Oral vaccine composition

Abstract

To provide a method for easily producing immunogenic pupae for oral administration, and to provide pupae for oral administration produced thereby.SOLUTION: Provided are a method for producing oral pupae, comprising: infecting baculovirus-infectible insect larvae or pupae 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.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Baculovirus is a nucleopolyhedrovirus (NPV) that infects insects as the main host, and forms a protein with a crystal structure called polyhedrin (also known as polyhedron) in the nucleus of infected cells during the growth process. Therefore, as one method for producing a target protein using the baculovirus-silkworm system, a gene encoding the target protein is introduced into the baculovirus, and the recombinant baculovirus is inoculated into silkworm larvae or pupae to produce the target protein in silkworms (Non-Patent Document 1). When producing a vaccine 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 in silkworms are usually required. Therefore, development of a method for more simply producing a vaccine using the baculovirus-silkworm system is desired.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention aims to provide a method for simply producing pupae for oral administration having immunogenicity and the pupae for oral administration produced thereby. The present invention also aims to provide an oral vaccine composition containing the above-mentioned pupae for oral administration and a method for producing the same.

Means for Solving the Problems

[0006] The present inventors have found that, surprisingly, by infecting pupae of silkworms with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced and then freeze-drying them, immunogenicity can be maintained, and the present invention has been completed.

[0007] That is, the present invention relates to, for example, the following inventions. (1) A pupa of a baculovirus-infected insect that has been subjected to an infection treatment and a freeze-drying treatment with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced. (2) A cell having baculovirus infectivity that has been subjected to an infection treatment and a freeze-drying treatment with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced. (3) An oral vaccine composition containing the pupa according to (1) and / or the cell according to (2). (4) The oral vaccine composition according to (3), further containing a solution containing an adjuvant. (5) The oral vaccine composition according to (3) or (4), wherein the pupa is contained in an amount of at least 20% by weight based on the total weight of the composition. (6) A functional food containing the pupa according to (1) and / or the cell according to (2). (7) A method for producing pupae for oral use, comprising infecting a larva or pupa of a baculovirus-infected insect with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced, and freeze-drying the pupa pupated from the infected larva or the infected pupa after the infection. (8) A method for producing cells for oral use, comprising infecting a cell having baculovirus infectivity with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced, and freeze-drying the infected cell after the infection. A method for producing an oral vaccine, comprising infecting a recombinant baculovirus into which DNA encoding an antigen protein has been introduced into larvae or pupae of baculovirus-infectious insects, and freeze-drying the pupae pupated from the infected larvae or the infected pupae. (10) A method for producing an oral vaccine, comprising infecting a recombinant baculovirus into which DNA encoding an antigen protein has been introduced into baculovirus-infectious cells, and freeze-drying the infected cells. (11) The method according to (7) or (9), wherein the insect is a silkworm. (12) The method according to (8) or (10), wherein the cells are derived from Bombyx mori, Spodoptera frugiperda, silkworm, Trichoplusia ni or Eriogyna pyretorum. (13) A method for inducing immunity against an antigen protein in a subject by administering to the subject the pupa according to (1), the cell according to (2), the oral vaccine composition according to any one of (3) to (5), or the food according to (6). [Effect of the Invention]

[0008] The present invention provides a method for easily producing an orally administrable pupa having immunogenicity and an oral vaccine composition containing the same. In addition, the produced orally administrable pupa can be used for administration as it is, and thus does not require, for example, extraction and purification of an antigen protein and treatment with a drug. [Brief Description of the Drawings]

[0009]

Figure 1

Figure 2

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Mode for Carrying Out the Invention

[0010] 1. Overview When producing a vaccine antigen against an infectious disease using a silkworm heterologous protein expression system, a recombinant baculovirus into which a vaccine antigen gene derived from a target pathogenic microorganism (such as a virus) is inserted is prepared, and this is inoculated into silkworm larvae or pupae. As a result, the virus proliferates within the silkworm, and the vaccine antigen is produced. For vaccines administered by ordinary injection, it is necessary to purify some kind of vaccine antigen protein. And for purification, it is necessary to combine multiple chromatograms such as affinity purification, ion exchange purification, and ammonium sulfate precipitation. However, if the silkworm pupae themselves expressing the vaccine antigen can be administered as the vaccine antigen, it becomes possible to reduce the cost of purification, and a reduction in the labor involved in injection administration can be expected.

