Oral vaccine composition against feline viral infection virus

Abstract

This oral vaccine composition against feline viral infection virus comprises pupae or cells of a baculovirus-infectable insect that has been: infected with recombinant baculovirus into which DNA that encodes viral proteins causing feline viral infections has been introduced; and dried.

Description

Oral vaccine composition against feline viral infectious disease virus

[0001] The present invention relates to an oral vaccine composition against feline viral infectious disease virus and a method for producing the same.

[0002] There are various types of feline viral infectious diseases, each with different symptoms and courses. In particular, kittens and cats with low immunity are likely to develop severe symptoms and may even be life-threatening, so caution is required. For example, feline panleukopenia virus (FPV) is a parvovirus of cats called feline panleukopenia virus. The peak incidence of diseases caused by this virus (FPV-related diseases) is 12 to 20 weeks of age. In particular, kittens and young cats are prone to infection, and symptoms appear suddenly within a short incubation period after infection with the virus. Since FPV is a highly contagious virus, kittens from the same litter may become ill one after another.

[0003] In addition to FPV, feline calicivirus, feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, etc. exist as causative viruses that cause feline viral infectious diseases. Therefore, it is necessary to develop effective preventive or treatment methods for infections caused by these viruses.

[0004] By the way, baculovirus is a nucleopolyhedrovirus (NPV) that infects insects as the main host, and forms a protein with a crystal structure called polyhedrin in the nucleus of infected cells during the growth process. Therefore, as one method for producing a target protein using the baculovirus-silkworm system, there is a method of introducing a gene encoding the target protein into baculovirus and inoculating the recombinant baculovirus into silkworm larvae or pupae to produce the target protein in silkworms (Non-Patent Document 1). When a vaccine is produced using the baculovirus-silkworm system in this way, it is useful in that a large amount of the target protein can be produced.

[0005] Maeda et al., “Production of human α-interferon in silkworm using a baculovirus vector.” Nature, 315, 592-594 (1985)

[0006] Given the above background, there is a need to develop a method for producing oral vaccines against feline viral infection viruses more simply and in larger quantities using baculovirus-silkworm strains.

[0007] In one embodiment, the inventors discovered that by infecting silkworm pupae with a recombinant baculovirus into which DNA encoding the FPV protein has been introduced, and then drying them, they were able to remarkably maintain immunogenicity, thus completing the present invention.

[0008] In other words, the present invention is, for example, as follows: [1] An oral vaccine composition against feline viral infection viruses, comprising pupae or cells of baculovirus-infecting insects that have been infected and dried with recombinant baculovirus into which DNA encoding a viral protein that causes feline viral infection has been introduced. [2] The oral vaccine composition according to [1], wherein the feline viral infection is at least one selected from the group consisting of feline parvovirus infection, feline calicivirus infection, feline herpesvirus infection, feline leukemia virus infection, feline immunodeficiency virus infection, and feline coronavirus infection. [3] The oral vaccine composition according to [1], wherein the viral protein that causes feline viral infection is feline calicivirus infection virus protein or feline parvovirus infection virus protein. [4] The oral vaccine composition according to [3], wherein the DNA encoding the feline parvovirus infection virus protein is shown in SEQ ID NO: 1. [5] The oral vaccine composition according to [3], wherein the DNA encoding the feline calicivirus infection virus protein is shown in SEQ ID NO: 3. [6] The oral vaccine composition according to [1], further comprising a solution containing an adjuvant. [7] The oral vaccine composition according to [1], wherein pupae are present in an amount of at least 20% by weight of the total weight of the composition. [8] The oral vaccine composition according to [1] for feline parvovirus-related disease or feline calicivirus-related disease. [9] The oral vaccine composition according to [8], wherein the feline parvovirus-related disease is at least one selected from the group consisting of feline panleukopenia (feline distemper), feline infectious enteritis, and cerebellar hypoplasia.

[10] The oral vaccine composition according to [8], wherein the feline calicivirus-related disease is at least one selected from the group consisting of feline calicivirus infection and feline viral upper respiratory tract infection.

[11] The oral vaccine composition according to [1], which is used to be administered to felines after administering an inactivated vaccine or a live attenuated vaccine of a virus that causes feline viral infection.

[12] A method for producing an oral vaccine against the virus, comprising the steps of infecting the larva or pupa of a baculovirus-infectious insect with a recombinant baculovirus into which DNA encoding a viral protein that causes feline viral infection has been introduced, and drying the pupa that has pupated from the infected larva, or the pupa that has been infected.

[13] A method for producing an oral vaccine against the virus, comprising the steps of infecting baculovirus-infectious cells with a recombinant baculovirus into which DNA encoding a viral protein that causes feline viral infection has been introduced, and drying the cells that have been infected.

[14] The method according to

[11] or

[12] , wherein the viral protein that causes feline viral infection is feline calicivirus infection virus protein or feline parvovirus infection virus protein.

[15] The method according to

[13] , wherein the DNA encoding the feline parvovirus infection virus protein is shown in Sequence ID No. 1.

[16] The method according to

[13] , wherein the DNA encoding the feline calicivirus infection virus protein is shown in Sequence ID No. 3.

[17] The method according to

[11] , wherein the insect is a silkworm.

[18] The method according to

[12] , wherein the cells are derived from the mulberry spotted fly moth, the sax moth, the silkworm, the armyworm or the nettle moth.

[19] A pet food product comprising the vaccine composition according to any one of [1] to

[10] .

[20] A method for preventing or treating a feline viral infection, comprising the step of administering the vaccine composition according to any one of [1] to

[10] to a feline.

[21] A method for preventing or treating a feline viral infection, comprising administering an inactivated vaccine or a live attenuated vaccine of a virus that causes a feline viral infection to a feline, and then administering the vaccine composition according to any one of [1] to

[10] to the cat.

[0009] The present invention provides an oral vaccine composition against feline viral infection viruses (e.g., FPV) and a method for producing the same. The manufactured oral vaccine composition can be used directly for administration and can therefore be included, for example, in the feed of felines.

[0010] This is a diagram showing the test schedule. This is a diagram showing the results of measuring salivary HI antibody titers (HI method) for vaccines against FPV. This is a diagram showing the results of measuring serum FPV antigen-specific IgG antibody response (ELISA method) for vaccines against FPV. This is a diagram showing the results of measuring serum HI antibody titers (HI method) for vaccines against FPV. This is a diagram showing the test schedule. This is a diagram showing the results of measuring salivary IgA antibody titers (ELISA method) for inactivated vaccines against FPV. This is a diagram showing the results of measuring salivary IgA antibody titers (ELISA method) for live attenuated vaccines against FPV. This is a diagram showing the results of measuring salivary HI antibody titers (HI method) for inactivated vaccines against FPV. This is a diagram showing the results of measuring salivary HI antibody titers (HI method) for live attenuated vaccines against FPV. This is a diagram showing the results of measuring serum IgG antibody titers (ELISA method) for inactivated vaccines against FPV. This is a diagram showing the results of measuring serum IgG antibody titers (ELISA method) for live attenuated vaccines against FPV. This is a diagram showing the results of measuring serum HI antibody titers (HI method) for inactivated vaccines against FPV. This is a diagram showing the results of measuring serum HI antibody titers (HI method) for live attenuated vaccines against FPV. This figure shows the results of measuring rectal IgA antibody titers (ELISA method) for inactivated vaccines against FPV. This figure shows the results of measuring rectal IgA antibody titers (ELISA method) for live attenuated vaccines against FPV. This figure shows the results of measuring serum IgG antibody titers (ELISA method) for inactivated vaccines against FCV from company A. This figure shows the results of measuring serum IgG antibody titers (ELISA method) for inactivated vaccines against FCV from company B. This figure shows the results of measuring salivary IgA antibody titers (ELISA method) for inactivated vaccines against FCV from company A. This figure shows the results of measuring salivary IgA antibody titers (ELISA method) for inactivated vaccines against FCV from company B. This figure shows the results of measuring rectal IgA antibody titers (ELISA method) for inactivated vaccines against FCV from company A. This figure shows the results of measuring rectal IgA antibody titers (ELISA method) for inactivated vaccines against FCV from company B.

