.

[0008] Influenza A viruses are a genetically diverse group of pathogens that infect a variety of hosts, including birds, pigs, and humans. Over the past three decades, vaccines and vaccination have become essential tools for controlling influenza. Using these tools in poultry can increase resistance to infection, prevent illness and mortality, reduce viral replication and shedding, and decrease transmission to birds and mammals, including humans.

[0009] Low pathogenic avian influenza (LPAI) viruses typically cause few or no clinical signs in infected poultry. LPAI viruses are shed in the feces and respiratory secretions of infected birds. The virus is primarily transmitted through direct contact between healthy and infected birds. The virus can also be spread indirectly through contact with contaminated equipment and materials. LPAI strains occur naturally in wild migratory waterfowl and shorebirds and do not cause illness. Highly pathogenic avian influenza (HPAI) is a serious disease that requires a rapid response because it is highly contagious and lethal. HPAI infects poultry, such as chickens, turkeys, pheasants, quail, ducks, and geese, as well as wild birds, especially waterfowl. HPAI is spread between birds through direct contact or through contact with contaminated water, fertilizers, equipment, vehicles, crates, and on the clothing or shoes of people who have been exposed to the virus.

[0010] DIVA stands for Differentiation between Vaccinated and Infected Animals. In the case of avian influenza, this can be achieved by using vaccines based on strains different from the current field strains (such as H5N2) and by using serological tests that can differentiate between vaccine-induced antibodies (such as anti-N2 antibodies) and antibodies against the field virus (N1).

[0011] New influenza virus strains evolve rapidly and can spread between species, necessitating the continuous production of new vaccines. The constant emergence and re-emergence of different influenza strains necessitates a continuous need for new influenza vaccines. These vaccines are often made using antigenic portions of newly emerging viral strains. Therefore, peptides and polynucleotides (particularly antigenic gene sequences) from novel, newly emerging, or re-emerging viral strains are highly desirable. Summary of the Invention

[0012] Provided herein are influenza vaccine viruses produced by reverse genetics that provide protection against current H5N1 field strains and / or provide DIVA (distinguishing between vaccinated and infected animals) capabilities.

[0013] The present disclosure relates to avian influenza viruses produced by reverse genetics and used as vaccines against highly pathogenic avian influenza (HPAI), methods of producing such viruses, and methods of using such vaccines to protect poultry.

[0014] In one embodiment, the present disclosure relates to a chimeric polynucleotide comprising a non-pathogenic high-growth influenza virus hemagglutinin (HA) non-coding sequence and a polynucleotide encoding an HA protein from a highly pathogenic avian influenza virus or a variant thereof, and / or a non-pathogenic high-growth influenza virus neuraminidase (NA) non-coding sequence 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 HA protein variant has at least about 80% identity with 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 NA protein variant has at least about 80% identity with 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.

[0015] In one embodiment, the present disclosure relates to a rescued influenza virus comprising a polynucleotide encoding PR8 M1 / M2, NP, PB1, PA, PB2 and NS1 / NS2, encoding A / turkey / Indiana / 22-003707-003 / 2022 HA, and encoding at least one N1, N2, N3, N4, N5, N6, N7, N8 or N9 NA.

[0016] In one embodiment, the present disclosure relates to a method for producing influenza virus, the method comprising introducing into a host cell population 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 / 2022HA, and at least one polynucleotide encoding one of N1, N2, N3, N4, N5, N6, N7, N8, or N9; culturing the host cell population at a temperature of less than or equal to 35°C, and recovering the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Depicted is a schematic diagram of the influenza A virus, showing the virion shape, lipid bilayer, the location of the membrane-bound proteins HA, NA, M1, and M2; and the genome segments PB2, PB1, PA, HA, NP, NA, M, and NS within the virion.

[0018] Figure 2The eight gene segments present in influenza A virions are depicted. 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.

[0019] Description of Sequence Listing

[0020] The nucleotide sequences disclosed in this specification are listed in Table 1 below.

[0021] DETAILED DESCRIPTION

[0022] The present disclosure relates to avian influenza vaccines for the prevention of HPAI produced by reverse genetics, kits containing such vaccines, and methods of using such vaccines to protect poultry.

