Oral vaccine composition

Abstract

[Problem] To provide: a method for simply producing an immunogenic chrysalis for oral administration; and a chrysalis which is for oral administration and produced by said method. [Solution] This method for producing a chrysalis for oral use comprises a step for infecting a larva or chrysalis of a baculovirus infectious insect with a recombinant baculovirus, into which DNA encoding an antigen protein has been introduced, and freeze-drying a chrysalis pupated from the infected larva or the infected chrysalis.

Description

[Technical field]

[0001] The present invention relates to pupae for oral administration, an oral vaccine composition, and methods for producing the same. [Background technology]

[0002] Baculovirus is a nuclear polyhedrosis virus (Nucleopolyhedrovirus: NPV) that infects insects as its main host, and forms a crystalline protein called polyhedrin (polyhedron) in the nucleus of infected cells during the proliferation process. One method for producing a target protein using the baculovirus-silkworm system is to introduce a gene encoding the target protein into the baculovirus, and inoculate the recombinant baculovirus into silkworm larvae or pupae to cause the silkworm to produce the target protein (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.

[0003] However, when producing a vaccine, steps such as extraction and / or purification of the antigen protein produced by silkworms are usually required. Therefore, there is a need for the development of a method for more easily producing vaccines using the baculovirus-silkworm system. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Maeda et al., “Production of human α-interferon in silkworm using a baculovirus vector.” Nature, 315, 592-594 (1985) Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a method for easily producing immunogenic pupae for oral administration and the pupae produced thereby. It also aims to provide an oral vaccine composition containing the pupae for oral administration and a method for producing the same. [Means for solving the problem]

[0006] The present inventors have surprisingly found that by infecting silkworm pupae with a recombinant baculovirus carrying DNA encoding an antigenic protein and then freeze-drying the pupae, it is possible to maintain the immunogenicity, thereby completing the present invention.

[0007] That is, the present invention relates to, for example, the following inventions. (1) Baculovirus-infected insect pupae that have been infected with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced and then freeze-dried. (2) Baculovirus-infected cells that have been infected with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced and then freeze-dried. (3) An oral vaccine composition comprising the pupa described in (1) and / or the cell described in (2). (4) The oral vaccine composition according to (3), further comprising a solution containing an adjuvant. (5) The oral vaccine composition according to (3) or (4), wherein the pupae constitute at least 20% by weight of the total weight of the composition. (6) A functional food comprising the pupa described in (1) and / or the cell described in (2). (7) A method for producing oral pupae, comprising the steps of infecting larvae or pupae of a baculovirus-infectible insect with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced, and freeze-drying the pupae that have developed from the infected larvae or the infected pupae. (8) A method for producing cells for oral administration, comprising the steps of infecting baculovirus-infectable cells with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced, and freeze-drying the infected cells. (9) A method for producing an oral vaccine, comprising the steps of infecting larvae or pupae of a baculovirus-infectible insect with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced, and freeze-drying the pupae that have developed from the infected larvae or the infected pupae. (10) A method for producing an oral vaccine, comprising the steps of infecting a baculovirus-infectable cell with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced, and freeze-drying the infected cell. (11) The method according to (7) or (9), wherein the insect is a silkworm. (12) The method according to (8) or (10), wherein the cell is derived from Butler moth, Anemone persica, Bombyx mori, Mamestra brassicae, or Polypodium nigra. (13) A method for inducing immunity against an antigen protein in a subject by administering to the subject the pupa described in (1), the cell described in (2), the oral vaccine composition described in any one of (3) to (5), or the food described in (6). Effect of the Invention

[0008] The present invention provides a method for easily producing immunogenic pupae for oral administration and an oral vaccine composition containing the same. In addition, the produced pupae for oral administration can be used as is for administration, and therefore does not require, for example, extraction and purification of antigen proteins or treatment with drugs. [Brief description of the drawings]