[0011] 1. Overview When producing vaccine antigens against infectious diseases using a silkworm heterologous protein expression system, recombinant baculoviruses are created by inserting vaccine antigen genes derived from the target pathogenic microorganism (virus, etc.). These are then inoculated into silkworm larvae or pupae, allowing the virus to proliferate within the silkworm and produce vaccine antigens. In conventional vaccines administered by injection, some form of purification of the vaccine antigen protein is necessary. This purification requires a combination of multiple chromatograms, such as affinity purification, ion exchange purification, and ammonium sulfate precipitation. However, if the silkworm pupae expressing the vaccine antigen themselves can be administered as the vaccine antigen, the cost of purification can be reduced, and the labor involved in injection administration can be expected to decrease.

[0012] In this invention, we succeeded in obtaining pupae with high antigenicity by drying them, such as freeze-drying, after introducing DNA encoding the protein of the causative virus of feline viral infection (hereinafter also referred to as "feline viral infection virus protein"), particularly the FPV protein, into the pupae of recombinant baculovirus-infectious insects. We then verified whether the antibody production response was actually enhanced when these pupae were fed to cats. As a result, a significant enhancement of the antibody production response to feline viral infection virus antigen (FPV antigen) was observed. Furthermore, a significant enhancement of the antibody production response to the FCV antigen was also observed in recombinant baculovirus-infectious insect pupae introduced with DNA encoding feline calicivirus protein (FCV), similar to the FPV antigen. This invention is based on the above findings.

[0013] The present invention provides an oral vaccine composition against a baculovirus, comprising pupae or cells of baculovirus-infecting insects that have been infected and / or dried with recombinant baculovirus into which DNA encoding a feline viral infection protein has been introduced. The recombinant baculovirus may be subjected to infection treatment, freeze-drying, hot-air drying, and ventilation drying, as well as a combination of both.

[0014] The present invention also provides a method for producing an oral vaccine against the feline viral infection virus, comprising the steps of infecting the larvae or pupae of baculovirus-infectious insects with a recombinant baculovirus into which DNA encoding the feline viral infection virus protein has been introduced, and drying the pupae that have developed from the infected larvae, or the pupae after infection. Furthermore, the present invention provides a method for producing an oral vaccine against the feline viral infection virus, comprising the steps of infecting baculovirus-infectious cells with a recombinant baculovirus into which DNA encoding the feline viral infection virus protein has been introduced, and drying the cells after infection.

[0015] 2. Recombinant baculovirus into which DNA encoding a feline viral protein has been introduced. In this specification, the antigen is the viral antigen (viral antigen protein) of the feline viral virus. A more specific example of a viral antigen protein is the viral capsid protein.

[0016] Examples of feline viral infection viruses include, but are not limited to, feline parvovirus, feline calicivirus, feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline coronavirus, feline morbillivirus, feline papillomavirus, novel coronavirus, feline rustrella virus, feline circovirus, feline formyvirus, and feline paramyxovirus.

[0017] In one embodiment of the present invention, the base sequence of DNA encoding the FPV viral antigen (for example, the DNA described in accession number EU498681.1) can be optimized to kycocodon. The optimized base sequence is shown in Sequence ID No. 1. In another embodiment of the present invention, the base sequence of DNA encoding the FCV viral antigen (for example, the DNA described in accession number NC_075569.1) can be optimized to kycocodon. The optimized base sequence is shown in Sequence ID No. 3.

[0018] The size of the antigen is not particularly limited and may be, for example, 5–1000 kDa, 10–500 kDa, or 30–200 kDa. Immunogenicity refers to the property of an antigen to induce antibody production or cellular immunity. The presence or absence of immunogenicity can be evaluated, for example, by the presence or absence of antibody production after antigen administration. Antibody expression can be confirmed by methods known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, flow cytometry, and immunohistochemistry.

[0019] The method for introducing DNA encoding feline viral proteins into baculovirus can be carried out by methods known to those skilled in the art, for example, by the methods described in Maeda et al., Nature 315, 592-594 (1985) and Y. Matsuura et al., Virology, (1989) 173, 674-682. For example, the above DNA (including cDNA) is cloned and incorporated into a baculovirus transfer vector to obtain a recombinant baculovirus transfer vector. Next, recombinant baculovirus DNA is obtained using this recombinant baculovirus transfer vector by homologous recombination or by transposon transfer. This recombinant baculovirus DNA is introduced into insect culture cells by known methods such as lipofection to obtain recombinant baculovirus.

[0020] Baculovirus transfer vectors are obtained by subcloning a DNA fragment containing the polyhedrin gene from the baculovirus genomic DNA into a plasmid. They can be prepared by known methods or commercially available vectors can be used. Examples of commercially available vectors include pAcYM1, pAcG2T, pAcGP67, and VL1392 (all from Farmingen), pDEST8 (Invitrogen), and pFastBac (Thermo Fisher Scientific).

[0021] In the present invention, examples of baculoviruses used to produce recombinant baculoviruses include Autographa californica multiple nucleopolyhedrovirus (AcNPV), Bombyx mori nucleopolyhedrovirus (BmNPV), Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpNPV), and Lymantria disper multiple nucleopolyhedrovirus (LdNPV), with Bombyx mori nucleopolyhedrovirus (BmNPV) being preferred.

[0022] 3. Infection of baculovirus-infected insect larvae or pupae In this invention, recombinant baculovirus is used to infect baculovirus-infected insect larvae or pupae or baculovirus-infected cells (collectively referred to as "hosts") that serve as hosts.

[0023] Suitable host insects include lepidopteran insects, and are not particularly limited as long as they are suitable for protein expression and are baculovirus infective. Examples include the mulberry moth (Spilosoma imparilis), the sax moth (Antheraea pernyi), silkworms (Mulberry / Bombyx mori, Eri / Samia ricini, Muga / Antheraea assamensis, Antheraea Mylitta, Oak Tasar / Antheraea proylei, etc.), armyworms (Spodoptera frugiperda), and nettle moths (Trichoplusia ni). The insects may be in either pupal or larval form.

[0024] Furthermore, the host cell is not particularly limited as long as it is a baculovirus-infected cell line suitable for protein expression. One characteristic of baculovirus-infected cells is the expression of a glycoprotein called GP64 on the cell surface. Therefore, as long as the cell possesses this property, the type of cell is not limited, and cultured cells such as insect cells can be used.

[0025] Examples of insect cells include: SpIm (derived from Spilosoma imparilis), Anpe (derived from Antheraea pernyi), BmN, BmN4, Oyanagi-2, Bme21 (derived from Bombyx mori), Sf9, Sf21 (derived from Spodoptera frugiperda), and High Five (derived from Trichoplusia ni).

[0026] Methods for infecting host insects or cells with recombinant baculoviruses can be carried out by methods known to those skilled in the art. For example, the recombinant baculovirus obtained in the above step is injected into pupae or larvae. To infect host cells, a solution containing recombinant baculovirus can be added to the cell culture medium. After infecting host insects or host cells with recombinant baculoviruses, rearing or culturing them for 1 to 9 days will result in the expression of antigenic proteins in the host insects or host cells.

[0027] In conventional methods for producing target proteins using baculovirus-silkworm strains, the target protein is expressed within the pupae or larvae of silkworms infected with recombinant baculovirus. After this, the pupae are crushed or body fluids are collected from the larvae, and the target protein is purified through various purification processes. In contrast, the method of the present invention eliminates the need for such extraction and purification of the target protein. The pupae or cells expressing feline viral proteins (e.g., FPV protein, FCV protein, etc.) can be used directly after drying, such as by freeze-drying.

[0028] 4. Freeze-drying of Hosts Infected with Recombinant Baculovirus The timing for freeze-drying the pupae or cells of hosts infected with recombinant baculovirus is preferably after a sufficient amount of feline viral infection proteins (e.g., FPV protein, FCV protein, etc.) have been expressed within the host (e.g., within the body of a silkworm pupa). If larvae are infected with baculovirus, they should be allowed to grow until they become pupae. Therefore, the timing for freeze-drying is after pupation if the infected silkworm is a larva, and freeze-drying can be done at any time after infection if the infected silkworm is a pupa. Freeze-drying may be performed with the pupae intact without cutting or crushing the pupae, or it may be performed after cutting or crushing the pupae.