[0011] In the present invention, by freeze-drying pupae expressing a vaccine antigen or cells expressing a vaccine antigen, we successfully obtained pupae and cells with highly guaranteed antigenicity. Then, these pupae or cells were fed to mice to verify whether the antibody production response was actually enhanced. As a result, for two types of porcine virus antigens and one type of human virus antigen, a significant enhancement of the antibody production response was observed, and it was found that antibody induction in the blood and intestinal tract was also possible. The present invention is based on the above findings.

[0012] The present invention provides a method for producing pupae for oral use, which includes infecting larvae or pupae of baculovirus-infectious insects with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced, and freeze-drying the pupae pupated from the infected larvae or the infected pupae after infection. The present invention also provides a method for producing cells for oral use, which includes infecting cells with baculovirus infectivity with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced, and freeze-drying the infected cells after infection. These pupae and cells after freeze-drying treatment can both be used as oral vaccines.

[0013] 2. Recombinant baculovirus into which DNA encoding an antigen protein has been introduced In this specification, an antigen protein means a protein that exhibits antigenicity, that is, immunogenicity. The antigen is not particularly limited, and various antigens known as usable ones can be arbitrarily selected and used. For example, viral antigens, bacterial antigens, fungal antigens, parasitic antigens, etc. can be mentioned. Specific examples include circovirus antigens such as porcine circovirus antigen (PCV), parvovirus antigens such as porcine parvovirus antigen (PPV), norovirus antigen, influenza virus antigen, coronavirus antigens such as SARS-CoV2, herpes virus antigen, iridovirus antigen, rhabdovirus antigen, birnavirus antigen, malaria antigens such as transmission-blocking vaccine candidate antigens for tertian malaria (Pvs25, Pvs28), Staphylococcus aureus antigen, Aeromonas antigen, Mycobacterium tuberculosis antigen, Actinobacillus pleuropneumoniae antigen, Mycoplasma hyopneumoniae antigen, Bordetella bronchiseptica antigen, Pasteurella multocida antigen, Mycoplasma hyorhinis antigen, Haemophilus parasuis antigen, Escherichia coli antigen, Salmonella cholerasuis antigen, Streptococcus antigen, influenza virus antigen, AIDS virus antigen, porcine epidemic diarrhea virus antigen, transmissible gastroenteritis virus antigen, porcine reproductive and respiratory syndrome virus antigen, Japanese encephalitis virus antigen, Orf virus antigen, Trypanosoma antigen, Pasteurella multocida toxin, coccidium antigen, and Theileria antigen, etc. More specific examples of antigen proteins include ORF2 (1-233 (SEQ ID NO: 1)) of the isolate PCV2a (GenBank accession #AF055392) of porcine circovirus type 2 (PCV2), and VP2 (T3 d17) of porcine parvovirus (PPV), etc.

[0014] The size of the antigen is not particularly limited, and for example, it may be 5 to 1000 kDa, 10 to 500 kDa, or 30 to 200 kDa. Immunogenicity means the property of an antigen to induce the production of antibodies 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. The expression of antibodies 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 methods known to those skilled in the art. For example, it can be carried out by the methods described in Maeda et al., Nature 315, 592-594 (1985), Y. Matsuura et al., Virology, (1989) 173, 674-682, etc. 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 insect cultured cells by a known method such as the lipofection method to obtain a recombinant baculovirus.

[0016] A baculovirus transfer vector is 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 ones can be used. Examples of commercially available vectors include pAcYM1, pAcG2T, pAcGP67, and VL1392 (all from Pharmingen), and pDEST8 (from Invitrogen).

[0017] In the present invention, examples of the types of baculoviruses used for producing recombinant baculoviruses include Autographa californica multiple nucleopolyhedrovirus (AcNPV), Bombyx mori nucleopolyhedrovirus (BmNPV), Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpNPV), Lymantria disper multiple nucleopolyhedrovirus (LdNPV), etc., and Bombyx mori nucleopolyhedrovirus (BmNPV) is preferred.

[0018] 3. Infection of larvae or pupae of baculovirus-infected insects with baculovirus In the present invention, the recombinant baculovirus is infected into larvae or pupae of baculovirus-infected insects serving as hosts or baculovirus-infected cells (collectively also referred to as "hosts"). Examples of the insects serving as hosts include Lepidoptera insects, and are not particularly limited as long as they have baculovirus infectivity suitable for protein expression. For example, Spilosoma imparilis, Antheraea pernyi, Bombyx mori, Spodoptera frugiperda, etc. can be mentioned. The insects can be in either the pupal or larval form.