[0023] As used herein, the term "about" is defined as plus or minus 10% of the recited value. For example, about 1.0 g means 0.9 g to 1.1 g.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. 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.

[0025] As used herein, the singular forms "a," "an," and "the" include both the singular and the plural, unless the context clearly indicates otherwise.

[0026] The term "about" is intended to refer to a range that is substantially within the recited range without departing from the scope of the present disclosure. As used herein, "about" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If the use of the term in the context in which it is used is unclear to those of ordinary skill in the art, "about" means no more than plus or minus 10% of the recited value.

[0027] The terms "individual," "subject," and "animal" are used interchangeably herein to refer to a vertebrate that demonstrates infection with a negative-strand RNA virus, particularly influenza A virus.

[0028] Exemplary subjects may include vertebrates that are endangered and therefore of great importance to humans, vertebrates of economic significance (vertebrates raised on farms for human consumption), and / or vertebrates of social significance (e.g., animals kept as pets or in zoos). The methods and compositions of the present disclosure are particularly suitable for warm-blooded vertebrates, including but not limited to birds, canines, cetaceans, felines, mustelids, rodents, equines, bovines, swine, 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.

[0029] As used herein, the terms "isolated" and "purified" refer to material that is substantially or essentially free from components that normally accompany the material in its native state.

[0030] As used herein, "substantially free" means that when a standard detection method is used to detect a specific substance (such as unwanted nucleic acids, proteins, cells, viruses, infectious pathogens, etc.), the detection result is below the detection level of the substance.

[0031] As used herein, an "amino acid sequence" refers to a string of characters that represents a polymer of amino acid residues (eg, a protein, polypeptide, or polymer).

[0032] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to a single- or double-stranded deoxyribonucleotide or ribonucleotide polymer, chimera, or analog thereof.

[0033] As used herein, the terms "polynucleotide sequence" and "nucleic acid sequence" are used interchangeably to refer to a string of characters representing a polynucleotide or nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence disclosed herein optionally includes a complementary sequence in addition to the sequence explicitly indicated. Based on any specified polynucleotide sequence, a given nucleic acid or complementary polynucleotide sequence (e.g., a complementary nucleic acid) can be determined.

[0034] As used herein, the term "gene" broadly refers to any nucleic acid associated with a biological function. Thus, a gene includes a coding sequence and / or regulatory sequences required for its expression. The term "gene" applies to a specific genomic sequence, as well as the cDNA or mRNA encoded by that genomic sequence.

[0035] Genes also include non-expressed nucleic acid segments, such as those that form recognition sequences for other proteins. Non-expressed regulatory sequences include "promoters" and "enhancers," to which regulatory proteins (such as transcription factors) bind to induce transcription of adjacent or neighboring sequences. "Tissue-specific" promoters or enhancers are promoters or enhancers that regulate transcription in specific tissue types or cell types.

[0036] An "open reading frame" or "ORF" is a possible translational reading frame of a DNA or RNA (e.g., a gene) that is capable of being translated into a polypeptide. That is, the reading frame is not interrupted by a stop codon. However, it should be noted that the term ORF does not necessarily indicate that a polynucleotide is actually translated into a polypeptide.

[0037] As used herein, "gene expression" or "nucleic acid expression" refers to the transcription of DNA into RNA (optionally including modification of the RNA, such as splicing), the translation of RNA into polypeptides (possibly including subsequent modification of the polypeptides, such as post-translational modifications), or both transcription and translation as indicated by the context.

[0038] The term "vector" herein refers to a means by which nucleic acids can be propagated and / or transferred between organisms, cells, or cell components. Vectors include plasmids, viruses, phages, proviruses, phagemids, transposons, artificial chromosomes, and the like that replicate autonomously or can be integrated into the chromosome of a host cell. Vectors can also be non-autonomously replicating naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides consisting of DNA and RNA in the same chain, polylysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, and the like. In most (but not all) common embodiments, the vector of the present disclosure is a plasmid.

[0039] An "expression vector" is a vector, such as a plasmid, that promotes 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.