[0029] In this specification, the expressions "using the pupa as is" and "in the shape of the pupa" mean using or including the pupa in a substantially pupa-like form. For example, even if a part of the pupa is unintentionally damaged during the manufacturing process, it may be judged as being in the shape of the pupa if 90% or more, 80% or more, 70% or more, 60% or more, or 50% or more of the pupa's shape is maintained.

[0030] Freeze-drying is a method of drying by reducing pressure (e.g., maintaining a vacuum) while frozen. Freeze-drying can be carried out by methods known to those skilled in the art, for example, by using a commercially available freeze-dryer. The freezing temperature can be set appropriately by those skilled in the art, for example, -90°C to -5°C, -80°C to -10°C, or -50°C to -10°C. The reduced pressure conditions can also be adjusted appropriately by those skilled in the art, for example, 2 Pa to 20 Pa, 3 Pa to 20 Pa, or 10 Pa to 20 Pa. The freeze-drying time is, for example, 8 hours to 50 hours.

[0031] Drying treatments such as freeze-drying remove most of the water from the pupa or cells, resulting in a dry state. The water content in the pupa after drying may be, for example, 1% or less by weight, 0.5% or less by weight, 0.1% or less by weight, 0.01% or less by weight, or 0.001% or less by weight relative to the total weight of the pupa or cells. From the viewpoint of maintaining immunogenicity, it is preferable that the water content in the pupa is as close to 0% by weight as possible.

[0032] In this invention, pupae or cells can be optionally treated with a liquid agent, immersion, light, gas, plasma, heat, or pressurized heat. The timing of the liquid agent treatment, immersion, light, gas, plasma, heat, or pressurized heat can be before or after a drying treatment such as freeze-drying, and it is preferable that the treatment be performed after the pupae or cells have been sufficiently dried by freeze-drying. The process of freeze-drying a host infected with recombinant baculovirus and the process of liquid agent treatment, immersion, light, gas, plasma, heat, or pressurized heat treatment can be performed consecutively, that is, liquid agent treatment, immersion, light, gas, plasma, heat, or pressurized heat treatment can be performed after freeze-drying without going through any other treatment steps. The timing after freeze-drying can be adjusted as appropriate depending on the condition of the dried silkworm pupae, but for example, it can be performed within 24 hours, 12 hours, 3 hours, or 1 hour after freeze-drying. It is preferable to treat freeze-dried pupae in their original shape without cutting or crushing them, by applying liquid treatment, immersion treatment, light treatment, gas treatment, plasma treatment, heat treatment, or pressurized heat treatment.

[0033] The pupae and cells produced by this method are immunogenic. Therefore, in this invention, there is no need to extract feline viral proteins (e.g., FPV protein, FCV protein, etc.), and they can be administered orally as is.

[0034] Furthermore, in one embodiment of the present invention, since freeze-dried pupae are dry and spongy, they can be easily penetrated by immersing them in a liquid. Therefore, the manufacturing method according to this embodiment may further include a step of immersing freeze-dried pupae, or pupae that have been pressurized and heated as necessary, in a liquid. The liquid to be penetrated is preferably a solution containing an adjuvant, as this can easily improve the vaccine's effectiveness. By immersing freeze-dried pupae in a solution containing an adjuvant, the solution containing the adjuvant can be easily penetrated into the pupae. When cultured cells are freeze-dried, they become a powdered composition, so if cells are used, the freeze-dried cells can be immersed in a solution containing an adjuvant. Also, the pupae produced by this method may be mixed with other solids or liquids. For example, a solid containing viral proteins or an adjuvant is preferred. In addition, the pupae produced by this method may have the adjuvant co-expressed with viral proteins within the silkworm body.

[0035] The pupae or cells produced according to the present invention may be used as is, cut, crushed, or liquefied before use, or a crude extract may be recovered from the pupae and then re-impregnated into the pupae, or as described above, they may be used after being permeated with a liquid, or mixed with other solids. Examples of forms in which they can be used as is include orally administering the pupae as a vaccine, and consuming the pupae as food.

[0036] Examples of forms in which the pupae are used after being cut or crushed include administering the cut or crushed pupae as they are, administering the crushed pupae mixed with other orally administrative materials, and mixing the crushed pupae into feed for consumption. For example, to adjust the dosage considering the immunogenicity of the pupae, they may be cut into, for example, 1 / 2, 1 / 3, 1 / 4, etc. The degree of crushing is not particularly limited as long as immunogenicity is maintained, and may be made into a powder, for example. Cutting can be done with, for example, food scissors and laboratory animal scissors, and crushing can be done with, for example, a mixer, hand mill, and grinding device. When using cells, they can be processed in the same way as when the pupae are crushed.

[0037] Oral administration includes, for example, intraoral and sublingual administration. In this specification, administration also includes cases where felines ingest the substance themselves. When administering pupae in their original form, intraoral administration is preferred, or they may be included in feed so that felines ingest them themselves.

[0038] Examples of orally administered additives include food ingredients, beverage ingredients, excipients, thickeners, stabilizers, preservatives, pH adjusters, sweeteners, colorants, emulsifiers, flavorings, and pharmaceutical additives described later. They can also be mixed with food ingredients or beverage ingredients and administered as functional foods or beverages. The liquid containing the orally administered material is not particularly limited as long as the orally administered pupa is immunogenic and suitable for oral administration; for example, it may be water or an aqueous solution.

[0039] 5. Oral vaccine composition The pupae or cells obtained as described above contain feline viral proteins (e.g., FPV protein, FCV protein, etc.) and are immunogenic, and are therefore used as an oral vaccine composition.

[0040] The oral vaccine of the present invention is intended for infectious diseases caused by feline viral infection viruses. Examples of diseases caused by each virus are listed below. Diseases caused by FPV (FPV-related diseases) include feline panleukopenia (feline distemper), feline infectious enteritis, and reproductive disorders such as cerebellar hypoplasia, and one or more of these can be targeted.

[0041] Diseases caused by feline viral infection (FCV-related diseases) include feline calicivirus infection and feline viral upper respiratory tract infection, and one or more of these may be targeted. Diseases caused by feline herpesvirus (feline herpesvirus-related diseases) include feline viral rhinotracheitis, feline herpesvirus conjunctivitis, and feline herpesvirus stomatitis, and one or more of these may be targeted.

[0042] Diseases caused by feline leukemia virus (feline leukemia virus-related diseases) include feline leukemia virus infection, feline lymphoma related to feline leukemia, and myeloid leukemia, etc., and one or more of these can be targeted. Diseases caused by feline immunodeficiency virus (feline immunodeficiency virus-related diseases) include chronic gingivostomatitis, lymphadenopathy, rhinitis, and immune-mediated glomerulonephritis, etc., and one or more of these can be targeted.

[0043] Diseases caused by feline coronavirus (feline coronavirus-related diseases) include feline infectious peritonitis and digestive system diseases such as diarrhea and ascites, etc., and one or more of these can be targeted. Diseases caused by feline morbillivirus (feline morbillivirus-related diseases) include interstitial nephritis, chronic kidney disease, and lower urinary tract diseases, etc., and one or more of these can be targeted. Diseases caused by feline papillomavirus (feline papillomavirus-related diseases) include cutaneous papilloma, viral plaque, multicentric intraepidermal squamous cell carcinoma (Bowenoid in situ carcinoma: BISC, pre-cancerous lesion of cutaneous SCC), and squamous cell carcinoma of the skin and oral cavity, etc., and one or more of these can be targeted.

[0044] Diseases caused by novel coronavirus (novel coronavirus-related diseases) include upper and lower airway inflammation, bronchopneumonia, myocarditis, etc., and one or more of these can be targeted. Diseases caused by feline rhabdovirus (feline rhabdovirus-related diseases) include ataxia associated with non-suppurative encephalitis / meningoencephalitis (so-called "staggering disease"), etc., and one or more of these can be targeted. Diseases caused by feline sarcovirus (feline sarcovirus-related diseases) include digestive symptoms such as diarrhea and enteritis, etc., and one or more of these can be targeted.

[0045] Diseases caused by feline herpesvirus (feline herpesvirus-related diseases) include oral inflammatory diseases and the like, and one or more of these can be targeted. Diseases caused by feline paramyxovirus (feline paramyxovirus-related diseases) include feline viral upper respiratory infections and the like, and one or more of these can be targeted.

