Chimeric virus expressing avian influenza antigen and vaccine composition comprising same

Abstract

The present specification relates to a chimeric virus expressing an avian influenza antigen and a vaccine composition comprising same.

Description

Chimeric virus expressing avian influenza antigen and vaccine composition comprising the same

[0001] The present disclosure relates to a chimeric virus expressing an avian influenza antigen and a vaccine composition comprising the same, and more particularly, to a chimeric virus produced by inserting an antigen gene of an avian influenza virus using CRISPR gene editing technology and a vaccine composition comprising the same.

[0002] Avian influenzavirus (AIV) is a causative agent classified as an Influenza A virus species in the Orthomyxoviridae family, Alphainfluenzavirus genus, and enveloped eight-segmented negative-sense single-stranded RNA [(-)ssRNA] genome. Among the major structural proteins of the virus, hemaglutinin (HA) and neuraminidase (NA) are divided into 16 HA and 9 NA subtypes, respectively. These contribute to immunogenicity and antigenicity, such as the formation of neutralizing antibodies and anti-hemagglutination antibodies, and are used for genotype identification. In addition, AIV pathogenic types are divided into low pathogenicity and highly pathogenic avian influenza based on pathogenicity in chickens. Among these, highly pathogenic avian influenza causes neurological symptoms and high mortality in chickens and birds, spreads very quickly, and is a disease that occurs widely worldwide.

[0003] Among low-pathogenic avian influenza strains, H9N2 has shown continuous mutation. Since the first report of H9N2 LPAIV of the Y280 lineage in China in the 1990s, it has circulated in countries bordering China (including Vietnam, Cambodia, and Myanmar), and recently in Japan and eastern Russia, which do not border China. In Korea, H9N2 LPAIV of the Y439 lineage was dominant since the first report in 1996, but the occurrence of H9N2 in farms decreased with the introduction of the H9N2 inactivated vaccine in 2007. However, in July and December 2020, a total of nine new cases of H9N2 LPAIV of the Y280 lineage occurred, and in 2021, H9N2 LPAIV of the Y280 lineage was confirmed in the Jeonnam / Gyeongbuk regions in domestic LBM. Analysis of the relatedness determined that it originated from China, and it was confirmed that it spread domestically after its initial introduction. Accordingly, the development of a vaccine that can control the new Y280 is necessary.

[0004] Recently used live vaccines have several problems. Commercialized vaccines still have some pathogenicity and, in some cases, can cause adverse reactions. Furthermore, existing vaccine development methods designed to overcome the rapidly changing genetic mutations of each pathogen require complex manufacturing processes and significant time and labor. A vaccine production system capable of responding to pathogen antigenic mutations while maintaining high immune response and safety is needed. Furthermore, a vaccine capable of distinguishing between field and vaccine strains, a rapid and simple manufacturing process, and a recombinant vaccine production system capable of simultaneously expressing multiple genes are required.

[0005]

[0006] An example of the present disclosure provides a chimeric virus comprising a heterologous virus-derived protein or a gene encoding said protein.

[0007] Specifically, the chimeric virus comprises a protein derived from the heterologous virus or a gene encoding the protein,

[0008] It may include an HA protein derived from an avian influenza virus or a gene encoding the HA protein.

[0009] Another example provides a vaccine composition comprising the chimeric virus.

[0010] Another example provides a method of immunizing against avian influenza virus, comprising administering the vaccine composition to a subject in need of immunization against avian influenza.

[0011] Another example provides a composition for preventing, ameliorating, or treating avian influenza, comprising the chimeric virus or vaccine composition.

[0012] Another example provides a method for preventing, ameliorating, or treating avian influenza, comprising administering the vaccine composition to a subject in need of preventing, ameliorating, or treating avian influenza.

[0013] The chimeric virus of the present invention may be a recombinant turkey herpesvirus comprising one or more heterologous antigens (proteins or peptides) and / or genes encoding said antigens.

[0014] In other words, the chimeric virus provided herein may be a recombinant turkey herpes virus in which one or more foreign antigens (proteins or peptides) or genes encoding said antigens are introduced into the turkey herpes virus (host cell (virus)).

[0015] More specifically, the chimeric virus

[0016] It may be a recombinant turkey herpesvirus comprising an HA protein derived from an avian influenza virus or a gene encoding the HA protein.

[0017] The HA protein derived from the above avian influenza virus may include, but is not limited to, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having a sequence homology of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to the amino acid sequence of SEQ ID NO: 1. The gene encoding the above HA protein may include, but is not limited to, the base sequence of SEQ ID NO: 2 or a base sequence having a sequence homology of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to the base sequence of SEQ ID NO: 2.

[0018] The above chimeric virus can be used as a vaccine strain against avian influenza virus.

[0019] Another example provides a vaccine composition comprising the chimeric virus. The vaccine composition may have an immunizing activity (e.g., antibody formation, etc.) against avian influenza virus.

[0020] Another example is

[0021] A set of recombinant vectors is provided, comprising a recombinant vector comprising a gene encoding an HA protein derived from an avian influenza virus.

[0022] The above recombinant vector set can be used for the production of the chimeric virus described above or a vaccine composition containing the same.

[0023]

[0024] Another example provides a method for producing a chimeric virus and / or a vaccine composition comprising the same, comprising the step of inserting (introducing) a gene encoding an HA protein derived from an avian influenza virus into a turkey herpes virus (more specifically, a turkey herpes virus genome).

[0025] The above chimeric virus and / or vaccine composition may have an immunizing activity (e.g., antibody formation, etc.) and / or a protective activity (e.g., prevention, improvement, and / or treatment activity of a disease (infection) caused by infection with the above virus, etc.) against the avian influenza virus.

[0026] Accordingly, another example provides a method of immunizing against avian influenza virus, comprising administering the chimeric virus or vaccine composition to a subject in need of immunization (e.g., antibody formation) against the avian influenza virus.

[0027] Another example provides a composition for preventing, ameliorating or treating avian influenza comprising the chimeric virus or vaccine composition.

[0028] Another example provides a method for preventing, ameliorating or treating avian influenza, comprising administering the chimeric virus or vaccine composition to a subject in need of preventing, ameliorating or treating avian influenza.

[0029]

[0030] Hereinafter, the present invention will be described in more detail.

[0031]

[0032] chimeric virus

[0033] An example of the present disclosure provides a chimeric virus comprising an HA protein derived from an avian influenza virus or a gene encoding the HA protein. Specifically, the chimeric virus may be a chimeric turkey herpes virus in which the gene encoding the HA protein is inserted into the genome of a turkey herpes virus.

[0034] As used herein, "full-length gene" refers to the entirety of the DNA bases that store the genetic information of a species. For example, the turkey herpes virus full-length gene refers to the entirety of the DNA bases that store the genetic information of the turkey herpes virus. The turkey herpes virus may have Genbank Accession No. NC_002641.1, AF282130.1, or AF282130.1, but is not limited thereto.

[0035] As used herein, the expressions “comprising (a given ingredient)” or “consisting of (a given ingredient)” mean essentially including the described ingredient, and may not exclude the inclusion of additional and / or auxiliary ingredients as long as they exhibit an effect equivalent to the desired effect.