[0040] As used herein, the term "polypeptide" refers to a polymer (e.g., a peptide or protein) comprising two or more amino acid residues. The polypeptide may optionally include modifications such as glycosylation. The amino acid residues of the polypeptide may be natural or non-natural, and may be unsubstituted, unmodified, substituted, or modified.

[0041] As used herein, the term "DIVA" stands for differentiating vaccinated animals from infected animals. In the case of avian influenza, this can be achieved by using vaccines based on strains different from the current field strains (e.g., H5N2) and by using serological tests that can distinguish between vaccine-induced antibodies (e.g., antibodies to N2) and antibodies to the field virus (N1). Because unvaccinated animals die and do not develop a serological response, the DIVA vaccines of the present disclosure can be used to distinguish between vaccinated and infected animals and animals that have only been vaccinated.

[0042] As used herein, "recombinant virus" refers to a virus that has been manipulated in vitro, for example, using recombinant nucleic acid technology to introduce changes in the viral genome, or to a virus that has been artificially generated.

[0043] As used herein, the terms "recombinant nucleic acid," "recombinant fragment," and "recombinant polynucleotide" are used interchangeably to refer to a nucleic acid that has been altered in vitro. The sequence of a recombinant polynucleotide is not naturally occurring, does not correspond to a naturally occurring sequence, or is located differently from its position in the native genome.

[0044] A "pharmaceutical composition" refers to a composition containing the recombinant mutant influenza gene segments or recombinant influenza viruses disclosed herein, and suitable for administration to a subject. The pharmaceutical composition is useful for preventing, treating, alleviating, or ameliorating one or more influenza symptoms in a subject. For purposes of the present disclosure, pharmaceutical compositions include vaccines.

[0045] As used herein, "diluent," "excipient," "carrier," and "adjuvant" are used interchangeably to refer to a diluent, excipient, carrier, or adjuvant that is physiologically acceptable to a subject while retaining the therapeutic properties of the pharmaceutical composition with which it is administered. Physiologically acceptable diluents, excipients, carriers, or adjuvants and their formulations are known to those skilled in the art (see, for example, U.S. Patent No. 9,017,691; Chaudhari SP et al., 2012, "A Review of Pharmaceutical Excipients," IJAPBC, Vol. 1 (1)). Reed SG et al. (2013, "The Critical Role of Adjuvants in Modern Medicine," Nature Medicine, 19 (12): 1597-1608) reviewed adjuvants used in vaccines.

[0046] As used herein, the term "attenuated" used in conjunction with a virus refers to a virus that is less toxic or pathogenic than a non-attenuated counterpart, but still viable or live. Typically, attenuation renders an infectious pathogen (e.g., a virus) less harmful or virulent to an infected subject than a non-attenuated virus.

[0047] The terms "inoculation" and "vaccination" are used interchangeably herein to refer to the act of introducing the influenza virus vaccines of the present disclosure into poultry.

[0048] Orthomyxoviruses belong to the RNA virus family and include seven genera: influenza A virus, influenza B virus, influenza C virus, influenza D virus, infectious salmon anemia virus (Isavirus), Thogotovirus, and Quaranjavirus. Influenza A virus is one of the world's major uncontrollable pathogens, causing seasonal epidemics as well as global pandemics. Influenza A virus can infect a variety of vertebrate hosts, including birds and mammals. Vertebrate hosts of influenza include birds, canines, cetaceans, felines, mustelids, rodents, equines, bovines, swine, 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.

[0049] The influenza A virus genome is a negative-sense, single-stranded, segmented RNA. Influenza A subtypes are designated (HxNy) based on 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.

[0050] Influenza virus particles (also called virions) consist of a viral envelope surrounding a central core. The outer layer of the influenza virus particle is a lipid membrane taken from the host cell where the virus is propagating. Inserted into the lipid membrane are the hemagglutinin (HA) protein, the neuraminidase (NA) protein, and the matrix 2 (M2) protein. The four M2 proteins form a proton-selective ion channel, the unit of which is a helix stabilized by two disulfide bonds. Beneath the lipid membrane is a layer of matrix protein (M1), which forms the shell. Inside the virion are eight negative-strand viral RNA segments, composed of RNA linked to the nucleoprotein (NP) and three polymerase subunits: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). Nonstructural proteins 1 (NS1) and 2 (NS2) are present in infected cells but are not packaged within the virion.