[0046] In the present invention, the animals to be administered the oral vaccine are felids, and the breed is not limited (it may be a hybrid or a purebred). Examples of felids include domestic cats, wild cats, lions, tigers, leopards, cheetahs, saber-toothed tigers, ocelots, pumas, snow leopards, etc.

[0047] The oral vaccine composition can be in the shape of a pupa, or can be in any dosage form such as a liquid agent (syrup, jelly, etc.), powder, granule, tablet, powder, capsule, mash feed, pellet feed, crumble feed, expandable feed, and flake feed. When cells are used as the oral vaccine composition, it can also be in any dosage form such as powder, granule, tablet, powder, capsule, mash feed, pellet feed, crumble feed, expandable feed, and flake feed.

[0048] The above dosage forms are formulated with pharmaceutical additives by methods commonly used in the art. Examples of pharmaceutical additives include adjuvants, carriers for oral administration, diluents, excipients, disintegrants, binders, lubricants, fluidizing agents, coating agents, solubilizers, solubilization aids, thickeners, dispersants, stabilizers, preservatives, pH adjusters, tonicity adjusters, wetting agents, sweeteners, and fragrances. From the perspective of improving the effect as a vaccine, it is preferable that the pharmaceutical additive contains an adjuvant.

[0049] Examples of adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvant, Bordetella pertussis adjuvant, Ribi adjuvant, Lipid A, liposomes, aluminum hydroxide, silica, enterotoxins (cholera toxin, cholera toxin α subunit, cholera toxin β subunit), Toll-like receptor (TLR) ligands (Poly(I:C), lipopolysaccharide, flagellin, CpG), mucosal adhesives (chitosan, lectin), cytokines, synthetic adjuvants, oil adjuvants, and high molecular weight polysaccharides.

[0050] The present invention may further include a step of mixing immunogenic pupae or cells with pharmaceutical additives or a liquid containing them. In the oral vaccine composition of the present invention, the pupae or cells contain expressed FPV protein. Therefore, the immunogenic pupae or cell components (e.g., powder of freeze-dried pupae) may be included in an amount of, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more by weight of the total weight of the vaccine composition.

[0051] The liquid used as a pharmaceutical additive is not particularly limited as long as the oral vaccine composition is immunogenic and pharmaceutically suitable for oral administration. Examples include aqueous solutions of water, physiological saline, or phosphate buffer (PBS).

[0052] Immunogenic pupae may be used as is, cut, crushed, liquefied, or impregnated with a crude extract recovered from the pupae. Cells can also be crushed. In this invention, when pupae are used in their original form, the step of mixing them with a liquid containing pharmaceutical additives may include the step of immersing the dried pupae in the liquid. For example, by immersing the pupae in a solution containing an adjuvant, the solution containing the adjuvant can be easily penetrated into the interior. The pupae, which have been penetrated by the liquid, may be further cut or crushed to form an oral vaccine composition. Cultured cells can also be used in a similar manner to crushed pupae.

[0053] When using cut or crushed pupae, the step of mixing them with a liquid containing pharmaceutical additives may include a step of dispersing the cut or crushed pupae in the liquid.

[0054] The oral vaccine of the present invention exhibits immunogenicity even when administered orally in the form of a pupa, but it may also be administered after being cut or crushed, or, as described above, after being impregnated with liquid. Examples of administration in the form of a pupa include administering the oral-administered pupa as a vaccine orally, and ingesting the oral-administered pupa as food.

[0055] Examples of forms of administration (including cell lysates) that involve cutting or crushing include administering the cut or crushed pupae or cells orally as they are, administering the cut or crushed pupae or cells mixed with other orally administrative materials, and mixing the crushed material into feed for consumption. For example, to adjust the dosage considering the immunogenicity of the pupae, they may be cut into, for example, 1 / 2, 1 / 3, 1 / 4, etc. The degree of crushing is not particularly limited as long as immunogenicity is maintained, and may, for example, be in powder form.

[0056] The dosage can be appropriately determined considering the target of administration and the amount of antigen protein expressed in the pupa or cells. For example, 0.01 to 100 pupae (0.001 g to 30 g) may be administered per dose, or 0.1 to 10 pupae (0.01 g to 3 g) may be administered per dose. The amount of feline viral infection protein expressed (e.g., FPV protein, FCV protein, etc.) is 0.2 mg to 160 mg per 800 mg of dried pupa weight, which is 0.025% to 20.0% by weight per pupa.

[0057] The number and duration of administration can be appropriately determined, taking into consideration the target of administration and the feline viral infection protein (FPV protein, etc.) expressed in the pupa or cells. For example, it may be administered once to five times per day, once to three times per day, or once per day. Also, for example, it may be administered for one to seven days per week, one to five days per week, or one to four days per week. Furthermore, these administrations may be repeated for, for example, two to 24 weeks, one to seven weeks, two to five weeks, or two to four weeks. When administering the vaccine, it is advisable to fast for about one to three hours before administration (before feeding).

[0058] Specifically, for example, it can be administered to felines as follows: Dosage: 0.1 to 5 pupae's worth (0.01 g to 1.5 g) once a day (by feeding) Duration: 3 to 5 doses per week for 3 to 4 weeks. Thereafter, booster immunization may be performed by administering the same dose every 1 to 4 weeks, preferably every 1 to 2 weeks. Accordingly, the present invention provides a method for preventing or treating feline viral infections, comprising the step of administering the oral vaccine composition to felines.

[0059] In one embodiment of the present invention, an inactivated vaccine or a live attenuated vaccine may be injected before the oral administration described above. By administering the vaccine orally for the second and subsequent doses after this injection (which is considered the first administration), the vaccine effect (booster effect) can be further enhanced. Accordingly, the present invention provides a method for preventing or treating feline viral infections, comprising the steps of administering an inactivated vaccine or a live attenuated vaccine of a virus that causes feline viral infections to a feline animal, and then administering the oral vaccine composition to the cat.

[0060] The vaccine composition of the present invention can also be used in combination with or in use with other vaccines. The other vaccines may be immunogenic against FPV proteins, or they may be immunogenic against different antigen proteins. If the other vaccines are immunogenic against the same antigen protein as the pupae or cells, the pupae of the present invention may be administered, for example, as a booster immunization for the other vaccines.

[0061] 5. Pet Food Products In the present invention, the vaccine composition can also be incorporated into pet food to create a pet food product. "Pet food" means a composition suitable for consumption by pets (companion animals, etc.). Pet food is, for example, a nutritionally balanced composition suitable for daily feeding, and includes nutritionally balanced supplements, treats, etc. The overall composition of the pet food may be a dry composition, a wet composition, or any mixture thereof. For example, it may be a powder, granules, suspension, paste, or any other delivery form.

[0062] In one embodiment, the pet food contains an antigen protein present in an amount of approximately 0.001% or more (e.g., 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, etc.) based on the weight of the composition comprising it. The weight of the composition and the additives may be determined according to the provisions described in section 5, "Oral Vaccine Compositions."

[0063] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the scope of these examples.

[0064] [Example 1] Efficacy study of a novel vaccine against feline panleukopenia virus (FPV) by oral administration to cats <Materials and Methods> (1) The preparation of silkworm culture cells and silkworm strain culture cells was carried out in accordance with the method described in the prior literature (Masuda et al., 2018). The silkworm pupa strain used was Kinshu Showa (Atsumaru HD, Kumamoto, Japan).

[0065] (2) Recombinant baculovirus production Based on publicly available information (GenBank accession number, EU498681.1), the genotype of the FPV capsid protein was determined by optimizing the region corresponding to the full length of ORF2 (amino acids 1 to 584) using cycocodons, and then synthesizing the DNA of the optimized base sequence (SEQ ID NO: 1). After amplifying the synthesized DNA by PCR, it was cloned into the pDEST8 vector to produce pDEST8-FPV ORF2(1-584). The cycocodon-optimized base sequence is shown as SEQ ID NO: 1, and the amino acid sequence encoded by this base sequence is shown as SEQ ID NO: 2.