[0036] In this specification, the expression "a nucleic acid molecule (which may be used interchangeably with a "gene") or a polypeptide (which may be used interchangeably with a "protein") "comprises a specific nucleic acid sequence or amino acid sequence, consists of a specific nucleic acid sequence or amino acid sequence, or is expressed by a specific nucleic acid sequence or amino acid sequence" is an equivalent and interchangeable expression, which may mean that the nucleic acid molecule or polypeptide essentially includes the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including (or not excluding) a "substantially equivalent sequence" in which a mutation (deletion, substitution, modification, and / or addition) is added to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or the desired function of the nucleic acid molecule or polypeptide is maintained.

[0037] As used herein, “avian influenza” means H9N2, H5N1, H5N6, HlNl, H1N2, H1N3, H1N6, H1N9, H2N1, H2N2, H2N3, H2N5, H2N7, H2N8, H2N9, H3N1, H3N2, H3N3, H3N4, H3N5, H3N6, H3N8, H3N9, H4N1, H4N2, H4N3, H4N4, H4N5, H4N6, H4N8, H4N9, H5N2, H5N3, H5N4, H5N7, H5N8, H5N9, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N8, H6N9, It may be, but is not limited to, H7N1, H7N2, H7N3, H7N4, H7N5, H7N7, H7N8, H7N9, H8N4, H8N5, H9N1, H9N3, H9N5, H9N6, H9N7, H9N8, H9N9, H10N4, H10N5, H10N7, H10N8, H10N9, Hl1Nl, Hl1N13, Hl1N2, Hl1N4, Hl1N6, Hl1N8, Hl1N9, H12N1, H12N4, H12N5, H12N8, H13N2, H13N3, H13N6, H13N7, H14N5, H14N6, H15N8, H15N9 and / or H16N3. no.

[0038] The chimeric virus (more specifically, the virus that becomes the host cell into which the foreign antigen (protein and / or gene) is introduced) may be, but is not limited to, turkey herpesvirus, canarypox virus, bovine viral diarrhea virus, adenovirus (e.g., adenovirus 5), vaccinia virus, feline parvovirus, flaviviruses, vesicular stomatitis virus, lentivirus (e.g., integrase-defective lentiviral vectors), paramyxovirus, or fowlpox virus.

[0039] The above chimeric virus can be produced by simultaneously inserting a gene encoding the HA protein and a luminescent gene, but is not limited thereto.

[0040] The gene encoding the above HA protein is U of turkey herpes virus L 2 Unique long region and / or U S 5 can be included (inserted) in the region (Unique short region), and in one example, the gene encoding the HA protein is included in the U of the turkey herpes virus. L 2 Part and / or U S 5The gene encoding the HA protein is included in the region, or is located at the 140100th to 140300th, 140100th to 140250th, 140100th to 140240th, 140100th to 140235th, 140150th to 140300th, 140150th to 140250th, 140150th to 140240th, 140150th to 140235th, 140200th to 140300th, 140200th to 140250th, 140200th to 140240th, 140200th to It may be included at the 140235th, 140230th to 140300th, 140230th to 140250th, 140230th to 140240th or 140230th to 140235th nucleotide, for example, but is not limited thereto, at the position following the 140231st nucleotide.

[0041] The above chimeric virus has a gene encoding the HA protein that is a U of the turkey herpes virus. L 2 Unique long region and / or U S 5 It can be manufactured by inserting into the unique short region, and specifically, the gene encoding the HA protein is U of the turkey herpes virus. L 2 Part and / or U S 5 It can be manufactured by inserting into the part, but is not limited thereto.

[0042] The above chimeric virus has a gene encoding the HA protein at positions 5' from the full-length gene of turkey herpes at positions 140100 to 140300, 140100 to 140250, 140100 to 140240, 140100 to 140235, 140150 to 140300, 140150 to 140250, 140150 to 140240, 140150 to 140235, 140200 to 140300, 140200 to 140250, 140200 to 140240, 140200 to It can be prepared by inserting at the position following the 140235th, 140230th to 140300th, 140230th to 140250th, 140230th to 140240th or 140230th to 140235th nucleotide, for example, the 140231st nucleotide, but is not limited thereto.

[0043] U of the above turkey herpes virus L 2 The portion may include, but is not limited to, the base sequence of SEQ ID NO: 18 or a base sequence having a sequence identity of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with the base sequence.

[0044] U of the above turkey herpes virus S 5 The portion may include, but is not limited to, the base sequence of SEQ ID NO: 9 or a base sequence having a sequence identity of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with the base sequence.

[0045] The above chimeric virus may be a virus deposited at the Biological Resource Center on June 27, 2023 and having the accession number KCTC 15489BP.

[0046]

[0047] Immunization uses of chimeric viruses

[0048] The chimeric virus described above can be used as a vaccine strain against avian influenza viruses. Furthermore, the chimeric virus can have immunogenic activity (e.g., antibody formation, etc.) and / or protective activity (e.g., preventive, ameliorating, and / or therapeutic activity against diseases (infections) caused by infection with the virus, etc.) against avian influenza viruses.

[0049] Accordingly, another example provides a vaccine composition comprising the chimeric virus. The vaccine composition may have an immunizing activity (e.g., antibody formation, etc.) against avian influenza virus.

[0050] Another example provides a method of immunizing against avian influenza virus, comprising administering the chimeric virus or vaccine composition to a subject in need of immunization (e.g., antibody formation) against the avian influenza virus.

[0051] Another example provides the use of the chimeric virus as a vaccine against avian influenza virus or for the manufacture of such a vaccine.

[0052] Another example provides a composition for preventing, ameliorating or treating avian influenza comprising the chimeric virus or vaccine composition.

[0053] Another example provides a method for preventing, ameliorating or treating avian influenza, comprising administering the chimeric virus or vaccine composition to a subject in need of preventing, ameliorating or treating avian influenza.

[0054] Another example provides a use of the chimeric virus or vaccine composition for preventing, ameliorating or treating avian influenza or for the manufacture of a composition for preventing, ameliorating or treating said composition.

[0055] In this specification, “vaccine” means a biological preparation containing an antigen that induces immunity in a living organism, and refers to an immunogen or antigenic substance that induces immunity in a living organism by administering it to a human or animal to prevent infection.

[0056] The above vaccine composition may be, but is not limited to, a live vaccine, an inactivated vaccine, a subunit vaccine, a vector vaccine, a chimeric vaccine, or a DNA vaccine.

[0057] The above vaccine composition may additionally include, but is not limited to, one or more selected from the group consisting of an antigen adjuvant, an efficacy enhancer, a preservative, a buffer, a surfactant, a carrier, an osmotic agent, an antioxidant, and a stabilizer.

[0058] As used herein, “adjuvant” means a substance used to quantitatively or qualitatively change the immune response to an administered antigen.

[0059] As used herein, "adjuvant (or potentiator or immunopotentiator)" refers to a substance used to increase the immunogenicity of the immunogenic composition of the present invention. Such adjuvants are often provided to enhance immune responses, and this is well known to those skilled in the art.

[0060] In the present invention, "preservative (or preservative)" means a substance having an anti-viral and / or antibacterial action that inhibits the growth of microorganisms in the vaccine composition, and may be, for example, one or more (one, two, or three) selected from the group consisting of thimerosal, phenoxyethanol (2-phenoxyethanol), formaldehyde, etc., but is not limited thereto, and all conventional preservatives used in the art may be used.