[0009] [Figure 1] The immunogenicity of pupae expressing PCV2a and pupae expressing PPV VP2 was confirmed by ELISA using serum from mice that had eaten the pupae. The top of Figure 1 shows the results for PBS coating, and the bottom of Figure 1 shows the results for bicarbonate buffer coating. "No dilution" shows the results when the primary antibody was used as is, and "1 / 50 dilution" shows the results when the primary antibody was diluted 50 times. [Diagram 2]This is a diagram showing antigen-specific IgG antibody or IgA antibody production response against PCV2 ORF2 antigen. Upper left: IgG antibody production response (OD450 nm) of serum (undiluted) and intestinal lavage fluid (undiluted). Lower left: IgG antibody production response (OD450 nm) of serum (50-fold dilution) and intestinal lavage fluid (50-fold dilution). Upper right: IgA antibody production response (OD450 nm) of serum (undiluted) and intestinal lavage fluid (undiluted). Lower right: IgA antibody production response (OD450 nm) of serum (50-fold dilution) and intestinal lavage fluid (50-fold dilution). Values ​​show the average value of N=3. Serum and intestinal lavage fluid of the non-administered group are described as Naive. [Diagram 3] This is a diagram showing antigen-specific IgG antibody or IgA antibody production response to PPV VP2 antigen. Upper left: IgG antibody production response (OD450 nm) of serum (undiluted) and intestinal lavage fluid (undiluted). Lower left: IgG antibody production response (OD450 nm) of serum (50-fold dilution) and intestinal lavage fluid (50-fold dilution). Antigen-specific production response to PPV VP2 antigen. Upper right: IgA antibody production response (OD450 nm) of serum (undiluted) and intestinal lavage fluid (undiluted). Lower right: IgA antibody production response (OD450 nm) of serum (50-fold dilution) and intestinal lavage fluid (50-fold dilution). Values ​​show the average value of N=3. Serum and intestinal lavage fluid of the non-administered group are described as Naive. [Figure 4] This is a diagram showing antigen-specific antibody production responses to the NV VP1 antigen. Left: IgG antibody production responses (OD450 nm) of serum (50-fold dilution) and intestinal lavage fluid (50-fold dilution). Right: IgA antibody production responses (OD450 nm) of serum (50-fold dilution) and intestinal lavage fluid (50-fold dilution). Values ​​show the average values ​​of N=6. Serum and intestinal lavage fluid from the non-administration group are referred to as "Naive." The group administered with pupae twice a week is referred to as the "twice a week administration group," and the group administered with pupae five times a week is referred to as the "five times a week administration group." DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1. Overview When producing vaccine antigens against infectious diseases using the silkworm heterologous protein expression system, a recombinant baculovirus is prepared by inserting a vaccine antigen gene derived from a target pathogenic microorganism (such as a virus) and inoculating this into silkworm larvae or pupae, causing the virus to grow within the silkworm and produce the vaccine antigen. In conventional vaccines administered by injection, some form of purification of the vaccine antigen protein is required. To purify the vaccine, a combination of multiple chromatograms, such as affinity purification, ion exchange purification, and ammonium sulfate precipitation, is required. However, if silkworm pupae expressing the vaccine antigen could be administered as the vaccine antigen, it would be possible to reduce the purification cost and the labor involved in administering by injection.

[0011] In the present invention, pupae expressing vaccine antigens or cells expressing vaccine antigens were freeze-dried to successfully obtain pupae and cells with high antigenicity. These pupae or cells were then fed to mice to verify whether antibody production responses were actually enhanced. As a result, a significant enhancement of antibody production responses was observed against two types of porcine virus antigens and one type of human virus antigen, and it was found that antibodies could even be induced in the blood and intestinal tract. The present invention is based on the above findings.

[0012] The present invention provides a method for producing pupae for oral use, which comprises the steps of infecting baculovirus-infectable insect larvae or pupae with a recombinant baculovirus having DNA encoding an antigen protein introduced therein, and freeze-drying the pupae that have developed from the infected larvae or the infected pupae.The present invention also provides a method for producing cells for oral use, which comprises the steps of infecting baculovirus-infectable cells with a recombinant baculovirus having DNA encoding an antigen protein introduced therein, and freeze-drying the infected cells. Both the pupae and the cells after freeze-drying can be used as oral vaccines.