[0019] Also, the cells serving as hosts are not particularly limited as long as they are baculovirus-infected cell lines suitable for protein expression. One of the characteristics of having baculovirus infectivity is that a glycoprotein called GP64 is expressed on the cell surface. Therefore, as long as it has such properties, the cell type is not limited, and cultured cells such as insect cells can be mentioned. Examples of the insect cells include the following. Cells derived from Spilosoma imparilis: SpIm Cells derived from Antheraea pernyi: Anpe Cells derived from Bombyx mori: BmN, BmN4, Oyanagi-2, Bme21 Cells derived from Spodoptera frugiperda: Sf9, Sf21

[0020] The method of infecting a recombinant baculovirus into an insect or cell serving as a host can be carried out by a method known to those skilled in the art. For example, the recombinant baculovirus obtained in the above step is injected into a pupa or larva. When infecting host cells, a liquid containing the recombinant baculovirus may be added to the cell culture medium. When the host insect or host cell is infected with the recombinant baculovirus and then reared or cultured for 5 to 8 days, the larva pupates into a pupa. And in the host insect or host cell, the antigen protein is expressed.

[0021] Here, in the case of producing a target protein using the conventional baculovirus - silkworm system, after the target protein is expressed in the body of a silkworm pupa or larva infected with the recombinant baculovirus, the pupa is crushed or the body fluid is recovered from the larva, and the target protein is purified by performing various purifications. In contrast, in the method of the present invention, it is not necessary to perform such extraction and purification treatment of the target protein. After the pupa or cell in which the antigen protein is expressed is freeze - dried, it can be used as it is.

[0022] 4. Freeze - drying of the host infected with the recombinant baculovirus As for the timing of freeze-drying the pupa or cells of the host infected with the recombinant baculovirus, it is preferably after a sufficient amount of antigen protein has been expressed in the host (for example, inside the pupa of the silkworm). Also, when the larvae are infected with the baculovirus, they are allowed to grow until they become pupae. Therefore, the timing of freeze-drying is after pupation when the infected silkworm is in the larval stage, and if the infected silkworm is a pupa, it can be freeze-dried at any time after infection. Freeze-drying may be performed while keeping the pupa in the shape of the pupa without cutting or crushing the pupa infected with the recombinant baculovirus, or it may be performed after cutting or crushing the pupa. In this specification, the expressions of using the pupa "as it is" and "in the shape of the pupa" mean using or including it substantially in the shape of the pupa. For example, even if a part is missing accidentally during the manufacturing process, if 90% or more, 80% or more, 70% or more, 60% or more, or 50% or more of the shape of the pupa is maintained, it may be judged that it is in the shape of the pupa.

[0023] Freeze-drying is a method of drying under reduced pressure (for example, 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, it can be performed using a commercially available freeze-dryer. The freezing treatment temperature can be appropriately set by those skilled in the art, for example, -90°C to -5°C, -80°C to -10°C, or -50°C to -10°C. Also, the reduced pressure conditions can be appropriately adjusted 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 36 hours.

[0024] By freeze-drying, most of the moisture in the pupa or cells is removed, and it becomes a dry state. The moisture contained in the pupa after freeze-drying 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 pupa or cells. From the viewpoint of easily maintaining immunogenicity, the closer the moisture contained in the pupa is to 0% by weight, the more preferable it is.

[0025] Here, in the present invention, after the above freeze-drying, it is also possible to optionally perform pressure-heat treatment on the pupa or cells. As for the timing of pressure-heating, it is preferably after the pupa or cells are sufficiently dried. The step of freeze-drying the host infected with the recombinant baculovirus and the step of pressure-heat treatment are continuously performed, that is, after freeze-drying, the pressure-heat treatment can also be performed without passing through other treatment steps. The timing after freeze-drying is appropriately adjusted according to the situation of the dried silkworm pupa, etc. For example, it can be performed within 24 hours, within 12 hours, within 3 hours or within 1 hour after freeze-drying. It is preferable to perform the pressure-heat treatment on the freeze-dried pupa in the shape of the pupa without cutting or crushing it.

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

[0027] The pupa and cells subjected to the pressure-heat treatment maintain immunogenicity. Therefore, in the present invention, it is not necessary to extract the antigen protein, and it can be directly used for oral administration.