[0061] Additionally, the vaccine composition may include one or more physiologically acceptable buffers. For example, when the vaccine composition is an infusion or injection, the buffer may have a buffering capacity at a pH of 4.0 to 10.0, specifically, at a pH of 5.0 to 9.0, and more specifically, at a pH of 6.0 to 8.0. The buffer may be at least one selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, and triethanolamine buffers.

[0062] In particular, when the vaccine composition of the present invention is intended for parenteral administration, the buffer may be selected from buffers suitable for the USP. For example, the buffer may be at least one selected from the group consisting of monobasic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, and lactic acid; dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; polybasic acids such as citric acid and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

[0063] In addition, the vaccine composition provided herein may further comprise a non-ionic surfactant. Examples of non-ionic surfactants that may be further comprised include polyoxyethylene sorbitan ester compounds (Polysorbate 20 and Polysorbate 80), copolymers of ethylene oxide (EO), propylene oxide (PO), and butylene oxide (BO) (e.g., DOWFAX™); oxtoxynols having different numbers of repeating ethoxy (oxy-1,2-ethanediyl) groups, particularly ostoxynol-9 (Triton-100); ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as lecithin; nonylphenol ethoxylates such as the NP series; polyoxyethylene fatty acid ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols (Brij surfactants), particularly triethylene glycol monolauryl ether (Brij 30); Sorbitan ethers known as SPANs, particularly one or more selected from the group consisting of sorbitan trioleate (Span 85) and sorbitan monolaurate, but are not limited thereto. Specifically, polyoxyethylene sorbitan ester compounds (polysorbate 20 and polysorbate 80) may be present in an amount of from 0.001 % (w / v) to 20 % (w / v), for example from 0.001 % (w / v) to 10 % (w / v), especially from 0.001 % (w / v) to 1 % (w / v) or about 0.5 % (w / v), but are not limited thereto.

[0064] Physiologically acceptable carriers used in liquid formulations include aqueous or non-aqueous solvents, suspensions, emulsions, and oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and ethyl oleate. Aqueous carriers include water, alcohol / aqueous solvents, emulsions or suspensions, saline, and buffered solutions. Examples of oils include vegetable or animal oils, peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, synthetic oils such as marine oil, and lipids derived from milk or eggs. The vaccine composition of the present invention may be isotonic, hypertonic, or hypotonic. For pharmaceutical compositions administered by infusion or injection, isotonicity is generally preferred, but is not limited thereto. Meanwhile, isotonicity or hypertonicity may be advantageous for the storage of the composition. If the vaccine composition is hypertonic, it may be diluted to make it isotonic before administration. The isotonic agent for dilution may be an ionic isotonic agent, such as a salt, or a non-ionic isotonic agent, such as a carbohydrate. Ionic tonicity agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, and magnesium chloride. Nonionic tonicity agents include, but are not limited to, sorbitol and glycerol.

[0065] The vaccine compositions provided herein may include antioxidants. Exemplary reducing agents include mercaptopropionyl glycine, N-acetylcysteine, β-mercaptoethylamine, glutathione, ascorbic acid and its salts, sulfites, or sodium metabisulfite, or similar compounds. Additionally, antioxidants may include, but are not limited to, natural antioxidants such as vitamins E and C, lecithin, xanthine, and beta-carotene, as well as substances such as zinc and selenium.

[0066] The vaccine compositions provided herein may include stabilizers, wetting agents, emulsifiers, dispersants, and additional substances such as monosaccharides and polysaccharides. They may also include immunostimulatory molecules to enhance vaccine efficacy. These molecules may enhance the immune response, induce inflammation, or be specific lymphocytes or cytokines. Cytokines may include, but are not limited to, interleukin (IL)-1, IL-2, IL-3, IL4, IL-12, IL-13, granulocyte-macrophage colony-stimulating factor (GMCSF), and macrophage inflammatory factor.

[0067] The above vaccine composition may be administered by one or more routes selected from the group consisting of, but not limited to, in ovo, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival, and subcutaneous.

[0068] The vaccine composition according to the present disclosure may be administered in a pharmaceutically effective amount to a subject in need of prevention of infection with an avian influenza virus and / or development of a disease and / or symptom due to an avian influenza virus infection (e.g., a subject at risk of infection with an avian influenza virus and / or development of a disease / symptom due to an avian influenza virus infection) for the purpose of preventing a disease and / or symptom due to an avian influenza virus infection.

[0069] As used herein, the term "prevention" may refer to any action that inhibits or delays infection by the avian influenza virus and / or the occurrence of diseases / symptoms caused by avian influenza virus infection by administration of the vaccine composition of the present invention. The term "pharmaceutically effective amount" may refer to the dosage required to induce antibodies to a degree that can significantly reduce the probability of infection by the avian influenza virus or the severity of the infection. The term "administration" refers to introducing a predetermined substance into an individual by any appropriate method. The vaccine composition may be administered by injection, for example, subcutaneous injection, intravenous injection, intramuscular injection, or the like.

[0070] For example, a single dose of the vaccine composition may be, based on the weight of the antigen (e.g., the chimeric virus), 0.1 to 1000 μg, 0.1 to 900 μg, 0.1 to 800 μg, 0.1 to 700 μg, 0.1 to 600 μg, 0.1 to 500 μg, 0.1 to 400 μg, 0.1 to 350 μg, 0.1 to 300 μg, 0.1 to 250 μg, 0.1 to 200 μg, 0.1 to 150 μg, 0.1 to 100 μg, 0.1 to 80 μg, 0.1 to 60 μg, 0.1 to 40 μg, 0.1 to 20 μg, , 0.5 to 1000 μg, 0.5 to 900 μg, 0.5 to 800 μg, 0.5 to 700 μg, 0.5 to 600 μg, 0.5 to 500 μg, 0.5 to 400 μg, 0.5 to 350 μg, 0.5 to 300 μg, 0.5 to 250 μg, 0.5 to 200 μg, 0.5 to 150 μg, 0.5 to 100 μg, 0.5 to 80 μg, 0.5 to 60 μg, 0.5 to 40 μg, 0.5 to 20 μg, 1 to 1000 μg, 1 to 900 μg, 1 to 800 μg, 1 to 700 μg, 1 to 600 μg, 1 to 500 μg, 1 to 400 μg, 1 to 350 μg, 1 to 300 μg, 1 to 250 μg, 1 to 200 μg, 1 to 150 μg, 1 to 100 μg, 1 to 80 μg, 1 to 60 μg, 1 to 40 μg, 1 to 20 μg, 5 to 1000 μg, 5 to 900 μg, 5 to 800 μg, 5 to 700 μg, 5 to 600 μg, 5 to 500 μg, 5 to 400 μg, 5 to 350 μg, 5 to 300 μg, 5 to 250 μg, 5 to 200 μg, 5 to 150 μg, 5 to 100 μg, 5 to 80 μg, 5 to 60 μg, 5 to 40 μg, 5 to 20 μg, 10 to 1000 μg, 10 to 900 μg, 10 to 800 μg, 10 to 700 μg, 10 to 600 μg, 10 to 500 μg, 10 to 400 μg, 10 to 350 μg, 10 to 300 μg, 10 to 250 μg, 10 to 200 μg, 10 to 150 μg, 10 to 100 The dose may be, but is not limited to, 10 to 80 μg, 10 to 60 μg, 10 to 40 μg, or 10 to 20 μg, and may be appropriately adjusted in consideration of the condition of the recipient, desired effect, etc.