[0013] 2. Recombinant baculovirus into which DNA encoding an antigen protein has been introduced As used herein, an antigenic protein refers to a protein that exhibits antigenicity, i.e., immunogenicity. The antigen is not particularly limited, and various known antigens that can be used can be arbitrarily selected and used. Examples include virus antigens, bacterial antigens, fungal antigens, and parasitic antigens. Specific examples include circovirus antigens such as porcine circovirus antigen (PCV), parvovirus antigens such as porcine parvovirus antigen (PPV), norovirus antigens, influenza virus antigens, coronavirus antigens such as SARS-CoV2, herpesvirus antigens, iridovirus antigens, rhabdovirus antigens, birnavirus antigens, malaria antigens such as Plasmodium vivax transmission blocking vaccine candidate antigens (Pvs25, Pvs28), Staphylococcus aureus antigens, Aeromonas antigens, Mycobacterium tuberculosis antigens, Actinobacillus pleuropneumoniae antigens, Mycoplasma hydatidis ... Examples of antigen proteins include B. onymoniea antigen, B. bronchiseptica antigen, B. pasteurella multocida antigen, B. mycoplasma hyorhinis antigen, B. parasuis antigen, B. escherichia coli antigen, B. cholera suis antigen, B. streptococcus antigen, B. influenza virus antigen, B. AIDS virus antigen, B. epidemic diarrhea virus antigen, B. transmissible gastroenteritis virus antigen, B. porcine reproductive and respiratory syndrome virus antigen, B. Japanese encephalitis virus antigen, B. pseudorabies virus antigen, B. trypanosoma antigen, B. pasteurella multocida toxin, B. coccidia antigen, and B. theileria antigen. More specific examples of antigen proteins include ORF2 (1-233 (SEQ ID NO: 1)) of the isolated strain PCV2a (GenBank accession #AF055392) of porcine circovirus type 2 (PCV2), and VP2 (T3 d17) of porcine parvovirus (PPV).

[0014] The size of the antigen is not particularly limited and may be, for example, 5 to 1000 kDa, 10 to 500 kDa, or 30 to 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 antibody 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 immunohistochemical staining.

[0015] The method of introducing DNA encoding an antigen protein into a baculovirus can be carried out by a method known to those skilled in the art, for example, the method described in Maeda et al., Nature 315, 592-594 (1985) or Y. Matsuura et al., Virology, (1989) 173, 674-682. For example, DNA (including cDNA) encoding an antigen protein is cloned and incorporated into a baculovirus transfer vector to obtain a recombinant baculovirus transfer vector. Next, using this recombinant baculovirus transfer vector, recombinant baculovirus DNA is obtained by homologous recombination or transposon transfer. This recombinant baculovirus DNA is introduced into cultured insect cells by a known method such as lipofection to obtain a recombinant baculovirus.

[0016] Baculovirus transfer vectors are obtained by subcloning a DNA fragment containing the polyhedrin gene of the baculovirus genomic DNA into a plasmid, and can be prepared by known methods or commercially available vectors such as pAcYM1, pAcG2T, pAcGP67, and VL1392 (all manufactured by Pharmingen) and pDEST8 (manufactured by Invitrogen).

[0017] In the present invention, examples of the type of baculovirus used to prepare a recombinant baculovirus include Autographa californica multiple nucleopolyhedrovirus (AcNPV), Bombyx mori nucleopolyhedrovirus (BmNPV), Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpNPV), and Lymantria disper multiple nucleopolyhedrovirus (LdNPV), and are preferably Bombyx mori nuclear polyhedrosis virus (BmNPV).

[0018] 3. Infection of baculovirus-infected insect larvae or pupae In the present invention, the recombinant baculovirus is infected into host baculovirus-infectable insect larvae or pupae or baculovirus-infectable cells (collectively referred to as "host"). Host insects include lepidopteran insects, and are not particularly limited as long as they are suitable for protein expression and have baculovirus infectivity, and examples thereof include Spilosoma imparilis, Antheraea pernyi, Bombyx mori, Spodoptera frugiperda, etc. Insects may be in either the pupal or larval form.