[0028] Furthermore, in one aspect of the present invention, since the freeze-dried pupa is dried and spongy, it can easily penetrate the liquid inside by being immersed in the liquid. Therefore, the manufacturing method according to the present embodiment may further include a step of immersing the freeze-dried pupa or the pupa subjected to pressure heating treatment if necessary in a liquid. As the liquid to be penetrated, a solution containing an adjuvant is preferably used because it can easily improve the effect as a vaccine. By immersing the freeze-dried pupa in a solution containing an adjuvant, the solution containing the adjuvant can be easily penetrated inside. Since culturing cells become a powdery composition when freeze-dried, when using cells, the freeze-dried cells may be immersed in a solution containing an adjuvant.

[0029] The pupa or cell produced by the present invention may be used as it is, may be used after being cut or crushed, or may be used after infiltrating a liquid as described above. Examples of the form of using as it is include a form of orally administering the pupa as it is as a vaccine and a form of ingesting the pupa as it is as food.

[0030] Examples of the form of using after cutting or crushing include a form of directly administering the cut or crushed pupa, a form of mixing the crushed pupa with other orally administrable materials and administering, and a form of mixing the crushed pupa with feed and ingesting it as food. For example, in order to adjust the dosage in consideration of the immunogenicity of the pupa, it 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 in powder form, for example. Cutting can be performed, for example, with edible scissors and scissors for experimental animals, and crushing can be performed, for example, with a mixer, a hand mill, and a pulverizer. When using cells, the same treatment as when crushing the pupa can be performed.

[0031] Examples of oral administration include buccal administration and sublingual administration. In this specification, administration includes cases where the subject ingests by himself / herself. When administering the pupa in its original form, buccal administration is preferable, and the subject may be allowed to eat it by himself / herself.

[0032] Examples of orally administrable additives include, for example, food materials, beverage materials, excipients, thickeners, stabilizers, preservatives, pH adjusters, sweeteners, colorants, emulsifiers, fragrances, pharmaceutical additives described below, etc. It can also be administered as a functional food or beverage by mixing with a food material or beverage material. The liquid containing an orally administrable material is not particularly limited as long as the pupa for oral administration has immunogenicity and is a liquid suitable for oral administration, and may be, for example, water or an aqueous solution.

[0033] 5. Oral vaccine composition The pupa or cells obtained as described above contain a foreign antigen protein and have immunogenicity, and thus are used as an oral vaccine composition. The oral vaccine composition can be in any dosage form such as, for example, a liquid preparation (syrup, jelly, etc.), powder, granule, tablet, powder, or capsule, in addition to the shape of the pupa as it is. When using cells as an oral vaccine composition, it can also be in any dosage form such as powder, granule, tablet, powder, or capsule.

[0034] The above dosage forms are formulated with pharmaceutical additives by methods commonly used in the art. Examples of pharmaceutical additives include, for example, adjuvants, carriers for oral administration, diluents, excipients, disintegrants, binders, lubricants, fluidizing agents, coating agents, solubilizing agents, solubilization aids, thickeners, dispersants, stabilizers, preservatives, pH adjusters, tonicity adjusters, wetting agents, sweeteners, and fragrances, etc. From the perspective of improving the effect as a vaccine, it is preferable that the pharmaceutical additive contains an adjuvant.

[0035] Examples of adjuvants include, for example, Freund's complete adjuvant, incomplete Freund's adjuvant, pertussis adjuvant, Ribi adjuvant, lipid A, liposomes, aluminum hydroxide, silica, etc.

[0036] In the present invention, a step of mixing an immunogenic pupa or cell with a pharmaceutical additive or a liquid containing them can be further included. In the oral vaccine composition of the present invention, the expressed antigen protein is contained in the pupa or cell. Therefore, the components of the immunogenic pupa or cell (for example, the powder of the pupa after freeze-drying) can be contained 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 the pharmaceutical additive is not particularly limited as long as the oral vaccine composition has immunogenicity and is a liquid pharmaceutically suitable for oral administration. Examples thereof include aqueous solutions such as water, physiological saline, or phosphate buffer solution (PBS).