[0051] The eight RNA segments of the influenza A virus genome are Figure 1As shown. Gene segment 1, also known as the PB2 gene segment, encodes the cap-binding transcriptase PB2. Gene segment 2, also known as the PB1 gene segment, encodes the elongation-associated proteins PB1, PB1-F2, and PB1-N40 using an alternative translation start site. Gene segment 3, also known as the PA gene segment, encodes the polymerase acidic proteins PA and PA-X via ribosomal frameshifting and PA-N155 and PA-N182 using an alternative translation site and N-terminal truncation. Gene segment 4, also known as the HA gene segment, encodes the hemagglutinin protein HA. Gene segment 5, also known as the NP gene segment, encodes the RNA-binding nuclear protein NP. Gene segment 6, also known as the NA gene segment, encodes the neuraminidase protein NA. Gene segment 7, also known as the matrix gene, encodes two proteins, M1 and M2. Gene segment 8, also known as the nonstructural gene, encodes the nonstructural protein NS1 and, through alternative RNA splicing, encodes NS3 and the nuclear export protein NS2 / NEP.

[0052] Avian influenza is caused by influenza A virus (influenza A). Avian influenza viruses are broadly classified based on the combination of two proteins on the surface of the influenza A virus: the hemagglutinin, or "H," protein, of which there are 16 types (H1-H16), and the neuraminidase, or "N," protein, of which there are nine types (N1-N9). The "H" and "N" proteins can occur in many different combinations. Each combination is considered a different subtype, and related viruses within a subtype are called a lineage. Avian influenza viruses are classified as either "low pathogenic" or "high pathogenic" based on their genetic characteristics and the severity of the disease they cause in chickens. Most viruses are low pathogenic, meaning they cause no or only mild clinical symptoms in poultry.

[0053] In the process of producing influenza seed virus by reverse genetics, tissue culture cells are transfected with cDNA clones derived from the apathogenic, high-growth A / Puerto Rico / 8 / 34 (PR8) backbone and donor antigenic proteins.

[0054] The inventors have prepared recombinant influenza viruses expressing modified hemagglutinin (HA) according to A / turkey / Indiana / 2022. The modified HA has changed in its cleavage site, so that the rescued virus has the phenotype of low pathogenicity HA. The disclosed recombinant influenza viruses express the neuraminidase (NA) of PR8 (N1), the NA of A / turkey / Indiana / 2022 (N1) or the NA of A / BlueWingedTeal / WY / AH0099021 / 2016 (N9). All three viruses have been confirmed by sequencing to have low pathogenicity cleavage sites and correct NA, and all grow at sufficiently high titers to become viable vaccine candidates. All of these vaccine variants are used as inactivated adjuvant vaccines.

[0055] As with human influenza vaccines, a close match between the influenza strain in the vaccine and the circulating virus is necessary for optimal protection. Several RG vaccines have been produced over the past 20 years for H5 influenza, but there are currently no commercial vaccines that are closely matched to the HPAI viruses currently circulating in the United States. The advantage of these proposed vaccines is that they will be closely matched to the field strains and should provide better protection than unmatched vaccines. Matching the hemagglutinin protein is crucial for protection, and matching the neuraminidase protein may provide some additional protection.

[0056] The inventors have prepared several vaccine viruses that contain attenuated hemagglutinin proteins that are homologous to field strains (belonging to antigenic clade 2.3.4.4b viruses) circulating in the 2022 North American H5N1 highly pathogenic avian influenza virus outbreak. This vaccine provides better protection than non-clade 2.3.4.4b vaccines (vaccines against antigenic clades 2.3.4.4a, 2.3.4.4c, 2.2, 2.3.2.1, and related H5 vaccine viruses).