[0066]

[0067] Sequence ID 2 MSDGAVQPDGGQPAVRNERATGSGNGSGGGGGGGSGGVGISTGTFNNQTEFKFLENGWVEITANSSRLVHLNMPESENYKRVVVNNMDKTAVKGNMALDDTHVQIVTPWSLVDANAWGVWFNPGDWQLIVNTMSELHLVSFEQEIFNVVLKTVSESATQPPTKVYNNDLTASLMVALDSNNTMPFTPAAMRSETLGFYPWKPTIPTPWRYYFQWDRTLIPSHTGTSGTPTNIYHGTDPDDVQFYTIENSVPVHLLRTGDEFATGTFFFDCKPCRLTHTWQTNRALGLPPFLN SLPQSEGATNFGDIGVQQDKRRGVTQMGNTDYITEATIMRPAEVGYSAPYYSFEASTQGPFKTPIAAGRGGAQTDENQAADGDPRYAFGRQHGQKTTTTGETPERFTYIAHQDTGRYPEGDWIQNINFNLPVTNDNVLLPTDPIGG KTGINYTNIFNTYGPLTALNNVPPVYPNGQIWDKEFDTDLKPRLHVNAPFVCQNNCPGQLFVKVAPNLTNEYDPDASANMSRIVTYSDFWWKGKLVFKAKLRASHTWNPIQQMSINVDNQFNYLPNNIGAMKIVYEKSQLAPRKLY

[0068] For recombinant baculovirus, we used the BmNPV-Q4 strain from Kyushu University, which was derived from the standard BmNPV-T3 strain by deleting six genes (chitinase A, cathepsin, egt, p26, p10, and p74) to improve protein expression and extend the survival period after silkworm infection.

[0069] (3) Expression of FPV capsid protein in insect cultured cells and silkworm pupae. FPV capsid protein expression was observed in silkworm pupae infected with recombinant baculovirus, resulting in 1 × 10⁶ expression per pupa. 5 The procedure was performed using a plaque-forming unit (PFU), with an infection period of 4 days and an infection temperature of 27°C.

[0070] (4) Preparation of oral vaccine antigen from silkworm pupae expressing FPV capsid protein Infected silkworm pupae were cryopreserved at -80°C. For the preparation of the powder, the silkworm pupae were removed from -80°C, freeze-dried, and powdered using a mill mixer to prepare oral vaccine antigen from silkworm pupae expressing FPV capsid protein for use in feline testing.

[0071] (5) Preparation of recombinant baculovirus-free silkworm pupa powder Baculovirus-free silkworm pupae were freeze-dried and powdered using a mill mixer to prepare recombinant baculovirus-free silkworm pupa powder for use in the feline test.

[0072] (6) Immunization Schedule and Sample Collection for the Cat Test A total of eight cats (sex: male, breed: mixed, age: 14 weeks) whose FPV negativity had been confirmed in advance by PCR and ELISA kits were introduced to the Sanwa Farm of Kyoto Animal Inspection Center Co., Ltd., and were subjected to the test after a 7-day acclimatization period. The cats were randomly divided into four groups: T01 group (oral vaccine administration group), T02 group (commercial injectable vaccine + oral vaccine administration group), T03 group (commercial injectable vaccine administration group), and T04 group (recombinant baculovirus-free silkworm pupa oral administration group).

[0073] The test design and test schedule are as shown in Table 1 and Figure 1.

[0074] 1) Administration of the test substance: In the T01 group, a paste-like feed containing silkworm pupa oral vaccine antigen was orally administered once a day for three consecutive days at week 0 of the study. Four weeks after the initial administration, the same paste-like feed containing silkworm pupa oral vaccine antigen was orally administered once a day for three consecutive days. The amount of silkworm pupa oral vaccine antigen administered was 0.125 g per day (of which 0.025 g was FPV capsid protein-expressing silkworm pupa powder).

[0075] In the T02 group, a commercially available injectable vaccine was administered subcutaneously at a dose of 0.5 mL in week 0 of the study, according to the package insert. Four weeks after the injection, a paste-like feed containing pupal oral vaccine antigen was orally administered once a day for three consecutive days. The oral vaccine antigen dose was 0.125 g per day (of which 0.025 g was FPV capsid protein-expressing silkworm pupa powder).

[0076] In the T03 group, the first dose of the commercially available injectable vaccine was administered subcutaneously at week 0 of the study, in accordance with the package insert, at a rate of 0.5 mL. Four weeks after the first dose, the second dose of the commercially available injectable vaccine was administered subcutaneously at a rate of 0.5 mL, in accordance with the package insert.

[0077] In the T04 group, a paste-like feed supplemented with recombinant baculovirus-free silkworm pupa powder was orally administered once a day for three consecutive days during week 0 of the study. Four weeks after the initial administration, the same paste-like feed supplemented with non-infected silkworm pupa powder was orally administered once a day for three consecutive days. The dosage of non-infected silkworm pupa powder was 0.125 g per dose per day.

[0078] 2) During the breeding trial period, the test cats were fed Purina One [manufactured by Nestlé Co., Ltd.]. 3) The observation period was 9 weeks from the start of administration.

[0079] 4) Saliva collection and antibody titer measurement: Saliva was collected once a week from week 0 to week 9 of the study, and antibody titers were measured using the hemagglutinin inhibition test (HI method). 5) Blood collection and antibody titer measurement: Blood was collected once a week from week 0 to week 9 of the study, and antibody titers were measured using ELISA and the HI method.

[0080] <Evaluation Test (1): Results of Salivary HI Antibody Titer Evaluation> 1) Salivary HI Antibody Titer Measurement was performed on salivary HI antibody titers in groups T01 and T04. Salivary samples were serially diluted from 1:3 to 2x, and the maximum dilution factor that showed inhibition of hemagglutination was defined as the HI antibody titer. The HI antibody titer at week 0 of immunization was set to 1, and the relative values ​​from week 1 to week 9 of immunization were calculated and evaluated as the relative HI antibody titer. In group T01, the relative HI antibody titer was 4 at weeks 4 and 5 of immunization, indicating an increase in HI antibody titer after immunization (Figure 2).

[0081] In the T04 group, the relative HI antibody titer from week 1 to week 9 of immunization was 1 or less, except for 2 in week 1 of immunization (Figure 2). Therefore, HI antibody titers in saliva were confirmed in the T01 group.

[0082] <Evaluation Test (2): Results of Evaluation of FPV Antigen-Specific Antibody Titer and HI Antibody Titer in Serum> 1) Serum FPV Antigen-Specific IgG Antibody Response To evaluate the amount of antibodies in the serum of groups T01, T02, T03, and T04, antigen-specific IgG detection was performed using the ELISA method. Serum samples were serially diluted from 500 to 2 times, and the highest dilution factor showing an absorbance of 0.1 or higher was evaluated as the antibody titer.

[0083] In groups T01 and T04, antibody titers from week 1 to week 9 after immunization were at the lowest dilution ratio (Figure 3). In group T02, antibody titers increased from week 4 after immunization, reaching 64,000 at week 6, and were maintained at a high level until week 9 (Figure 3). In group T03, antibody titers increased from week 4 after immunization, reaching 64,000 at week 5, and were maintained at a high level until week 9 (Figure 3). Based on these results, antigen-specific IgG responses in serum were confirmed in groups T02 and T03.

[0084] 2) Serum HI antibody titers were measured in the T01, T02, and T03 groups. Serum samples were serially diluted from 1:8 to 2-fold, and the maximum dilution factor showing inhibition of hemagglutination was evaluated as the HI antibody titer. In the T01 group, the HI antibody titer from week 0 to week 9 after immunization was the value at the lowest dilution factor (Figure 4). In the T02 group, the HI antibody titer increased from week 4 after immunization, reaching 1:80 at week 6 after immunization, and maintaining a high level of immunity until week 9 (Figure 4).

[0085] In the T03 group, HI antibody titers increased from the 4th week after immunization, reaching 1:128 at the 6th week, and maintaining a high level of immunity until the 9th week (Figure 4). Therefore, serum HI antibody titers were confirmed in both the T02 and T03 groups.

[0086] <Summary> The results of evaluating antigen-specific IgG antibodies by ELISA and HI antibody titers by HI using serum from each test group showed that mucosal immunity was acquired in the T01 group, and sufficient systemic immunity was acquired in the T02 group through a combination of injection and oral administration.