[0071] The "subject" to which the vaccine composition of the present specification is administered may mean a living organism that can be infected with a pathogenic substance (avian influenza virus) or a cell, tissue or culture thereof isolated therefrom, and the subject and / or organism may be an animal including a bird, a bovine (e.g., a cow), a swine (e.g., a pig), a canine (e.g., a dog, a wolf, a fox, a coyote, a jackal, etc.), a feline (e.g., a lion, a tiger, a domestic cat, a wild cat, other big cats, a cheetah, a lynx, etc.), an equine (e.g., a horse, etc.), an ovine (e.g., a sheep, a goat, a lamb, a bison, etc.), a primate (e.g., a human, a prosimian, a tarsier, a monkey, a gibbon, a monkey), and the like, and specifically, may be a bird including a chicken, a duck, a goose, a turkey, a quail, a pheasant, a parrot, a finch, a hawk, a crow, an ostrich, an emu and a cassowary. However, the term "animal" may include, but is not limited to, individual animals at any stage of development, including embryonic and fetal stages. Furthermore, the term "animal" may exclude humans.

[0072]

[0073] Creation of chimeric viruses

[0074] Another example is

[0075] A set of recombinant vectors is provided, comprising a first recombinant vector comprising a gene encoding an HA protein derived from an avian influenza virus.

[0076] The above recombinant vector set, in addition to the first recombinant vector,

[0077] U in the turkey herpes virus genome S 5 A second recombinant vector comprising a polynucleotide targeting the site; and

[0078] The present invention may further include, but is not limited to, one or more selected from the group consisting of a third recombinant vector comprising a polynucleotide targeting a gene encoding an HA protein derived from an avian influenza virus within the above recombinant vector.

[0079] Each of the above vectors (mother vectors) may be at least one selected from the group consisting of, but is not limited to, a pGEM T vector (e.g., pGEM-T-easy-vector), a pcDNA3 vector (e.g., pcDNA3.1 / Hygro (+)), and a vector used for backbone cloning for sgRNA (e.g., pSpCas9 (BB)-2A-Puro (PX459)).

[0080] In this specification, "targeting a predetermined nucleic acid sequence (such as a gene or nucleic acid sequence portion; hereinafter referred to as "target portion")" may mean, but is not limited to, specifically binding to the target portion, and / or inducing a desired mutation (e.g., deletion, insertion of nucleotides, etc.) and / or truncation (e.g., single-strand break, double-strand break, etc.) in the target portion, and / or inserting a desired nucleic acid molecule (e.g., gene, etc.) into the truncated position within the target portion. For example, in this specification, "U of the turkey herpes virus genome" L 2 Part and / or U S 5 “Targeting a site” means inserting a target gene (e.g., a foreign antigen gene) into the target site (U L 2 Part and / or U S 5This may mean inducing a mutation (cleavage) for insertion of a target gene into the target site (site), inducing a mutation (cleavage) for insertion of a target gene into the target site, and / or inserting the target gene into the target site (more specifically, the cleavage site). In addition, “targeting a gene encoding an HA protein in a recombinant vector” may mean cutting both ends of the gene site in the recombinant vector to isolate the gene from the recombinant vector.

[0081] The above “polynucleotide targeting a predetermined gene” may include a polynucleotide that encodes, expresses, and / or produces a protein (e.g., a target-specific nuclease, etc.) and / or a nucleic acid molecule (e.g., a guide RNA, etc.) for the above “targeting of a target site.”

[0082] In one example,

[0083] In the second recombinant vector, the U of the turkey herpes virus S 5 A polynucleotide targeting the site comprises (i) a gene encoding a target-specific nuclease (e.g., Cas9, etc.) and (ii) the U of the turkey herpes virus. S 5 It may include a guide RNA capable of specifically binding / hybridizing to a site or DNA encoding (generating) the same,

[0084] In the third recombinant vector, the polynucleotide targeting the gene encoding the HA protein derived from avian influenza virus in the recombinant vector may include (i) a gene encoding a target-specific nuclease (e.g., Cas9, etc.) and (ii) a guide RNA capable of specifically binding / hybridizing to a gene region encoding the HA protein derived from influenza virus in the second recombinant vector (e.g., both terminal regions of the gene) or a DNA encoding (producing) the same.

[0085] Another example provides a method for producing a chimeric virus and / or a vaccine composition comprising the same, comprising the step of inserting (introducing) a gene encoding an HA protein derived from an avian influenza virus into a turkey herpes virus (more specifically, a turkey herpes virus genome).

[0086] More specifically, the method for producing the chimeric virus and / or vaccine composition comprising the same comprises inserting a gene encoding an HA protein derived from an avian influenza virus into the U of the turkey herpes virus genome. S 5 region (e.g., 140100th to 140300th, 140100th to 140250th, 140100th to 140240th, 140100th to 140235th, 140150th to 140300th, 140150th to 140250th, 140150th to 140240th, 140150th to 140235th, 140200th to 140300th, 140200th to 140250th, 140200th to 140240th, 140200th to 140235th, It may include a step of inserting at the 140230th to 140300th, 140230th to 140250th, 140230th to 140240th or 140230th to 140235th nucleotide, for example, at the position following the 140231st nucleotide.

[0087] The above insertion target virus (host cell) may be, for example, turkey herpes virus, but may also be, but is not limited to, Canarypox virus, Bovine viral diarrhea virus, Adenovirus (e.g., Adenovirus 5), Vaccinia virus, Feline Parvovirus, Flaviviruses, Vesicular stomatitis virus, Lentivirus (e.g., Integrase-defective lentiviral vectors), Paramyxovirus, or Fowlpox virus.

[0088] Another example provides a method for producing a chimeric virus and / or a vaccine composition comprising the same, comprising the step of inserting the recombinant vector set into a turkey herpes virus.

[0089] The above manufacturing method can insert the first to fourth recombinant vectors of the above recombinant vector set simultaneously.

[0090] In the chimeric virus provided herein, the gene encoding the HA protein derived from the avian influenza virus can be inserted by a target-specific nuclease, such as the CRISPR-Cas9 system.

[0091] The term "target-specific nuclease" in this specification is also called a programmable nuclease, and refers to all types of endonucleases that can recognize and cut a specific location on a target genomic DNA.

[0092] For example, the target-specific nuclease may be at least one selected from all nucleases that recognize a specific sequence of a target gene and have nucleotide cleavage activity to cause insertion and / or deletion (Indel) in the target gene.

[0093] For example, the target-specific nuclease

[0094] TALEN (transcription activator-like effector nuclease) fused with a cleavage domain and a transcription activator-like effector domain derived from a plant pathogenicity gene, which is a domain that recognizes a specific target sequence in the genome;

[0095] zinc-finger nuclease;

[0096] meganuclease;

[0097] RGEN (RNA-guided engineered nuclease; e.g., Cas9, Cpf1, etc.) derived from the microbial immune system CRISPR;

[0098] Ago homolog (DNA-guided endonuclease)

[0099] It may be one or more selected from the group consisting of, but is not limited to.