[0057] The use of a heterologous neuraminidase protein is motivated by DIVA considerations. DIVA stands for "differentiation between vaccinated and infected animals," but for trade purposes, DIVA is often understood to mean differentiating between vaccinated, unexposed poultry and vaccinated, infected poultry. Currently, avian influenza vaccination has a significant trade impact, as countries are reluctant to import poultry or poultry products from countries that have vaccinated against avian influenza. A simple serological test that could reliably distinguish vaccinated from infected animals could potentially overcome the negative trade impact of avian influenza. The use of vaccines containing the N9 neuraminidase protein allows for serological monitoring of the N1 neuraminidase protein present in the wild. Previous studies have shown that the use of a heterologous neuraminidase protein only slightly reduces protection.

[0058] The construction of the three viruses reported herein uses a unique feature that has not been reported before. All influenza gene segments have protein coding regions with non-coding sequences flanking both ends. It is known that non-coding sequences are very important in regulating gene expression and packaging genes into viral particles. The non-coding region (NCR) has a small area of ​​highly conserved sequences, but also has a variability area. As part of the construction of the plasmid for RG rescue, different non-coding regions were used for the N9 neuraminidase gene. These included sequences found in the original virus, as well as coding sequences identical to the non-coding regions of the PR8 virus. The inventors hypothesized that if the virus had a PR8 non-coding region that was compatible with the rest of the virus, the virus would replicate better. This change resulted in an increase of almost two logarithmic units in the titer of the natural NCR to the PR8 NCR. This effectively transformed viruses that grew poorly and were not sufficient to be used as commercial vaccine seed strains into viruses that could be used as excellent seed strains. Previously, the use of the PR8 NCR had not been found to be an important factor affecting viral titer.

[0059] The vaccine virus is prepared by combining an attenuated chimeric construct of the HA coding sequence from a prevalent field strain and a non-coding sequence from the PR8 virus, or a chimeric construct of the NA coding sequence from a prevalent field strain and a non-coding sequence from the PR8 virus. The use of the chimeric construct increases the expression of the hemagglutinin and neuraminidase proteins, thereby increasing the titer of the rescued virus. Better growth in chicken embryos makes the vaccine more economical to produce, thereby reducing the production cost of the vaccine.

[0060] The DIVA principle has also been discussed for years but has not been routinely used for avian influenza, partly due to the lack of a convenient companion diagnostic test. Published studies support the heterologous NADIVA strategy as a viable option (Avellaneda, G. et al., 2010, "A heterologous neuraminidase subtype strategy for differentiating vaccinated from infected animals using a more flexible neuraminidase inhibition assay for avian influenza viruses (DIVA strategy)," 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). The vaccine virus is prepared to contain a neuraminidase gene that is heterologous to the field strain. The inclusion of a neuraminidase gene of a different subtype from the field virus allows serological testing to distinguish between vaccinated and uninfected birds and vaccinated and subsequently infected birds. Complete serological surveillance requires easy-to-use and cost-effective companion diagnostic tests.

[0061] In one embodiment, the present disclosure relates to a chimeric polynucleotide comprising a hemagglutinin (HA) non-coding sequence from a non-pathogenic high-growth influenza virus and a polynucleotide encoding an attenuated HA protein from a highly pathogenic avian influenza virus or a variant thereof, or a chimeric polynucleotide comprising a neuraminidase (NA) non-coding sequence from 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 non-coding region in the chimeric polynucleotide is from A / PuertoRico / 8 / 34 (PR8). In some embodiments of the present disclosure, the HA protein or variant thereof encoded by the chimeric polynucleotide of the present disclosure is derived from a highly pathogenic avian influenza virus. In some embodiments of the present disclosure, the NA protein or variant thereof encoded by the chimeric polynucleotide of the present disclosure is derived from a highly pathogenic avian influenza virus. In some embodiments of the present disclosure, the HA protein or variant thereof encoded by the chimeric polynucleotide of the present disclosure is mutated to include an attenuated cleavage site. In some embodiments of the present disclosure, the HA protein or variant thereof is from A / turkey / Indiana / 22-003707-003 / 2022. In some embodiments of the present disclosure, the NA protein or variant thereof encoded by the chimeric polynucleotide is N1, N2, N3, N4, N5, N6, N7, N8 or N9. In some embodiments of the present disclosure, the NA protein or variant thereof encoded by the chimeric polynucleotide is from A / turkey / Indiana / 22-003707-003 / 2022. In some embodiments of the present disclosure, the NA protein or variant thereof encoded by the chimeric polynucleotide is from A / Blue WingedTeal / Wyoming / AH0099021 / 2016.