[0087] The HI method is a common vaccine efficacy evaluation method, and the Japanese Ministry of Agriculture, Forestry and Fisheries defines the criterion for FPV as an HI antibody titer of 1:64 or higher (Standards for Biological Products for Animals (Ministry of Agriculture, Forestry and Fisheries Notification No. 1567 of 2002)). On the other hand, the criterion used overseas is an HI antibody titer of 1:40 or higher (E. Jenkins et al., Viruses. 2020).

[0088] The T02 group showed HI antibody titers of 1:40 or higher from week 5 to week 9 of immunization, suggesting that its effectiveness was comparable to that of commercially available vaccines. The World Small Animal Veterinary Association (WSAVA) Vaccination Guidelines for Dogs and Cats states in the FPV vaccine fact sheet that immunity to inactivated vaccines is established only after the second dose, and it is known that immunity is not established after a single dose (M. Day et al., J Small Anim Pract. 2016). In other words, in the T02 group, oral administration of silkworm pupa oral vaccine antigen after injectable immunization resulted in an antigen-specific IgG antibody response and a sufficient increase in HI antibody titers that met the above criteria.

[0089] Based on the above, it was concluded that the silkworm pupa oral vaccine antigen expressing FPV capsid protein does not induce sufficient systemic immunity through oral administration alone, but does induce mucosal immunity. Furthermore, oral administration of this vaccine antigen showed a boosting effect after injection immunization, and induced antibody titers exceeding the vaccine efficacy criteria, similar to injection boosting. Therefore, it was concluded that this vaccine antigen is an oral vaccine that obtains effective immunity through the acquisition of mucosal immunity and boosting effect.

[0090] [Example 2] In Example 2, the vaccine effect against FPV was investigated when an inactivated vaccine or a live attenuated vaccine was injected followed by oral vaccine administration. Silkworm culture cells were prepared in the same manner as in Example 1.

[0091] 1) Administration of test substance In the T01 group, a commercially available injectable vaccine was administered subcutaneously at 0.5 mL in week 0 of the study, according to the package insert. Four weeks after the injection, a paste-like feed containing pupal oral vaccine antigen was orally administered once a day for three consecutive days. The oral vaccine antigen dosage was 0.225 g per dose per day (of which 0.025 g was FPV capsid protein-expressing silkworm pupa powder and 0.2 g was FCV capsid protein-expressing silkworm pupa powder).

[0092] In the T02 group, the first dose of the commercially available injectable vaccine was administered subcutaneously at week 0 of the study, according to the package insert, at a rate of 0.5 mL. Four weeks after the first dose, the second dose of the commercially available injectable vaccine was administered subcutaneously at a rate of 0.5 mL, according to the package insert.

[0093] In the T03 group, the first dose of a commercially available injectable vaccine was administered subcutaneously at week 0 of the study, in accordance with the package insert, at a rate of 0.5 mL.

[0094] In the T04 group, 0.5 mL of a commercially available injectable vaccine was administered subcutaneously in week 0 of the study, according to the package insert. Four weeks after the injection, a paste-like feed containing pupal oral vaccine antigen was orally administered once a day for three consecutive days. The oral vaccine antigen dosage was 0.225 g per day (of which 0.025 g was FPV capsid protein-expressing silkworm pupa powder and 0.2 g was FCV capsid protein-expressing silkworm pupa powder).

[0095] In the T05 group, the first dose of the commercially available injectable vaccine was administered subcutaneously at week 0 of the study, according to the package insert, at a rate of 0.5 mL. Four weeks after the first dose, the second dose of the commercially available injectable vaccine was administered subcutaneously at a rate of 0.5 mL, according to the package insert.

[0096] In the T06 group, the first dose of a commercially available injectable vaccine was administered subcutaneously at week 0 of the study, in accordance with the package insert, at a rate of 0.5 mL.

[0097] The rearing and observation period of the test cats was carried out in the same manner as in Example 1.

[0098] 2) Saliva collection and antibody titer measurement Saliva was collected once a week from week 0 to week 9 of the study, and antibody titers were measured using ELISA and hemagglutinin inhibition (HI method).

[0099] 3) Blood collection and antibody titer measurement: Blood was collected once a week from week 0 to week 9 of the study, and antibody titers were measured using ELISA and HI methods. 4) Rectal swab collection and antibody titer measurement: Rectal swab fluid was collected once a week from week 0 to week 9 of the study, and antibody titers were measured using ELISA.

[0100] The genotype of the FCV capsid protein is based on publicly available information (NCBI Reference Sequence: NC_075569.1). The base sequence of the DNA encoding ORF2 is shown in Sequence ID No. 3, and the amino acid sequence encoded by that base sequence is shown in Sequence ID No. 4. From the preparation of recombinant baculovirus to the preparation of the silkworm pupa oral vaccine antigen expressing the FPV capsid protein, the procedure was the same as in Example 1, except that the viral antigen was changed to the FCV capsid protein.

[0101]

[0102] Sequence ID 4 MADDGSITAPEQGTMVGGVIAEPSAQMSTAADMATGKSVDSEWEAFFSFHTSVNWSTSETQGKILFKQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSMLQYPHVLFDARQVEPVIFCLPDLRSTLYHLMSDTDTTSLVIMVYNDLINPYANDANSSGCIVTVETKPGPDFKFHLLKPPGSMLTHGSIPSDLIPKTSSLWIGNRYWSDITDFVIRPFVFQANRHFDFNQETAGWSTPRFRPISVTITE QNGAKLGIGVATDYIVPGIPDGWPDTTIPGELIPAGDYAITNGTGNDITTATGYDTADIIKNNTNFRGMYICGSLQRAWGDKKISNTAFITTATLDGDNNKINPCNTIDQSKIVVFQDNHVGKKAQTSDDTLALLG YTGIGEQAIGSDRDRVVRISTLPETGARGGNHPIFYKNSIKLGYVIRSIDVFNSQILHTSRQLSLNHYLLPPDSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSGFGKLEFPLSASYMGIQLAKIRLASNIRSPMTKL

[0103] The test design is shown in Table 2 below, and the test schedule is shown in Figure 5.

[0104] Salivary antibody titers and serum antibody titers were measured in the same manner as in Example 1. Antibody titers in rectal swab fluid were measured as follows: <Method for measuring FPV antigen-specific antibody titers in rectal swab fluid> Antigen-specific IgA detection was performed using the ELISA method to evaluate the amount of antibodies in the serum of groups T01, T02, and T03. Serum samples were serially diluted from 5 to 2 times, and the highest dilution factor showing an absorbance of 0.1 or higher was evaluated as the antibody titer.

[0105] The results are shown in Figures 6-15. These figures show the following: <Evaluation Test (1): Results of Evaluation of FPV Antigen-Specific Antibody Titer and HI Antibody Titer in Saliva> 1) FPV Antigen-Specific IgA Antibody Response in Saliva (Inactivated Injectable Vaccine Group) In the T01 group, the antibody titer was 1,600 from 5 weeks post-immunization, and reached a maximum of 6,400 at 6 and 7 weeks post-immunization. The antibody titer at 9 weeks post-immunization was 1,600, maintaining a high antibody titer. In the T02 group, the antibody titer was 1,600 at 3 weeks post-immunization, but from 4 weeks post-immunization onward, the antibody titer was 400 or less, at the same level as before administration. In the T03 group, the maximum antibody titer during the observation period was 400, and no increase in antibody titer was observed (Figure 6). From the above, antigen-specific IgA response reactions in saliva were confirmed from 5 weeks post-immunization in the T01 group and at 3 weeks post-immunization in the T02 group.

[0106] 2) Salivary FPV antigen-specific IgA antibody response (attenuated live injection vaccine group) In the T04 group, the antibody titer was 3,200 from 2 weeks post-immunization, and reached a maximum of 32,000 at 6 weeks post-immunization. The antibody titer was 3,200 at 9 weeks post-immunization, maintaining a high antibody titer. In the T05 group, the antibody titer was 800 at 3 weeks post-immunization, but remained below 400 from 4 weeks post-immunization onward, at the same level as before administration. In the T06 group, the antibody titer was 1,600 at 2 weeks post-immunization, and reached a maximum of 3,200 at 3 weeks post-immunization. The antibody titer remained at 400 from 6 weeks post-immunization onward, at the same level as before administration (Figure 7). From the above, antigen-specific IgA response reactions in saliva were confirmed at 2 weeks post-immunization in the T04 and T05 groups.