[0100] In one embodiment, the target-specific nuclease may be at least one selected from the group consisting of endonucleases associated with type II and / or type V CRISPR systems, such as Cas proteins (e.g., Cas9 protein (CRISPR (Clustered regularly interspaced short palindromic repeats) associated protein 9)), Cpf1 protein (CRISPR from Prevotella and Francisella 1), etc.). The target-specific nuclease may further comprise a target DNA-specific guide RNA for guiding it to a target site of genomic DNA. The guide RNA may be transcribed in vitro, for example, but is not limited thereto, from an oligonucleotide duplex or a plasmid template. The above target-specific nuclease can be applied in the following forms, but is not limited thereto: an expression vector comprising a target-specific nuclease-encoding DNA and a guide RNA-encoding DNA, a mixture of a transcript (mRNA) of the target-specific nuclease-encoding DNA and a guide RNA, and a ribonucleic acid protein (RNP) (ribonucleic acid-protein complex) in which a target-specific nuclease and a guide RNA are bound.

[0101] The Cas9 protein is a major protein component of the CRISPR / Cas system, and is a protein that can form an active endonuclease or nickase. Cas9 protein or genetic information can be obtained from known databases such as GenBank of the National Center for Biotechnology Information (NCBI). For example, the Cas9 protein

[0102] Cas9 protein from Streptococcus sp., e.g., Streptococcus pyogenes (e.g., SwissProt Accession number Q99ZW2 (NP_269215.1)), Streptococcus thermophiles or Streptococcus aureus;

[0103] Cas9 protein from a Campylobacter genus, e.g., Campylobacter jejuni;

[0104] Cas9 protein from Neisseria meningitidis;

[0105] A Cas9 protein from the genus Pasteurella, e.g., Pasteurella multocida;

[0106] a Cas9 protein from the genus Francisella, e.g., Francisella novicida;

[0107] It may be one or more selected from the group consisting of, but is not limited to.

[0108] The Cpf1 protein is a novel CRISPR system endonuclease distinct from the aforementioned CRISPR / Cas system. Compared to Cas9, it is relatively small, does not require tracrRNA, and can be activated by a single guide RNA. Furthermore, it recognizes a thymine-rich protospacer-adjacent motif (PAM) sequence and cleaves the DNA double-strand, creating a cohesive end (a cohesive double-strand break).

[0109] For example, the Cpf1 protein may be derived from, but is not limited to, the genus Candidatus, the genus Lachnospira, the genus Butyrivibrio, the genus Peregrinibacteria, the genus Acidominococcus, the genus Porphyromonas, the genus Prevotella, the genus Francisella, the genus Candidatus Methanoplasma, or the genus Eubacterium.

[0110] The target-specific endonuclease may be isolated from a microorganism or may be non-naturally occurring, such as by a recombinant or synthetic method. In one example, the target-specific endonuclease (e.g., Cas9, Cpf1, etc.) may be a recombinant protein produced by recombinant DNA. Recombinant DNA (rDNA) refers to a DNA molecule artificially produced by a genetic recombination method such as molecular cloning to include heterologous or homologous genetic material obtained from various organisms. For example, when a recombinant DNA is expressed in an appropriate organism to produce a target-specific endonuclease (in vivo or in vitro), the recombinant DNA may have a nucleotide sequence that is reconstructed by selecting a codon optimized for expression in the organism from among the codons encoding the protein to be produced.

[0111] In one specific embodiment, the Cas protein is U of turkey herpes virus. S 5 It may include, but is not limited to, a single guide RNA (sgRNA) that targets the site.

[0112] In this specification, “guide RNA” is a general term for a nucleic acid molecule that includes a sequence (targeting sequence) complementary to a specific sequence (target sequence) of a gene to be corrected / edited (target gene), and that binds to the RNA-guided endonuclease (e.g., Cas9 nuclease in a CRISPR / Cas9 system) to help the endonuclease cleave the target site. The guide RNA includes crRNA and tracrRNA, and may be in a single-stranded or double-stranded form, and may be, for example, a single guide RNA (single guide RNA; sgRNA). The specific sequence of the guide RNA can be appropriately selected depending on the type (derived microorganism) of the endonuclease (e.g., Cas9 protein or Cpf1 protein, etc.), and this is a matter that can be easily known to a person of ordinary skill in the art to which this invention pertains. The expression “gene-targeting” means that it is capable of specific binding (hybridization) with a specific sequence (target sequence) of a gene (target gene) to be corrected / edited, and “gene-targeting guide RNA” means a guide RNA that is capable of specific binding with a target gene, including a sequence complementary to the target site sequence within the target gene.

[0113] The specific sequence of the above guide RNA can be appropriately selected depending on the type of target-specific endonuclease used or the microorganism from which it is derived, and this is something that can be easily known to a person having ordinary skill in the technical field to which this invention belongs.

[0114] In one specific example, the gene encoding the HA protein derived from avian influenza virus included in the target-specific nuclease may be introduced into the virus by the non-homologous end joining (NHEJ) principle and / or the homology-directed repair pathway (HDR) principle, specifically, but not limited to, by the non-homologous end joining (NHEJ) principle.

[0115] The above introduction process may occur in, but is not limited to, Primary chicken embryo fibroblasts (CEF) cells or DF-1 cells.

[0116] The present invention provides a chimeric virus expressing an avian influenza antigen and a vaccine composition comprising the same, which has an immune effect against avian influenza disease.

[0117] Figure 1 is a phylogenetic map showing the phylogenetic information of the MRA-003 strain isolated for preparation of the avian influenza Y280 HA protein encoding gene.

[0118] Figure 2 is a graph showing a schematic diagram of the pGEM-T-easy-sgI-RFP-HA-sgI plasmid manufacturing process.

[0119] Figure 3 is a diagram showing vector map information of the vector used to produce sgRNA and Cas9 protein expression plasmids.

[0120] Figure 4 is a schematic diagram showing the cloning process of a Cas9 protein expression plasmid produced for antigen expression of turkey herpes virus.

[0121] Figure 5 shows the results of PCR detection of a positive virus for a recombinant antigen-expressing chimeric virus produced in primary chicken embryo fibroblasts (CEF) cells.

[0122] Figure 6 shows the results of detecting the expression of fluorescent proteins using a fluorescence microscope for the detection of recombinant antigen-expressing chimeric viruses produced in primary chicken embryo fibroblasts (CEF) cells.

[0123] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited to the following examples.

[0124]

[0125] Example 1. Construction of related plasmids for the production of recombinant chimeric viruses expressing turkey herpesvirus antigens.

[0126] Example 1-1. Preparation of avian influenza Y280 HA protein encoding gene

[0127] Diagnostic PCR was performed on samples (trachea, cecal tonsils) submitted for pathological diagnosis due to the findings of respiratory symptoms such as 20 deaths per day, open mouth breathing, and bronchitis at a breeding farm in Iksan-si, Jeollabuk-do in 2021, with avian influenza H9N2 Y280 lineage. Using the primers (P3-For / Rev) listed in Table 2, the following conditions were performed: 1 cycle of pre-denaturation (94℃, 2 min), 35 cycles of denaturation (94℃, 30 s), annealing (58℃, 30 s), extension (72℃, 1 min), and 1 cycle of final extension (72℃, 5 min). The samples diagnosed with avian influenza were isolated and identified.