[0038] The immunogenic pupa may be used as it is, cut, or crushed. When using cells, they can also be crushed. In the present invention, when using the pupa in the shape of a pupa, the step of mixing with the liquid containing the pharmaceutical additive may include a step of immersing the freeze-dried pupa in the liquid. For example, by immersing the pupa in a solution containing an adjuvant, the solution containing the adjuvant can be easily penetrated inside. The pupa into which the liquid has penetrated may be further cut or crushed to obtain an oral vaccine composition. The same applies to cultured cells as in the case of the crushed pupa.

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

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

[0041] 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, and as described above, it may be used by infiltrating a liquid. Examples of the form of administration in the form of a pupa include a form in which the pupa for oral administration is directly orally administered as a vaccine, and a form in which the pupa for oral administration is directly ingested as food.

[0042] Examples of the form of administration after cutting or crushing (including cell lysates) include a form in which the cut or crushed pupa or cell is directly orally administered, a form in which the cut or crushed pupa or cell is mixed with other orally administrable materials and then administered, and a form in which the crushed material is mixed with feed and ingested as food. For example, in order to adjust the dosage in consideration of the immunogenicity of the pupa, it 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 it may be in powder form, for example.

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

[0044] The number of administrations and the period can be appropriately set in consideration of the administration target and the amount of antigen protein expressed in the pupa or cells. For example, it may be administered 1 to 5 times a day, 1 to 3 times a day, or 1 time a day. Also, for example, it may be administered 1 to 7 days a week, 1 to 5 days a week, or 1 to 4 days a week. Further, 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 feeding).

[0045] Specifically, for example, it can be administered as follows. Administration target: In the case of mice Dosage: Administer 1 / 4 to 1 pupa (0.2 g to 1.6 g) once a day (during feeding) Period: Repeat the administration 4 to 5 times a week for 3 to 4 weeks Subsequently, for example, booster immunization may be performed by administering in the same manner every 1 to 4 weeks, preferably every 1 to 2 weeks.

[0046] The pupa and cells of the present invention can also be used in combination or in combination with other vaccines. The other vaccines may have immunogenicity against the same antigen protein as the pupa or cells of the present invention, or may have immunogenicity against different antigen proteins. When it has immunogenicity against the same antigen protein as the pupa or cells, for example, the pupa of the present invention may be administered for booster immunization of the above other vaccines.

[0047] 6. Food The food of the present invention has immunogenicity because it contains pupa or cells. There are no particular restrictions on the food containing pupa for oral administration, and examples include animal foods and plant foods. Also, the food can be in the form of animal feed.

[0048] As a form of incorporating silkworm pupae for oral administration into food, there is no particular limitation as long as it exhibits immunogenicity. The silkworm pupae having immunogenicity may be used in their original form, in a cut form, or in a crushed form. The subject of food intake is not particularly limited, and examples include mammals, birds, insects, amphibians, fish, etc. as described above. Mammals include humans and non-human mammals such as mice, rats, pigs, monkeys, cows, horses, sheep, rabbits, dogs, cats, and goats.

[0049] 7. Method for inducing immunity By administering the above-mentioned silkworm pupae for oral administration, the oral vaccine composition containing the same, or food to a subject, immunity can be induced in the subject. Regarding administration and the like, it is as described above. That immunity has been induced in the subject and the degree of immunity can be confirmed by methods well known to those skilled in the art, such as measurement of antibody production or antibody titer in the subject.

Examples

[0050] Hereinafter, the present invention will be described more specifically by way of examples, but the technical scope of the present invention is not limited to these examples. [Example 1] Production of silkworm pupae for oral administration of a vaccine antigen for livestock virus DNA encoding ORF2 (1 - 233) of porcine circovirus type 2 (PCV2; GenBank accession LC381288) isolate PCV2a or VP2 (T3 d17) of porcine parvovirus (PPV) was introduced into silkworm baculovirus by a conventional method (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.). The resulting recombinant baculovirus was injected into silkworm pupae to infect them. Eight days after injection, the silkworm pupae were treated in a freeze dryer at - 40 °C for 720 minutes.

[0051] [Example 2] Feeding test Using 7 - week - old female BALB / c mice (n = 3), a feeding test of the pupae for oral administration prepared in Example 1 was conducted. They were fasted and water - deprived for 2 hours before feeding. Each mouse was isolated and allowed to freely consume the pupae for oral administration. No water was given during pupa consumption. The mice were allowed to consume the pupae according to the schedule in the following table. There was no food restriction other than the pupae. Each mouse consumed 14 pupae during the period.