[0107] 3) Salivary HI antibody titer (inactivated injection vaccine group): In the T01 group, the relative HI antibody titer was 8 or higher from week 5 of immunization, and the HI antibody titer increased after immunization. Furthermore, the relative HI antibody titer remained 8 or higher until week 9 of immunization, and was maintained after oral administration. In the T02 and T03 groups, the relative HI antibody titer was 2 or lower during the observation period (Figure 8).

[0108] 4) Salivary HI antibody titer (attenuated live injection vaccine group): In the T04 group, the relative HI antibody titer was 25 or higher from week 5 of immunization, and the HI antibody titer increased after immunization. Furthermore, the relative HI antibody titer remained 16 or higher until week 9 of immunization, and was maintained after oral administration. In the T05 and T06 groups, the relative HI antibody titer was 2 or lower during the observation period (Figure 9).

[0109] <Evaluation Test (2): Results of Evaluation of Serum FPV Antigen-Specific Antibody Titer and HI Antibody Titer> 1) Serum FPV Antigen-Specific IgG Antibody Response (Inactivated Injectable Vaccine Group) In the T01 group, the antibody titer was 5,657 from 3 weeks post-immunization, and was above 10,000 from 4 weeks post-immunization onward. The antibody titer at 9 weeks post-immunization was 50,000, maintaining a high antibody titer. In the T02 group, the antibody titer was 8,000 from 3 weeks post-immunization, and was above 16,000 from 4 weeks post-immunization after injection administration. The antibody titer at 9 weeks post-immunization was 128,000, maintaining a high antibody titer. In the T03 group, the antibody titer increased from 3 weeks post-immunization to 8,000. The antibody titer at 9 weeks post-immunization was 40,000 (Figure 10). From the above, antigen-specific IgG response reactions in serum were confirmed in the T01, T02, and T03 groups.

[0110] 2) Serum FPV antigen-specific IgG antibody response (attenuated live injection vaccine group) In the T04 group, the antibody titer was 256,000 from 2 weeks post-immunization, and the antibody titer was 512,000 or higher from 4 weeks post-immunization after oral administration. The antibody titer was 512,000 at 9 weeks post-immunization, maintaining a high antibody titer. In the T05 group, the antibody titer was 32,000 from 2 weeks post-immunization, and the antibody titer was 64,000 or higher from 4 weeks post-immunization after injection administration. The antibody titer was 64,000 at 9 weeks post-immunization, maintaining a high antibody titer. In the T06 group, the antibody titer increased from 2 weeks post-immunization to 128,000. The antibody titer was 128,000 at 9 weeks post-immunization (Figure 11). Based on the above, antigen-specific IgG response reactions in serum were confirmed in the T01, T02, and T03 groups.

[0111] 3) Serum HI antibody titer (inactivated injection vaccine group) In the T01 group, the HI antibody titer increased from 3 weeks post-immunization, reaching 1:91. High HI antibody titers were maintained thereafter, with the HI antibody titer remaining at a high level of 1:1823 from 7 weeks post-immunization. In the T02 group, the HI antibody titer increased from 3 weeks post-immunization, reaching 1:128. High HI antibody titers were maintained thereafter, with the HI antibody titer remaining at a high level of 1:2,734 from 7 weeks post-immunization. In the T03 group, the HI antibody titer increased from 3 weeks post-immunization, reaching 1:128. High HI antibody titers were maintained thereafter, with the HI antibody titer being 1:512 from 8 weeks post-immunization (Figure 12). From the above, serum HI antibody titers were confirmed in the T01, T02, and T03 groups.

[0112] 4) Serum HI antibody titer (live attenuated vaccine group) In the T04 group, the HI antibody titer increased from the first week after immunization, showing a titer of 1:102. High HI antibody titers were maintained thereafter, and the HI antibody titer remained at a high level of 1:2,734 from the 7th week after immunization. In the T05 group, the HI antibody titer increased from the first week after immunization, showing a titer of 1:64. High HI antibody titers were maintained thereafter, and the HI antibody titer remained at a high level of 1:810 from the 7th week after immunization. In the T06 group, the HI antibody titer increased from the first week after immunization, showing a titer of 1:256. High HI antibody titers were maintained thereafter, and the HI antibody titer was 1:1,823 from the 8th week after immunization (Figure 13). From the above, serum HI antibody titers were confirmed in the T04, T05, and T06 groups.

[0113] <Evaluation Test (3): Results of FPV Antigen-Specific Antibody Titer in Rectal Swab Fluid> 1) FPV Antigen-Specific IgA Antibody Response in Rectal Swab Fluid (Inactivated Injectable Vaccine Group) In the T01 group, the antibody titer was 80 from 6 weeks post-immunization. The antibody titer reached a maximum of 80 at 7 weeks post-immunization, and was 40 at 9 weeks post-immunization, maintaining a high antibody titer. In the T02 group, the antibody titer was 80 at 5 weeks post-immunization, but from 6 weeks post-immunization onward, the antibody titer was 5 or less, at the same level as before administration. In the T03 group, the maximum antibody titer during the observation period was 5, and no increase in antibody titer was observed (Figure 14). From the above, antigen-specific IgA response reactions in the rectum were confirmed from 6 weeks post-immunization in the T01 group and at 5 weeks post-immunization in the T02 group.

[0114] 2) Salivary FPV antigen-specific IgA antibody response (attenuated live injection vaccine group) In the T04 group, the antibody titer was 80 from 2 weeks post-immunization. The antibody titer was 20 at 9 weeks post-immunization, maintaining a high antibody titer. In the T05 group, the antibody titer was 20 at 3 weeks post-immunization, but from 4 weeks post-immunization onward, the antibody titer was 5 or less, at the same level as before administration. In the T06 group, the antibody titer was 20 at 2 weeks post-immunization. The antibody titer was 5 at 9 weeks post-immunization, at the same level as before administration (Figure 15). From the above, antigen-specific IgA response reactions in the rectum were confirmed from 2 weeks post-immunization in the T04 group, and for some periods in the T05 and T06 groups.

[0115] <Summary> The results of evaluating antigen-specific IgG antibodies by ELISA and HI antibody titers by HI using serum from each test group showed that sufficient systemic immunity could be acquired by combining injection and oral administration. Furthermore, systemic immunity could be acquired by combining oral administration with inactivated or attenuated live injection vaccines of different immunogen types, indicating that the boost effect of oral administration of this vaccine antigen after injection immunization is not limited to the type of vaccine immunogen.

[0116] The results of evaluating antigen-specific IgA antibodies using saliva and rectal swab fluid, as well as relative HI antibody titers using the HI method, showed that mucosal immunity was not acquired with one or two injections (T02, T03, T05, and T06 groups), but mucosal immunity was acquired with a combination of injection and oral administration. Furthermore, high antibody titers were observed for 5 weeks after oral administration, indicating that mucosal immunity was maintained. Since the main routes of feline parvovirus infection are the nose and mouth, it was considered effective in preventing infection, controlling its spread, and treating the virus by inactivating the virus in the nasal cavity and oral cavity. Based on these results, it was concluded that the silkworm pupa oral vaccine antigen expressing FPV capsid protein has the effect of boosting systemic immunity and mucosal immunity when administered orally after injection immunization, making it an effective oral vaccine for infection prevention and treatment.

[0117] [Example 3] In Example 3, the vaccine effect against FCV was investigated when an inactivated vaccine from Company A or Company B was injected followed by oral vaccine administration. The study design is shown in Table 3 below, and the study schedule was the same as in Example 2 (Figure 5).

[0118]

[0119] Salivary antibody titers, serum antibody titers, and rectal swab antibody titers were measured in the same manner as in Example 2. The results are shown in Figures 16 to 21. These figures illustrate the following points.