[0128] The isolated and identified strain was named A21-MRA-003 and selected as a donor strain of HA. The amino acid sequence and gene sequence of the full-length HA gene of the donor strain are shown in Table 1 below, and the lineage information of the isolated and identified A21-MRA-003 is shown in Figure 1.

[0129]

[0130] The HA full-length gene of the A21-MRA-003 HA donor strain isolated above was amplified using the primers (P4-For / Rev) listed in Table 2 to include a restriction enzyme recognition site (Not I) under the following conditions: 1 cycle of pre-denaturation (95°C, 2 min), 40 cycles of denaturation (95°C, 20 s), annealing (53°C, 40 s), extension (72°C, 3 min 22 s), and 1 cycle of final extension (72°C, 5 min).

[0131] Primer name sequence (5' -> 3') sequence number P3_ForCATCCCAGTGCTGGGAARGAYCCTAAGAA3 P3_RevAGAGCTCTTGTTCTCTGATAGGTG4 P4_ForCGAGCGGCCGCATGGAAGCAGTATCACTAATAACTA5 P4_RevGCGGCGGCCGCTTATATACAAATGTTGCATCTGCAG6

[0132] (The bolded portion in Table 2 above corresponds to the Not I restriction enzyme recognition site)

[0133] After the above amplification, both ends of the amplified HA full-length gene were cut with NotI restriction enzyme (SEQ ID NO: 7) and cloned into a cloning vector (Vector: pcDNA3.1 / Hygro(+), Manufacturer: Addgene, Cat.No: V87020) in which an antigen expression cassette was inserted, and then both ends of the HA full-length gene antigen expression cassette were cut with SfiI restriction enzyme (SEQ ID NO: 8) and cloned into pGEM-T-easy-sgI-RFP-sgI (basic vector: pGEM-T-easy-vector, Manufacturer: Promega, Cat.No: A1360) to complete the construction of the HA (Y280) donor plasmid (pGEM-T-easy-sgI-RFP-HA-sgI). The sequence information of the two types of restriction enzymes is shown in Table 3 below.

[0134] Name sequence (5' -> 3') Sequence number NotI_RE_enzymegcggccgc7SfiI_RE_enzymeggccnnnnnggcc8

[0135] A schematic diagram of the manufacturing process including the NotI restriction enzyme digestion of the HA full-length gene during the above cloning process and the SfiI restriction enzyme digestion of the HA full-length gene antigen expression cassette and pGEM-T-easy-sgI-RFP-sgI is shown in Figure 2.

[0136]

[0137] Example 1-2. Construction of a Cas9 protein expression plasmid for expression of turkey herpes virus antigens.

[0138] U of turkey herpes virus for expression of avian influenza Y280 antigen of turkey herpes virus S 5(Unique short region) For each of the sgRNA primer (P7) targeting the region and the sgRNA primer (P8) targeting the donor of H9N2 Y280 in the cloning vector prepared in Example 1-1, T4 Polynucleotide Kinase (PNK) (Manufacturer: New England BioLabs (NEB), Cat. No: M0201S) and T4 DNA Ligase buffer (Manufacturer: New England BioLabs (NEB), Cat. No: B0202S) were used to phosphorylate and anneale the two primer pairs P7_For / P7_Rev and P8_For / P8_Rev in the following reaction sequence: 37°C for 30 min, 95°C for 5 min, and room temperature for 1 hr, to construct each Oligo-duplex.

[0139] The above Oligo-duplex was cloned into a Cas9 protein expression plasmid (vector: pSpCas9 (BB)-2A-Puro (PX459) V2.0, manufacturer: Addgene, Cat.No: 62988) containing the Puromycin resistant gene cut by treating with BbsI restriction enzyme (NEW ENGLAND BioLabs; catalog#: R0539S and R0539L), and U constructed using the above P7 S 5 Part (specifically, U in Table 4 below) S 5 A plasmid expressing sgRNA and Cas9 targeting the sequence (sequences indicated in bold and underlined in the gene sequence) and a plasmid expressing sgRNA and Cas9 targeting the donor of H9N2 Y280 produced using the above P8 (specifically, the sgI sequences at the two terminal flanking regions of the donor on the vector produced in the above Example 1-1) were prepared. The above U S 5The sequence information of the site gene is shown in Table 4 below, the sequence information of the sgRNA primer used above is shown in Table 5 below, the vector map of the vector used above is shown in Figure 3, and the schematic diagram of the cloning process is shown in Figure 4.

[0140]

[0141] Primer name sequence (5' -> 3') sequence number P5_ForCACCGAAAACACAGTAACCGTTAG19 P5_RevAAACCTAACGGTTACTGTGTTTTC20 P7_ForCACCACACAAATTGCGTTTAGGTG10 P7_RevAAACCACCTAAACGCAATTTGTGT11 P8_ForCACCGAAGTCGAGTTCGGCTAGAC12 P8_RevAAACGTCTAGCCGAACTCGACTTC13

[0142] (In Tables 4 and 5 above, the sequences written in underline and bold letters correspond to sequences that are expressed as sgRNA and play a role in targeting the target object.)

[0143]

[0144] Example 2. Production and screening of recombinant antigen-expressing chimeric viruses.

[0145] To produce chimeric viruses expressing avian influenza antigens, primary chicken embryo fibroblasts (CEF) cells were prepared from 10-day-old SPF hatching chicken embryos, cultured in 5% M199 medium (10x) (Gibco, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) (Peak Serum, Wellington, CO, USA), 50% F-10 (1x) nutrient mixture (Sigma, St. Louis, MO, USA), 1% antibiotic-antimycotic (Gibco, Grand Island, NY, USA), 10% tryptose phosphate broth (TPB) (Becton, Dickinson and Company, Sparks, MD, USA), and 7.5% sodium bicarbonate solution at 37°C and 5% CO2.

[0146] The CEF cells prepared above were 4 X 10 5 Cells were cultured in a 24-well plate at a concentration of 10 cells / well for 2 days, and sgIRNA, U prepared in Example 1-2 were added using Lipofectamine 3000® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. S 5 A plasmid expressing sgRNA targeting the site and the above-constructed pGEM-T-easy-sgI-RFP-HA-sgI were simultaneously transfected.

[0147] After transfection, selection was performed using puromycin, and on the third day, the cells were infected with 0.01 MOI turkey herpesvirus (HVT; Fc126 strain; Genbank Accession No. AF282130.1) to insert the avian influenza antigen gene into the desired location in the turkey herpesvirus by the principle of non-homologous end-joining (NHEJ).

[0148] The above insertion site was confirmed by comparing the transformed turkey herpes virus with the non-transformed turkey herpes virus (turkey herpesvirus; HVT; Fc126 strain; Genbank Accession No. AF282130.1) through sequencing, and it was confirmed that the HA protein encoding gene was inserted at the position following the 140231st nucleotide from the 5' end of the full-length gene of turkey herpesvirus Fc126 (Genbank Accession No. AF282130.1). The chimeric virus in which the avian influenza antigen gene was inserted into the turkey herpes virus was deposited at the National Institute of Biological Resources of Korea on June 27, 2023, and was assigned the accession number KCTC 15489BP.