[0019] In addition, the host cells are not particularly limited as long as they are baculovirus-infectable cell lines suitable for protein expression. One of the characteristics of baculovirus infectivity is the expression of a glycoprotein called GP64 on the cell surface. Therefore, as long as the cells have such properties, the type of cells is not limited, and examples of such cells include cultured cells such as insect cells. Insect cells include the following: Cells derived from the mulberry butterfly (Spilosoma imparilis): SpIm Cells derived from Antheraea pernyi: Anpe Silkworm (Bombyx mori) derived cells: BmN, BmN4, Oyanagi-2, Bme21 Cells derived from the armyworm (Spodoptera frugiperda): Sf9, Sf21

[0020] The recombinant baculovirus can be infected into a host insect or cell by a method known to those skilled in the art. For example, the recombinant baculovirus obtained in the above step can be injected into a pupa or larva. When infecting a host cell, a liquid containing the recombinant baculovirus can be added to a cell culture medium. After infecting a host insect or host cell with the recombinant baculovirus and culturing it for 5 to 8 days, the larvae will pupate into pupae, and the antigen protein will be expressed in the host insect or host cell.

[0021] In the case of producing a target protein using a conventional baculovirus-silkworm system, the target protein is expressed in the bodies of silkworm pupae or larvae infected with a recombinant baculovirus, and then the pupae are crushed or the body fluids are collected from the larvae and various purification procedures are carried out to purify the target protein. In contrast, the method of the present invention does not require such extraction and purification procedures for the target protein, and the pupae or cells in which the antigen protein is expressed can be freeze-dried and then used as is.

[0022] 4. Freeze-drying of hosts infected with recombinant baculovirus The timing for freeze-drying the host pupae or cells infected with the recombinant baculovirus is preferably after a sufficient amount of antigen protein is expressed within the host (for example, within the body of a silkworm pupa). In addition, when a larva is infected with a baculovirus, it is allowed to grow until it becomes a pupa. Therefore, the timing for freeze-drying is after pupation if the infected silkworm is a larva, and freeze-drying can be performed at any time after infection if the infected silkworm is a pupa. Freeze-drying may be performed without cutting or crushing the pupae infected with the recombinant baculovirus while they are still in the pupal shape, or may be performed after cutting or crushing the pupae. In this specification, the expressions "as is" and "in pupal shape" refer to a pupa "as is" and "in pupal shape" mean using or including substantially the same pupa shape. For example, even if a part is unintentionally chipped during the manufacturing process, it may be determined that the pupa still retains its pupa shape as long as it maintains 90% or more, 80% or more, 70% or more, 60% or more, or 50% or more of the pupa shape.

[0023] Freeze-drying is a method of drying under reduced pressure (e.g., maintained in a vacuum state) in a frozen state. Freeze-drying can be performed by a method known to those skilled in the art, for example, using a commercially available freeze-dryer. The freezing temperature can be appropriately set by those skilled in the art, and is, 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 appropriately adjusted by those skilled in the art, and are, 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 36 hours.

[0024] By freeze-drying, most of the moisture in the pupae or cells is removed, resulting in a dry state. The moisture content of the freeze-dried pupae may be, for example, 1% by weight or less, 0.5% by weight or less, 0.1% by weight or less, 0.01% by weight or less, or 0.001% by weight or less, based on the total weight of the pupae or cells. From the viewpoint of easily maintaining immunogenicity, the moisture content of the pupae is preferably as close to 0% by weight as possible.

[0025] Here, in the present invention, after the freeze-drying, the pupae or cells can be optionally subjected to a pressurized heat treatment. The timing of pressurizing and heating is preferably after the pupae or cells have been sufficiently dried. The process of freeze-drying the host infected with the recombinant baculovirus and the process of pressurizing and heating can be performed consecutively, that is, the pressurized and heated treatment can be performed without any other treatment process after freeze-drying. The timing after freeze-drying is appropriately adjusted depending on the condition of the dried silkworm pupae, and can be performed, for example, within 24 hours, 12 hours, 3 hours, or 1 hour after freeze-drying. It is preferable to pressurize and heat the freeze-dried pupae while keeping them in the shape of pupae without cutting or crushing them.

[0026] The pressurized heat treatment means a treatment in which pressure is applied in a heated state. The pressurized heat treatment can be performed by a method known to those skilled in the art, for example, using a commercially available pressurized heat treatment device (for example, an autoclave device, etc.). The pressurizing conditions can be appropriately set by those skilled in the art, and may be performed, for example, by pressurizing to 0.1 MPa to 0.6 MPa, or 0.2 MPa to 0.5 MPa. The heating conditions can also be appropriately adjusted by those skilled in the art, and may be performed, for example, at 100°C to 150°C, or 110°C to 140°C. The time for which the pressurized heat treatment is performed is not particularly limited, and may be, for example, 3 minutes to 90 minutes, 5 minutes to 60 minutes, or 10 minutes to 30 minutes.