[0062] In some embodiments, the present disclosure relates to a polynucleotide encoding a variant attenuated HA protein from A / turkey / Indiana / 22-003707-003 / 2022. The hemagglutinin (HA) or hemagglutinin glycoprotein of the influenza virus is a homotrimeric integral membrane protein present on the surface of the influenza virus. The HA protein has multifunctional activity as an attachment factor and a membrane fusion protein. The polynucleotide encoding the HA protein variant may have less than 100% identity with the polynucleotide encoding the attenuated A / turkey / Indiana / 22-003707-003 / 2022 HA protein, but may retain the multifunctional activity of the HA protein. For example, the 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 shown in SEQ ID NO:3, while retaining its multifunctional activity as an attachment factor and a membrane fusion protein.

[0063] In some embodiments, the present disclosure relates to a polynucleotide encoding an NA protein or a variant thereof from a highly pathogenic avian influenza virus. Influenza neuraminidase (NA) is present on the surface of virions and plays an important role in the release and spread of progeny virions from infected cells. The polynucleotide encoding the NA protein variant may have less than 100% identity with the polynucleotide encoding the A / turkey / Indiana / 22-003707-003 / 2022NA protein or the A / Blue Winged Teal / Wyoming / AH0099021 / 2016NA 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 shown in SEQ ID NO:4 or SEQ ID NO:5, while retaining its virus release and transmission activity.

[0064] In one embodiment, the present disclosure relates to a rescued influenza virus comprising polynucleotides encoding influenza A M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2 from a non-pathogenic high-growth influenza virus, polynucleotides encoding HA from A / turkey / Indiana / 22-003707-003 / 2022, modified to include a low pathogenicity cleavage site, and polynucleotides encoding N1, N2, N3, N4, N5, N6, N7, N8, or N9 NA. In some embodiments of the present disclosure, the polynucleotides encoding NA in the rescued influenza virus are from A / turkey / Indiana / 22-003707-003 / 2022 or A / BlueWingedTeal / Wyoming / AH0099021 / 2016. In some embodiments of the present disclosure, the polynucleotides encoding influenza A M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2 in the rescued influenza virus are from PR8.

[0065] In one embodiment, the present disclosure relates to a vector comprising a chimeric polynucleotide of the present disclosure. In some embodiments of the present disclosure, the vector is a plasmid, a cosmid, a phage, a virus or a viral fragment. In one embodiment, the present disclosure relates to a rescue influenza virus, comprising coding for influenza A M1 / M2, NP, PB1, PA, PB2 and NS1 / NS2 from a pathogenic high-growth influenza virus, coding for a polynucleotide of HA from A / turkey / Indiana / 22-003707-003 / 2022, modified to include a low pathogenicity cleavage site, and coding for a polynucleotide of N1, N2, N3, N4, N5, N6, N7, N8 or N9 NA. In some embodiments of the present disclosure, the polynucleotide encoding NA in the rescue influenza virus is from A / turkey / Indiana / 22-003707-003 / 2022 or A / Blue Winged Teal / Wyoming / AH0099021 / 2016. In some embodiments of the present disclosure, the polynucleotides encoding influenza A M1 / M2, NP, PB1, PA, PB2, and NS1 / NS2 in the rescued influenza virus are from PR8. In one embodiment, the present disclosure relates to an immunogenic composition comprising an immunogenic effective amount of the rescued influenza virus of the present disclosure.

[0066] In one embodiment, the present disclosure relates to a method for producing influenza virus, comprising introducing into a host cell population a plurality of vectors comprising polynucleotides corresponding to at least six internal segments of a non-pathogenic, high-growth influenza virus, chimeric polynucleotides comprising a non-coding sequence from 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 a chimeric polynucleotide comprising a non-coding sequence from 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; culturing the host cell population at a temperature of less than or equal to 35° C., and recovering the virus. In some embodiments of the present disclosure, influenza virus is produced using PR8 as the non-pathogenic virus with HA from A / turkey / Indiana / 22-003707-003 / 2022 or a variant thereof, and NA is N1, N2, N3, N4, N5, N6, N7, N8, or N9.