[0149] The transfection rate of the above-mentioned infected cells was confirmed by counting fluorescence-expressing cells using the Object count function of the NIS-Element BR program, and the fluorescence expression rate was determined based on 100% when 200 or more cells were counted. Puromycin was added to the cell culture medium containing cells confirmed to have a transfection rate of 100%, and through a selection process, only the desired cells transfected with the sgRNA expression plasmid containing the puromycin resistant gene were selected.

[0150] For half of the infected cells, P11 and P12 PCR was performed using the primers in Table 6 below (primers for Y280 HA confirmation: P11 and P12) under the conditions of 1 cycle of pre-denaturation (95°C, 2 min), 35 cycles of denaturation (95°C, 20 s), annealing (58°C, 40 s), extension (72°C, 1 min 32 s), and 1 cycle of final extension (72°C, 5 min) to detect positive virus strains.

[0151] After that, the virus strain expressing the marker (fluorescent protein) in the above virus strain was detected by fluorescence microscopy, and the pure chimeric virus was obtained by purifying the cells expressing the H9N2 HA protein in the turkey herpes virus using FACS (Flourescence Activated Cell Sorter) equipment and then mass-produced. The results of detecting the recombinant positive virus strain through the PCR performance are shown in Fig. 5, and the results of detecting the fluorescent protein expressing virus strain using a fluorescence microscope are shown in Fig. 6.

[0152] Primer name sequence (5' -> 3') sequence number P11_ForCTGTGATACACTTGGGAGCC14 P11_RevACTCCTTGATGACGTCCTCG15 P12_ForCTGTGATACACTTGGGAGCC16 P12_RevGACGTTTCCGATCTTCCACA17

[0153]

[0154] Example 3. Efficacy test of recombinant antigen-expressing chimeric virus vaccine

[0155] Example 3-1. Immunogenicity and protective capacity test against avian influenza in chickens 1

[0156] In order to confirm the efficacy of the recombinant antigen-expressing chimeric virus produced in the above Example 2 as a vaccine against avian influenza, Specific Pathogen Free (SPF) 3-day-old chicks were prepared, and the existing vaccine ABBN (manufacturer: Korea B&P) was prepared as a control group, and A21-MRA-003 (Y280) selected in the above Example 1-1 was prepared as a challenge inoculation virus.

[0157] Specifically, 12 chicks of the above 3-day-old age were vaccinated with 1 dose (1 X 10) of the recombinant antigen-expressing chimeric virus produced in Example 2. 3 PFU / ml; PFU: Plaque forming unit) was inoculated subcutaneously (experimental group 1), and 1 dose (1 X 10) of the ABBN was administered to 8 other chicks per chick. 5 The ABBN vaccine was administered intramuscularly at a dose of 50 (EID50 / ml; EID50: Egg infectious dose 50) (comparative group 1), and the sequence information of the HA protein included in the ABBN vaccine is shown in Table 7 below.

[0158]

[0159] After 3 weeks from the date of each vaccination, blood was collected and HI test (Hemagglutination inhibition assay) was performed on the obtained serum to confirm the antibodies induced by vaccination in experimental group 1 and control group 1, and 10 of the above A21-MRA-003 were administered to each chick vaccinated with each vaccine. 7.0 EID 50 Each chick was challenged intranasally. Clinical symptoms such as depression, feather upturning, and death were observed for 5 days from the date of challenge vaccination, and the results are shown in Table 8 below. A group of 8 chicks that received only challenge vaccination without vaccination was prepared as a positive control group (PC1), and a group of 2 chicks that received neither vaccination nor challenge vaccination was prepared as a negative control group (NC1).

[0160] In addition, in order to confirm virus shedding in oropharyngeal swabs (OP swab) and cloacal swabs (CL swab), swabs were collected on the 3rd and 5th days after challenge vaccination, and PCR was performed using an AIV M qRT-PCR kit (Manufacturer: Median diagnostics, Cat. No: NP-AIV-38) using a FAM probe under the conditions of 1 cycle of cDNA synthesis (50℃, 30min), 1 cycle of initial inactivation (95℃, 15min), 40 cycles of denaturation (95℃, 15s), and annealing & extension (60℃, 40s). Samples with a Ct value (Cycle threshold) of 40 or less in the FAM probe were judged positive.

[0161] On the fifth day after the challenge vaccination, all groups of chickens were euthanized, and the obtained organ samples (trachea, lungs, brain, F bursa, and cecal tonsils) were subjected to AIV M qRT-PCR testing using the same method as for the swab samples. The results of the PCR are shown in Table 8 below.

[0162] Group Experimental group 1 Comparative group 1 PC 1 NC 1 HI titer (log2) 3 wpv 6.8 (12 / 12) 6.3 (8 / 8) 0 (0 / 8) 0 (0 / 2) Virus shedding (log 10 EID 50 / 0.1ml)3 dpiOP0 / 12 (0)8 / 8 (3.9)8 / 8 (4.2)0 / 2 (0)CL0 / 12 (0)2 / 8 (0.6)6 / 8 (1.1)0 / 2 (0)5 dpiOP0 / 12 (0)8 / 8 (2.3)8 / 8 (3.2)0 / 2 (0)CL0 / 12 (0)2 / 8 (0.4)6 / 8 (1.8)0 / 2 (0)Virus replication in tissues (log 10 EID50 / 0.1ml)5 dpiTrachea0 / 12 (0)7 / 8 (2.6)8 / 8 (4.2)0 / 2 (0)Lung0 / 12 (0)4 / 8 (1.2)6 / 8 (1.5)0 / 2 (0)Brain0 / 12 (0)3 / 8 (0.6)5 / 8 (1.1)0 / 2 (0)Bursa0 / 12 (0)3 / 8 (0.9)4 / 8 (1.0)0 / 2 (0)Cecal Tonsil0 / 12 (0)2 / 8 (0.3)4 / 8 (1.1)0 / 2 (0)PI100%50%-

[0163] (In Table 8 above, HI titer (log2) means the titer value (positive number / number of samples) measured by performing the HI test, and each value means the number of chicks positive for the virus / total number of chicks in which the experiment was performed (titer), and PI is calculated as follows:

[0164] PI (Protection Index) (calculated from Cesar Tonsil) = [(Positive rate of control group (PC2) - Positive rate of vaccinated group) / Positive rate of control group (PC2)] X 100; Positive rate (%) = (Number of individuals found positive when performing cecal tonsil (CT) / Total number of individuals) X 100)

[0165] As a result of checking the PI related to the positivity rate, the comparison group 1 was measured at 50%, but the experimental group 1 was measured at 100%, confirming that the recombinant antigen-expressing chimeric virus is suitable for use as a vaccine against avian influenza.

[0166]

[0167] Example 3-2. Immunogenicity and protective capacity test against avian influenza in chickens 2

[0168] In order to confirm the efficacy of the recombinant antigen-expressing chimeric virus produced in Example 2 as a vaccine against avian influenza, the hemagglutination inhibition assay (HI) antibody titer of the vaccine was additionally confirmed.