[0027] The pupae and cells that have been subjected to pressure and heat treatment maintain their immunogenicity, and therefore, in the present invention, there is no need to extract the antigen protein, and they can be administered orally as is.

[0028] Furthermore, in one aspect of the present invention, the freeze-dried pupa is dry and spongy, so that the liquid can be easily permeated into the inside by immersing it in a liquid. Therefore, the manufacturing method according to this embodiment may further include a step of immersing the freeze-dried pupa, or the pressurized and heated pupa as necessary, in a liquid. The liquid to be permeated is preferably a solution containing an adjuvant, since it can easily improve the effect as a vaccine. The solution containing the adjuvant can be easily permeated into the inside by immersing the freeze-dried pupa in a solution containing an adjuvant. Since cultured cells are freeze-dried to form a powdered composition, when cells are used, the freeze-dried cells may be immersed in a solution containing an adjuvant.

[0029] The pupae or cells produced according to the present invention may be used as they are, or may be cut or crushed before use, or may be permeated with a liquid as described above. Examples of the form in which the pupae are used as they are include a form in which the pupae are orally administered as a vaccine, and a form in which the pupae are ingested as they are for food.

[0030] Examples of the form in which the pupae are cut or crushed include administering the cut or crushed pupae as is, administering the crushed pupae by mixing them with other orally administrable materials, and mixing the crushed pupae with feed to be ingested as food. For example, the pupae may be cut into 1 / 2, 1 / 3, 1 / 4, etc., in order to adjust the dosage in consideration of the immunogenicity of the pupae. The degree of crushing is not particularly limited as long as it shows immunogenicity, and may be, for example, powdered. Cutting can be performed, for example, with food scissors and laboratory animal scissors, and crushing can be performed, for example, with a mixer, hand mill, and grinding device. When cells are used, they can be treated in the same manner as when the pupae were crushed.

[0031] Oral administration includes, for example, oral administration and sublingual administration. In this specification, administration also includes the case where the subject ingests it by itself. When administering the pupae in their original form, oral administration is preferred, and the subject may eat them by themselves.

[0032] Orally administrable additives include, for example, food materials, beverage materials, excipients, thickeners, stabilizers, preservatives, pH regulators, sweeteners, colorants, emulsifiers, flavorings, pharmaceutical additives described below, etc. They can also be mixed with food materials or beverage materials and administered as functional foods or beverages. The liquid containing the orally administrable material is not particularly limited as long as the orally administrable pupae have immunogenicity and are a liquid suitable for oral administration, and may be, for example, water or an aqueous solution.

[0033] 5. Oral vaccine composition The pupae or cells obtained as described above contain the foreign antigen protein and have immunogenicity, and therefore can be used as an oral vaccine composition. The oral vaccine composition may be in the form of intact pupae or in any other form, such as liquid (syrup, jelly, etc.), powder, granules, tablets, powder, capsules, etc. When cells are used as an oral vaccine composition, they may also be in any form, such as powder, granules, tablets, powder, capsules, etc.

[0034] The above dosage forms are formulated with pharmaceutical additives by methods commonly used in the art. Examples of pharmaceutical additives include adjuvants, oral administration carriers, diluents, excipients, disintegrants, binders, lubricants, fluidizing agents, coating agents, solubilizers, solubilizing aids, thickeners, dispersants, stabilizers, preservatives, pH regulators, tonicity regulators, wetting agents, sweeteners, and flavors. From the viewpoint of improving the effect of the vaccine, it is preferable that the pharmaceutical additives contain adjuvants.

[0035] Examples of adjuvants include Freund's complete adjuvant, incomplete Freund's adjuvant, Bordetella pertussis adjuvant, Ribi adjuvant, lipid A, liposome, aluminum hydroxide, silica, and the like.

[0036] In the present invention, a step of mixing immunogenic pupae or cells with pharmaceutical additives or liquids containing them may be further included. In the oral vaccine composition of the present invention, the pupae or cells contain expressed antigen proteins. Thus, the vaccine composition may contain immunogenic pupae or cell components (e.g., freeze-dried pupae powder) in an amount of, for example, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more based on the total weight of the vaccine composition.