[0067] In one embodiment, the present disclosure relates to a method for producing influenza virus, the method comprising introducing into a host cell population a plurality of vectors comprising polynucleotides corresponding to at least six internal segments of a non-pathogenic high-growth influenza virus, a polynucleotide encoding A / turkey / Indiana / 22-003707-003 / 2022 HA or a variant thereof, and an NA polynucleotide of N1, N2, N3, N4, N5, N6, N7, N8 or N9; culturing the host cell population at a temperature of less than or equal to 35°C, and recovering the virus. In some embodiments of the present disclosure, the non-pathogenic virus in the method for producing influenza virus is PR8. In some embodiments of the present disclosure, the NA polynucleotide in the method for producing influenza virus is from A / turkey / Indiana / 22-003707-003 / 2022 or A / Blue Winged Teal / Wyoming / AH0099021 / 2016.

[0068] Embodiments of the present disclosure are shown and described herein. It will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present disclosure, those skilled in the art will make many changes, modifications, and substitutions. In practicing the present disclosure, various alternatives to the embodiments of the present disclosure described herein may be adopted. The claims included are intended to limit the scope of the present disclosure, and methods and structures within the scope of these claims and their equivalents are encompassed in the present disclosure. 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 were specifically and individually indicated as being incorporated by reference.

[0069] Example

[0070] Having generally described the present disclosure, it may be better understood by reference to certain specific examples which are included herein to further illustrate the present disclosure and are not intended to limit the scope of the present disclosure which is defined by the claims.

[0071] Example 1

[0072] Preparation of viruses and vaccines

[0073] Using reverse genetics, viruses comprising polynucleotides encoding HA and / or NA of A / turkey / Indiana / 22-003707-003 / 2022 virus, or NA of PR8 virus, N3 virus, N4 virus, or N9 virus were assembled.

[0074] The coding sequence of the A / turkey / Indiana / 22-003707-003 / 2022 (TK / IN / 22) virus was obtained from GISAID (Global Initiative on Sharing Influenza Data). The hemagglutinin (HA) gene sequence was altered to change the highly pathogenic cleavage site to a less pathogenic cleavage site. The HA coding sequence was modified to change the cleavage site from REKRRKR / GLF (as shown in SEQ ID NO: 1) to RETR / GLF (as shown in SEQ ID NO: 2), which included the deletion of three basic amino acids and the substitution of lysine with threonine at the -2 position of the cleavage site. The gene sequence was additionally modified to include non-coding gene sequences that are not included in the publicly available sequences. The sequence was synthesized de novo by IDT DNA and includes non-coding and coding sequences as well as additional non-influenza sequences (DNA assembly sites) to facilitate insertion of the sequence into a plasmid. The N1 neuraminidase gene sequence from A / turkey / Indiana / 22-003707-003 / 2022 was also synthesized de novo using coding, noncoding, and non-influenza sequences. The N9 neuraminidase gene from A / Blue Winged Teal / Wyoming / AH0099021 / 2016 was also synthesized de novo using coding, noncoding, 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.

[0075] Using DNA assembly enzymes, the sequence of each individual gene is inserted into a plasmid containing an RNA polymerase promoter and a transcription promoter. The HA and NA influenza coding segments are inserted between the PR8 HA and NA non-coding regions, and the PR8 internal genes are the same as normal. Using this reverse genetics plasmid, viral RNA can be transcribed as well as mRNA for expressing viral proteins required for viral packaging. In addition to the hemagglutinin (HA) gene, the neuraminidase (NA) gene and 6 other viral gene segments are also required for rescue. The 6 internal genes are derived from the PR8 vaccine virus. The neuraminidase gene is obtained from A / turkey / Indiana / 22-003707-003 / 2022, PR8 virus, or N3, N4 or N9 influenza virus.

[0076] Polynucleotides encoding the other six essential genes of the PR8 vaccine strain (PB2, PB1, PA, NP, M1 / M2, and NS) are introduced into a plasmid carrying the HA coding sequence and a neuraminidase (NA) gene plasmid and transfected into cell culture to rescue the live vaccine virus. A total of eight genes are required to rescue the virus.