[0169] Specifically, a hemagglutination inhibition assay (HI) was performed on the serum collected in Example 3-1, and serum with a Titer of 2 or higher was judged positive, and the HI measurement results are shown in Table 9 below.

[0170] Group1 wpv a 2 wpv3 wpvLog2HI titer (Mean)Positive rate (%)Log2HI titer (Mean)Positive rate (%)Log2HI titer (Mean)Positive rate (%)Experimental group 1003.81006.8100

[0171] (In Table 9 above, wpv means Weeks-post vaccination, Log2HI titer means the titer value measured by performing the HI test, and positive rate means the proportion of individuals measured positive among all measured individuals)

[0172] As a result of confirming the HI antibody titer, the HI antibody titer before challenge vaccination 3 weeks after the vaccination date of experimental group 1 showed a high positive rate (100%), confirming that the produced recombinant antigen-expressing chimeric virus is suitable for use as a vaccine against avian influenza.

[0173]

[0174] Example 4. Test of protective efficacy against avian influenza virus according to HA protein insertion location.

[0175] Example 4-1. Production of chimeric viruses with different HA protein insertion positions.

[0176] The gene encoding the HA protein produced in Example 2 above is U S 5 A chimeric virus expressing a recombinant antigen inserted into was prepared (Example).

[0177] In order to measure the protective efficacy against avian influenza virus according to the HA protein insertion position, U in the above Example 1-2 S 5 (Unique short region) Instead of sgRNA primer (P7) targeting the region, U L 2 (Unique long region) U in a substantially identical manner except that an sgRNA primer (P5) targeting the region was used. L 2 Part (specifically, U in Table 4 above) L 2 A plasmid expressing sgRNA and Cas9 targeting the sgRNA targeting the sequence (sequences indicated in bold and underlined in the gene sequence) was constructed, and a chimeric virus was constructed using the same method as in Example 2 (comparative example). The P5 primer sequence information is shown in Table 5, U L 2 The sequence information is shown in Table 4 above.

[0178] The insertion position of the above comparative example was confirmed by comparing the transfected turkey herpes virus with the non-transfected turkey herpes virus (turkey herpesvirus; HVT; Fc126 strain; Genbank Accession No. AF282130.1) through sequencing, and it was confirmed that the HA protein encoding gene was inserted at the 95335th nucleotide from the 5' end of the full-length gene of turkey herpesvirus Fc126 (Genbank Accession No. AF282130.1).

[0179]

[0180] Example 4-2. Evaluation of defensive efficacy against avian influenza virus

[0181] In order to evaluate the protective efficacy of the chimeric virus of Example 4-1 above against avian influenza virus, Specific Pathogen Free (SPF) 3-day-old chicks were prepared, and 10 chicks were each vaccinated with the vaccine of the example and comparative example prepared in Example 4-1 above at 1 dose per chick (1 X 10 3 The vaccine was administered subcutaneously at a dose of 100 PFU / ml (PFU: Plaque forming unit).

[0182] After 3 weeks from the date of each vaccination, 10 chicks were vaccinated with each of the above vaccines, A21-MRA-003 of Example 1-1. 7.0 EID 50 Each vaccine was administered intranasally.

[0183] In order to confirm virus shedding, cloacal swab samples (Cloacal swab; CL swab) were collected on the 3rd and 5th days after challenge vaccination, and PCR was performed using the AIV M qRT-PCR kit (Manufacturer: Median diagnostics, Cat. No: NP-AIV-38) using the FAM probe under the conditions of 1 cycle of cDNA synthesis (50℃, 30min), 1 cycle of initial inactivation (95℃, 15min), 40 cycles of denaturation (95℃, 15s), and annealing & extension (60℃, 40s). Samples with a Ct value (Cycle threshold) of 40 or less in the FAM probe were judged positive, and the protection rate was calculated, and the results are shown in Table 10 below.

[0184] Type of defense rate: Example 100%, Comparative example 60%

[0185] As a result of checking the defense rate, the gene encoding the HA protein was U S 5 The chimeric virus inserted into the had a better protective effect.

[0186]

[0187] [Accession number]

[0188] Name of depositor: National Institute of Biological Resources

[0189] Accession number: KCTC15489BP

[0190] Date of acceptance: 20230627

[0191]

[0192]

[0193]

Claims

1. A chimeric virus comprising an HA protein derived from an avian influenza virus or a gene encoding said HA protein.

2. A chimeric virus according to claim 1, wherein the HA protein comprises an amino acid sequence having at least 70% sequence homology with the amino acid sequence of sequence number 1.

3. A chimeric virus according to claim 1, wherein the gene encoding the HA protein comprises a base sequence having a sequence homology of 70% or more with the base sequence of sequence number 2.

4. The chimeric virus of claim 1, wherein the chimeric virus is turkey herpesvirus, canarypox virus, bovine viral diarrhea virus, adenovirus, vaccinia virus, feline parvovirus, flaviviruses, vesicular stomatitis virus, lentivirus, paramyxovirus, or fowlpox virus.

5. In the fourth paragraph, the gene encoding the HA protein is U of the turkey herpes virus. L 2 Unique long region or U S 5 A chimeric virus that is included in the unique short region.

6. In the fifth paragraph, the gene encoding the HA protein is U of the turkey herpes virus. S 5 A chimeric virus that is included in the unique short region.

7. A vaccine composition against avian influenza virus comprising a chimeric virus according to any one of claims 1 to 6.

8. In paragraph 7, the vaccine composition is a live vaccine, an inactivated vaccine, a subunit vaccine, a vector vaccine, a chimeric vaccine, or a DNA vaccine.

9. In paragraph 7, the vaccine composition further comprises at least one selected from the group consisting of an antigen adjuvant, an efficacy enhancer, a preservative, a buffer, a surfactant, a carrier, an osmotic agent, an antioxidant, and a stabilizer.

10. A vaccine composition according to claim 7, which is administered by at least one route selected from the group consisting of in ovo, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival, and subcutaneous.

11. A set of recombinant vectors, comprising a first recombinant vector comprising a gene encoding an HA protein derived from an avian influenza virus.

12. In paragraph 11, U of turkey herpes virus S 5 A second recombinant vector comprising a polynucleotide targeting the site; and A set of recombinant vectors, further comprising at least one selected from the group consisting of a third recombinant vector comprising a polynucleotide targeting a gene encoding an avian influenza virus derived HA protein in the first recombinant vector.

13. A method for producing a chimeric virus, comprising the step of inserting a gene encoding an HA protein derived from an avian influenza virus into a turkey herpes virus.

14. A method for immunizing against avian influenza virus, comprising the step of administering a chimeric virus of any one of claims 1 to 6 or a vaccine composition comprising the same to a subject in need of immunization against avian influenza virus.

15. A method for immunization in accordance with claim 14, wherein the subject requiring immunization against the avian influenza virus is a bird.

16. A method for preventing, ameliorating or treating avian influenza, comprising the step of administering a chimeric virus of any one of claims 1 to 6 or a vaccine composition comprising the same to a subject in need of preventing, ameliorating or treating avian influenza.

17. In paragraph 16, the individual in need of prevention, improvement or treatment of avian influenza is a method for prevention, improvement or treatment of avian influenza.

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