[0037] The liquid used as a pharmaceutical additive is not particularly limited as long as the oral vaccine composition has immunogenicity and is a liquid that is pharma- ceutical suitable for oral administration, and examples thereof include aqueous solutions such as water, physiological saline, and phosphate buffered saline (PBS).

[0038] The immunogenic pupae may be used as is, cut, or crushed. When cells are used, they may also be crushed. In the present invention, when the pupae are used in the form of pupae, the step of mixing with a liquid containing a pharmaceutical additive may include a step of immersing the freeze-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 inside of the pupae. The pupae into which the liquid has penetrated may be further cut or crushed to prepare an oral vaccine composition. In the case of cultured cells, they can be used in the same manner as crushed pupae.

[0039] When cut or crushed pupae are used, the step of mixing with a liquid containing a pharmaceutical additive may include a step of dispersing the cut or crushed pupae in the liquid.

[0040] The subjects to which the pupae for oral administration (vaccine) are administered are not particularly limited, and examples thereof include mammals, birds, reptiles, amphibians, and fish, etc. Mammals include humans and non-human mammals such as mice, rats, pigs, monkeys, cows, horses, sheep, rabbits, dogs, cats, and goats.

[0041] The orally administered vaccine of the present invention exhibits immunogenicity when orally administered in the form of a pupa, but may also be cut or crushed before administration, or may be used by permeating the pupa with a liquid, as described above. Examples of the form in which the pupae are administered in their pupal form include a form in which the pupae for oral administration are orally administered as they are as a vaccine, and a form in which the pupae for oral administration are ingested as they are as food.

[0042] Examples of the form of administration in which the pupae are cut or crushed (including cell crushed products) include a form in which the cut or crushed pupae or cells are orally administered as is, a form in which the cut or crushed pupae or cells are mixed with other orally administrable materials and administered, and a form in which the crushed pupae are mixed with feed and ingested as food. For example, in order to adjust the dosage taking into account the immunogenicity of the pupae, the pupae may be cut into, for example, 1 / 2, 1 / 3, 1 / 4, etc. The degree of crushing is not particularly limited as long as it exhibits immunogenicity, and may be, for example, powdered.

[0043] The dosage can be appropriately set in consideration of the subject of administration and the amount of antigen protein expressed in the pupa or cells. For example, 1 / 10 to 2 pupae (0.08 g to 1.6 g) may be administered per time, or 1 / 2 to 3 / 2 pupae (0.2 g to 1.2 g) may be administered per time. The amount of expressed antigen protein varies depending on the type of antigen protein, but when PCV2 and PPV are used, it is 0.2 mg to 10 mg per pupa weight of about 800 mg, which is 0.025% by weight to 1.25% by weight per pupa. This means that one pupa for oral administration contains 10 times or more of antigen virus protein than the amount of one dose of a normal injection vaccine.

[0044] The number and duration of administration can be appropriately set in consideration of the subject of administration, the amount of antigen protein expressed in the pupae or cells, etc. For example, administration may be performed 1 to 5 times per day, 1 to 3 times per day, or once per day. Furthermore, administration may be performed, for example, 1 to 7 days per week, 1 to 5 days per week, or 1 to 4 days per week. Furthermore, these administrations may be repeated, for example, for 2 to 10 weeks, 1 to 7 weeks, 2 to 5 weeks, or 2 to 4 weeks. When administering the vaccine, it is advisable to fast for about 1 to 3 hours before administration (before eating).

[0045] Specifically, for example, it can be administered as follows. Administration target: Mice Dosage: 1 / 4 to 1 pupa (0.2g to 1.6g) should be administered once per day (ingestion). Duration: Repeat 4-5 doses per week for 3-4 weeks Thereafter, booster immunization may be performed by similarly administering the vaccine every 1 to 4 weeks, preferably every 1 to 2 weeks.

[0046] The pupae and cells of the present invention can also be used in combination with other vaccines. The other vaccines may have the same immunogenicity against the antigenic protein as the pupae or cells of the present invention, or may have the immunogenicity against a different antigenic protein. In the case where the other vaccine has the immunogenicity against the same antigenic protein as the pupae or cells of the present invention, the pupae of the present invention may be administered, for example, for boosting the other vaccine.