[0077] The eight gene fragments were transfected into 293T cells using the commercially available TransIT-LT1 transfection reagent. Two days later, the cell supernatant was inoculated into embryonated chicken eggs. Five days later, the allantoic fluid was assayed for hemagglutination activity, which provided evidence of viral rescue.

[0078] The rescued viruses were sequenced to ensure the presence of the low pathogenicity cleavage site. To confirm that the rescued viruses were low pathogenicity, they were grown in cell culture with or without trypsin. Low pathogenicity viruses require trypsin for growth in cell culture.

[0079] Low pathogenicity viruses were titrated, and three viruses were selected for further study. The viruses selected for further study were: 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. For the original noncoding region, A / turkey / Indiana / 22-003707-003 / 2022 (HA-LPAI), A / Blue Winged Teal / Wyoming / AH0099021 / 2016 (N9), PR / 8 (MA, NS, PA, PB1, PB2, and NP) showed a value of 10^7.6 EID50 / mL. For the PR8 coding region, A / turkey / Indiana / 22-003707-003 / 2022 (HA-LPAI), A / Blue Winged Teal / Wyoming / AH0099021 / 2016 (N9), PR / 8 (MA, NS, PA, PB1, PB2, and NP) showed a value of 10^9.5 EID50 / mL.

[0080] Two candidate viruses were selected, one was an H5N1 virus comprising a polynucleotide encoding A / turkey / Indiana / 22-003707-003 / 2022 NA and attenuated HA, and the other was an H5N9 virus comprising a polynucleotide encoding A / turkey / Indiana / 22-003707-003 / 2022 attenuated HA and A / Blue Winged Teal / Wyoming / AH0099021 / 2016 NA(N9).

[0081] This example shows the rescue of candidate viruses comprising attenuated A / turkey / Indiana / 22-003707-003 / 2022 HA and NA, and candidate viruses comprising attenuated A / turkey / Indiana / 22-003707-003 / 2022 HA and A / Blue Winged Teal / Wyoming / AH0099021 / 2016 NA having a PR3 noncoding region or an A / Blue Winged Teal / Wyoming / AH0099021 / 2016 noncoding region.

Claims

1. A chimeric polynucleotide comprising: A hemagglutinin (HA) non-coding sequence from a non-pathogenic high growth influenza virus and a polynucleotide encoding the HA protein from a highly pathogenic avian influenza virus or a variant thereof, or Neuraminidase (NA) non-coding sequences from non-pathogenic high-growth influenza virus and polynucleotides encoding NA protein from highly pathogenic avian influenza virus or variants thereof.

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

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

4. The chimeric polynucleotide of claim 3, wherein the polynucleotide encoding the 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. The chimeric polynucleotide of claim 1, wherein the NA protein is from A / turkey / Indiana / 22-003707-003 / 2022, A / Blue Winged Teal / Wyoming / AH0099021 / 2016, or a variant thereof. A vector comprising the chimeric polynucleotide according to claim 1 .

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

9. A cell comprising the vector according to claim 7.

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

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

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

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

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

15. The rescued influenza virus of claim 10, wherein the polynucleotides encoding M1 / M2, NP, PB1, PA, PB2 and NS1 / NS2 are from PR8. 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. A method for producing influenza virus, the method comprising introducing into a host cell population 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 an HA non-coding sequence from a non-pathogenic high growth influenza virus and a polynucleotide encoding an attenuated HA protein from a highly pathogenic avian influenza virus or a variant thereof, and a chimeric polynucleotide comprising an NA non-coding sequence from 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; culturing the host cell population at a temperature of less than or equal to about 35°C, and recovering the virus.

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

21. A method for producing influenza virus, the method comprising introducing into a host cell population a plurality of vectors, the vectors comprising polynucleotides corresponding to at least six internal segments of a non-pathogenic high-growth influenza virus, polynucleotides encoding attenuated A / turkey / Indiana / 22-003707-003 / 2022HA or a variant thereof, and NA polynucleotides of N1, N2, N3, N4, N5, N6, N7, N8 or N9; culturing the host cell population at a temperature less than or equal to 35°C, and recovering the virus.

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

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

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