[0120] <Evaluation Test (1): Results of Evaluation of FCV Antigen-Specific Antibody Titer in Serum> 1) Serum FCV Antigen-Specific IgG Antibody Response (Company A Injectable Vaccine Group) In the T01 group, the antibody titer was 1,000 from 3 weeks post-immunization, and was 8,000 or higher from 5 weeks post-immunization onwards. The antibody titer at 9 weeks post-immunization was 8,000, maintaining a high antibody titer. In the T02 group, the antibody titer was 500 from 2 weeks post-immunization, and was 16,000 or higher from 5 weeks post-immunization onwards. The antibody titer at 9 weeks post-immunization was 16,000, maintaining a high antibody titer. In the T03 group, the antibody titer increased from 2 weeks post-immunization to 500, and was 4,000 or lower thereafter. The antibody titer at 9 weeks post-immunization was 4,000 (Figure 16). From the above, antigen-specific IgG response reactions in serum were confirmed in the T01, T02, and T03 groups.

[0121] 2) Serum FCV antigen-specific IgG antibody response (Company B injectable vaccine group) In the T04 group, the antibody titer was 500 from 4 weeks post-immunization. The antibody titer was 1,000 at 9 weeks post-immunization and was maintained. In the T05 group, the antibody titer was 4,000 from 5 weeks post-immunization. The antibody titer was 2,000 at 9 weeks post-immunization and was maintained. In the T06 group, the antibody titer increased from 6 weeks post-immunization to 500. The antibody titer was 1,000 at 9 weeks post-immunization and was maintained (Figure 17). From the above, antigen-specific IgG response reactions in serum were confirmed in the T04, T05, and T06 groups.

[0122] <Evaluation Test (2): Results of Evaluation of FCV Antigen-Specific Antibody Titer in Saliva> 1) FCV Antigen-Specific IgA Antibody Response in Saliva (Company A Injectable Vaccine Group) In the T01 group, the antibody titer was 500 from 5 weeks post-immunization, and reached a maximum of 3,200 at 6 and 7 weeks post-immunization. The antibody titer at 9 weeks post-immunization was 200, indicating that the antibody titer was maintained. In the T02 group, the antibody titer was 200 at 6 weeks post-immunization, but from 7 weeks post-immunization onward, the antibody titer was 50 or less, at the same level as before administration. In the T03 group, no increase in antibody titer was observed during the observation period (Figure 18). From the above, antigen-specific IgA response reactions in saliva were confirmed at 6 weeks post-immunization in the T01 and T02 groups.

[0123] 2) Salivary FCV antigen-specific IgA antibody response (Company B injectable vaccine group) In the T04 group, the antibody titer was 800 from 7 weeks post-immunization, and reached a maximum of 800 at 8 weeks post-immunization. The antibody titer at 9 weeks post-immunization was 200, the same level as before administration. No increase in antibody titer was observed in the T05 and T06 groups during the observation period (Figure 19). Based on the above, an antigen-specific IgA response reaction in saliva was confirmed in the T04 group.

[0124] <Evaluation Test (3): Results of Evaluation of FCV Antigen-Specific Antibody Titer in Rectal Swab Fluid> 1) FCV Antigen-Specific IgA Antibody Response in Rectal Swab Fluid (Company A Injectable Vaccine Group) In the T01 group, the antibody titer was 20 at 6 weeks post-immunization, but from 7 weeks post-immunization onward, the antibody titer was 5 or less, at the same level as before administration. No increase in antibody titer was observed in the T02 and T03 groups during the observation period (Figure 20). From the above, an antigen-specific IgA response reaction was confirmed in the rectal swab fluid at 6 weeks post-immunization in the T01 group.

[0125] 2) FCV antigen-specific IgA antibody response in rectal swab fluid (Company B injectable vaccine group) In the T04 group, the antibody titer was 5 at 6 weeks post-immunization, but from 7 weeks post-immunization onward, the antibody titer remained below 5, at the same level as before administration. In the T05 group, the antibody titer was 5 at 5 weeks post-immunization, but from 6 weeks post-immunization onward, the antibody titer remained below 5, at the same level as before administration. In the T06 group, no increase in antibody titer was observed during the observation period (Figure 20). Based on the above, antigen-specific IgA response reactions were confirmed in rectal swab fluid at 6 weeks post-immunization in the T04 group and at 5 weeks post-immunization in the T05 group.

[0126] <Summary> Evaluation of antigen-specific IgG antibody titers using ELISA with serum from each test group showed that sufficient systemic immunity could be acquired by combining injection and oral administration. Furthermore, although the feline calicivirus strains used as immunogens differed between Company A and Company B, systemic immunity could be acquired by combining oral administration with either strain. This indicates that the boost effect of oral administration of this vaccine antigen after injection immunization is not limited to the feline calicivirus strain.

[0127] Evaluation of antigen-specific IgA antibody titers using saliva and rectal swab fluid showed that mucosal immunity was not acquired with one or two injections (T02, T03, T05, and T06 groups), but mucosal immunity was acquired with a combination of injection and oral administration. Therefore, it was concluded that the silkworm pupa oral vaccine antigen expressing FCV capsid protein has the effect of boosting systemic and mucosal immunity when administered orally after injection immunization, making it an effective oral vaccine for infection prevention and treatment.

Claims

1. An oral vaccine composition against feline viral infection virus, comprising pupae or cells of baculovirus-infecting insects that have been infected and dried with recombinant baculovirus into which DNA encoding a viral protein that causes feline viral infection has been introduced.

2. The oral vaccine composition according to claim 1, wherein the feline viral infection is at least one selected from the group consisting of feline parvovirus infection, feline calicivirus infection, feline herpesvirus infection, feline leukemia virus infection, feline immunodeficiency virus infection, and feline coronavirus infection.

3. The oral vaccine composition according to claim 1, wherein the viral protein that causes feline viral infection is feline calicivirus infection virus protein or feline parvovirus infection virus protein.

4. The oral vaccine composition according to claim 3, wherein the DNA encoding the feline parvovirus infection virus protein is shown in Sequence ID No.

1.

5. The oral vaccine composition according to claim 3, wherein the DNA encoding the feline calicivirus infection virus protein is shown in Sequence ID No.

3.

6. The oral vaccine composition according to claim 1, further comprising a solution containing an adjuvant.

7. The oral vaccine composition according to claim 1, wherein the pupa is contained in an amount of at least 20% by weight relative to the total weight of the composition.

8. The oral vaccine composition according to claim 1 for feline parvovirus-related disease or feline calicivirus-related disease.

9. The oral vaccine composition according to claim 8, wherein the feline parvovirus-related disease is at least one selected from the group consisting of feline panleukopenia (feline distemper), feline infectious enteritis, and cerebellar hypoplasia.

10. The oral vaccine composition according to claim 8, wherein the feline calicivirus-related disease is at least one selected from the group consisting of feline calicivirus infection and feline viral upper respiratory tract infection.

11. The oral vaccine composition according to claim 1, which is used to be administered to felines after administering an inactivated vaccine or a live attenuated vaccine of a virus that causes feline viral infections.

12. A method for producing an oral vaccine against a baculovirus, comprising the steps of infecting the larva or pupa of a baculovirus-infectious insect with a recombinant baculovirus into which DNA encoding a viral protein that causes feline viral infection has been introduced, and drying the pupa that has developed from the infected larva, or the pupa that has been infected.

13. A method for producing an oral vaccine against a virus, comprising the steps of infecting baculovirus-infectious cells with a recombinant baculovirus into which DNA encoding a viral protein that causes feline viral infection has been introduced, and drying the infected cells.

14. The method according to claim 11 or 12, wherein the viral protein causing feline viral infection is feline calicivirus infection virus protein or feline parvovirus infection virus protein.

15. The method according to claim 13, wherein the DNA encoding the feline parvovirus infection virus protein is shown in Sequence ID No.

1.

16. The method according to claim 13, wherein the DNA encoding the feline calicivirus infection virus protein is shown in Sequence ID No.

3.

17. The method according to claim 11, wherein the insect is a silkworm.

18. The method according to claim 12, wherein the cells are derived from the mulberry spotted fly moth, the sax moth, the silkworm, the armyworm, or the nettle moth.

19. A pet food product comprising the vaccine composition according to any one of claims 1 to 10.

20. A method for preventing or treating feline viral infections, comprising the step of administering a vaccine composition according to any one of claims 1 to 10 to a feline animal.

21. A method for preventing or treating feline viral infections, comprising the step of administering an inactivated vaccine or a live attenuated vaccine of a virus that causes feline viral infections to a feline animal, and then administering the vaccine composition according to any one of claims 1 to 10 to the cat.

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