[0047] 6.Food The food of the present invention has immunogenicity because it contains pupae or cells. The food containing the pupae for oral administration is not particularly limited, and examples thereof include animal foods and plant foods. The food may also be in the form of animal feed.

[0048] The form in which the pupae for oral administration are included in food is not particularly limited as long as they exhibit immunogenicity, and the immunogenic pupae may be used in their original form, cut into pieces, or crushed. There are no particular limitations on the subjects that consume the food, and as mentioned above, examples of the subject include mammals, birds, reptiles, amphibians, fish, etc. Mammals include humans and non-human mammals such as mice, rats, pigs, monkeys, cows, horses, sheep, rabbits, dogs, cats, and goats.

[0049] 7. Methods for inducing immunity By administering the above-mentioned silkworm pupae for oral administration, or an oral vaccine composition or food containing the same to a subject, immunity can be induced in the subject. Administration etc. are as described above. The induction of immunity in a subject and the level of immunity can be confirmed by methods well known to those skilled in the art, for example, by measuring antibody production or antibody titer in the subject. EXAMPLES

[0050] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Example 1] Production of pupae for oral administration of vaccine antigens for livestock viruses DNA encoding ORF2 (1-233) of porcine circovirus type 2 (PCV2) isolate PCV2a (GenBank accession LC381288) or VP2 (T3 d17) of porcine parvovirus (PPV) was introduced into a silkworm baculovirus by standard methods (Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae. Journal of General Virology, 2018, volume 99, Issue 7, 917-926.), and the resulting recombinant baculovirus was injected into silkworm pupae to infect them. Eight days after the injection, the silkworm pupae were treated in a freeze-dryer at -40°C for 720 minutes.

[0051] [Example 2] Feeding test A feeding test of the pupae for oral administration prepared in Example 1 was carried out using 7-week-old female BALB / c mice (n=3). The animals were deprived of food and water for 2 hours before feeding. They were isolated individually and allowed to feed the pupae orally ad libitum. No water was given to them during feeding. The mice were fed pupae according to the schedule in the table below. There was no food restriction other than pupae. Each mouse was fed 14 pupae during the period.

[0052] [Table 1]

[0053] On day 22, blood was collected under anesthesia, and serum and intestinal lavage fluid were obtained.

[0054] [Example 3] Confirmation of specific antibody production response Using the serum and intestinal washing fluid obtained in Example 2, the immunogenicity of the pupae expressing PCV2a and the pupae expressing PPV VP2 was confirmed by ELISA, respectively. The serum from mice in the non-administered group was used as naive.

[0055] The coating antigen for the ELISA was prepared as follows. The dilution was carried out with PBS. PCV2a ORF2 (1-233): 0.44 mg / mL was diluted to 56.8 μL / 4943.2 μL (5 μg / mL), coated at 50 μL / well, and left to stand overnight at 4°C. PPV VP2: 0.34 mg / mL was diluted to 73.5 μL / 4926.5 μL (5 μg / mL), coated at 50 μL / well, and left to stand overnight at 4°C.

[0056] In addition, various antibodies, substrates, etc. were used as follows. Primary antibodies: Various immune sera and intestinal lavage fluid undiluted (undiluted) or 50-fold diluted (50 μL / well) Secondary antibody: 1 / 10,000 diluted Anti-Mouse IgG or IgA-HRP in 0.5% BSA was used at 50 μL / well and incubated at 37° C. for 1 hour. Blocking: 1% BSA in PBS was used at 200 μL / well and the plate was left to stand at 37° C. for 2 hours. Wash: 1 / 50 dilution with 0.5% BSA or undiluted solution was used at 50 μL / well and incubated at 37° C. for 2 hours. Substrate: 1step Turbo TMB-ELISA (Thermo Scientific, 100 μL / well) was used and the plate was left to stand at room temperature for 10 minutes. Stop solution: Treat with 2M H2SO4 (100 μL / well).

[0057] The results of measuring the absorbance at 450 nm (OD450 value) of each well are shown in Figures 1 to 3. In the results for PCV2, a high IgG antibody production response was observed in the serum and intestinal lavage fluid. In addition, when the IgA response was evaluated, antibody production was observed in the serum, but higher production was observed in the intestinal lavage fluid, and contrary to expectations, a sufficient antibody production response was observed not only in the original solution, but also in a 50-fold dilution (Figure 2). On the other hand, in the case of PPV, IgG and IgA antibody production responses were observed in the serum, but no antibody production response was observed in the intestinal lavage fluid (Figure 3). It is believed that these differences are due to differences in the properties of the viruses.

[0058] [Example 4] Production of pupae for oral administration of vaccine antigens for human viruses DNA encoding VP1 of norovirus (NV) genotype GII.4 (GenBank accession BAG70500.1) was introduced into a silkworm baculovirus by a standard method, and the resulting recombinant baculovirus was injected into silkworm pupae to infect them. Eight days after the injection, the silkworm pupae were treated in a freeze-dryer at -40°C for 720 minutes.

[0059] [Example 5] Feeding test A feeding test of the pupae for oral administration prepared in Example 1 was carried out using 7-week-old female BALB / c mice (n=6). The animals were deprived of food and water for 2 hours before feeding. They were isolated individually and allowed to feed the pupae orally ad libitum. No water was given to them during feeding. The mice were fed pupae according to the schedules in Tables 2 and 3 below. There was no food restriction other than pupae. During the period, each mouse was fed 14 or 6 pupae.

[0060] [Table 2] [Table 3]

[0061] On day 21, blood was collected under anesthesia, and serum and intestinal lavage fluid were obtained.

[0062] [Example 6] Confirmation of specific antibody production response Using the serum and intestinal washing fluid obtained in Example 5, the immunogenicity of the pupae expressing NV VP1 was confirmed by ELISA. The serum from mice in the non-administered group was used as naive. The coating antigen for the ELISA was prepared as follows. The dilution was carried out with PBS. NV VP2: 1.36 mg / mL was diluted to 18.4 μL / 4981.6 μL (5 μg / mL), coated at 50 μL / well, and left to stand overnight at 4°C.

[0063] In addition, various antibodies, substrates, etc. were used as follows. Primary antibody: Various immune sera and intestinal lavage fluid diluted 50-fold (50 μL / well) Secondary antibody: 1 / 10,000 diluted Anti-Mouse IgG or IgA-HRP in 0.5% BSA was used at 50 μL / well and incubated at 37° C. for 1 hour. Blocking: 1% BSA in PBS was used at 200 μL / well and the plate was left to stand at 37° C. for 2 hours. Wash: 50 μL / well of a 1 / 50 dilution with 0.5% BSA was used and incubated at 37° C. for 2 hours. Substrate: 1step Turbo TMB-ELISA (Thermo Scientific, 100 μL / well) was used and the plate was left to stand at room temperature for 10 minutes. Stop solution: Treat with 2M H2SO4 (100 μL / well).

[0064] The absorbance at 450 nm (OD450 value) of each well was measured, and the results are shown in Figure 4. From the results in Figure 4, a high IgG antibody production response was observed in the serum and intestinal lavage fluid. In addition, when the IgA response was evaluated, antibody production was also observed in the serum and intestinal lavage fluid. Compared to the injectable vaccine, an antibody production response was observed at a level comparable to that of the injectable vaccine, even without the concomitant administration of an adjuvant. When the antibody production response was evaluated by dividing the administration frequency into two groups, one five times a week and one twice a week, an enhanced antibody production response was observed in the five times a week administration group.

Claims

1. An inducer for the production of IgA and / or IgG against a parvovirus antigen protein, comprising freeze-dried pupae and / or cells of a baculovirus-infected insect, which contains a recombinant baculovirus containing DNA encoding a parvovirus antigen protein, said inducer being an oral formulation, and said IgA and / or IgG being produced in serum by oral administration.

2. The inducer of claim 1 , further comprising a solution containing an adjuvant.

3. The inducer according to claim 1 , wherein the pupae constitute at least 20% by weight of the total weight of the freeze-dried product.

4. The inducer according to claim 1, wherein the insect is a silkworm.

5. The inducer according to claim 1, wherein the cells are derived from Mackerel moth, Anemone persica, Bombyx mori, Mamestra brassicae or Arctium nigra.

6. A functional food comprising the inducer according to claim 1.

7. A feed comprising the inducer according to claim 1.

8. A method for inducing the production of IgA and / or IgG against a parvovirus antigen protein in the serum of a subject (excluding humans) by orally administering to the subject the inducer described in claim 1, the functional food described in claim 6, or the feed described in claim 7.

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