[0052]

Table 1

[0053] On Day 22, after blood collection under anesthesia, serum and intestinal lavage fluid were obtained.

[0054] [Example 3] Confirmation of specific antibody production response Using the sera and intestinal lavage fluids obtained in Example 2, the immunogenicities of pupae expressing PCV2a and pupae expressing PPV VP2 were confirmed by ELISA, respectively. The sera of non-administered group mice were regarded as Naive.

[0055] The coating antigens for the ELISA method were prepared as follows. Dilution was carried out with PBS. PCV2a ORF2(1 - 233): Dilute 0.44 mg / mL to 56.8 μL / 4943.2 μL (5 μg / mL), coat at 50 μL / well, and leave standing overnight at 4°C. PPV VP2: Dilute 0.34 mg / mL to 73.5 μL / 4926.5 μL (5 μg / mL), coat at 50 μL / well, and leave standing overnight at 4°C.

[0056] Also, various antibodies and substrates etc. were used as follows. Primary antibody: Various immune sera and undiluted intestinal lavage fluids (without dilution) or 50-fold dilution (both 50 μL / well) Secondary antibody: 1 / 10000 diluted Anti-Mouse IgG or IgA-HRP in 0.5% BSA was used at 50 μL / well and left standing at 37°C for 1 hour. Blocking: 1% BSA in PBS was used at 200 μL / well and left standing at 37°C for 2 hours. Wash: 1 / 50 dilution or undiluted solution with 0.5% BSA was used at 50 μL / well and left standing at 37°C for 2 hours. Substrate: 1step Turbo TMB-ELISA (Thermo Scientific, 100 μL / well) was used and left standing at room temperature for 10 minutes. Stop solution: Treated 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 case of PCV2, a high IgG antibody production response was observed in serum and intestinal lavage fluid. Also, when the IgA response was evaluated, antibody production was observed in serum, but higher production was observed in intestinal lavage fluid. A sufficient antibody production reaction was observed not only in the response at the undiluted solution but also, contrary to expectations, at a 50-fold dilution (Figure 2). On the other hand, in the case of PPV, an antibody production response of IgG and IgA was observed in serum, but no antibody production response was observed in intestinal lavage fluid (Figure 3). These differences are considered to be due to differences in the properties of the viruses.

[0058] [Example 4] Production of Silkworm Pupae for Oral Administration of a Human Virus Vaccine Antigen DNA encoding VP1 of norovirus (Norovirus, NV) genotype GII.4 (GenBank accession BAG70500.1) was introduced into silkworm baculovirus by a conventional method, and the obtained recombinant baculovirus was injected into silkworm pupae to infect them. Eight days after injection, the silkworm pupae were treated at -40°C for 720 minutes in a freeze dryer.

[0059] [Example 5] Feeding Test Using 7-week-old female BALB / c mice (n = 6), a feeding test of the orally administered pupae produced in Example 1 was conducted. They were fasted and water-deprived for 2 hours before feeding. Each mouse was isolated and allowed to freely ingest the orally administered pupae. Water was not given during pupae ingestion. The mice were allowed to ingest the pupae according to the schedules in Tables 2 and 3 below. There was no dietary restriction other than the pupae during the fasting period. Each mouse ingested 14 or 6 pupae during the period.

[0060]

Table 2

Table 3

[0061] On Day 21, after blood sampling under anesthesia, serum and intestinal lavage fluid were obtained.

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

[0063] Also, various antibodies and substrates etc. were used as follows. Primary antibody: Various immune sera and 50-fold diluted intestinal lavage fluid (both 50 μL / well) Secondary antibody: Anti-Mouse IgG or IgA-HRP diluted 1 / 10000 in 0.5% BSA was used at 50 μL / well and left standing at 37°C for 1 hour. Blocking: 1% BSA in PBS was used at 200 μL / well and left standing at 37°C for 2 hours. Wash: 1 / 50 dilution with 0.5% BSA was used at 50 μL / well and left standing at 37°C for 2 hours. Substrate: 1step Turbo TMB-ELISA (Thermo Scientific, 100 μL / well) was used and left standing at room temperature for 10 minutes. Stop solution: Treated with 2M H2SO4 (100 μL / well).

[0064] The results of measuring the absorbance at 450 nm (OD450 value) of each well are shown in Figure 4. From the results in Figure 4, a high IgG antibody production response was observed in serum and intestinal lavage fluid. Also, when the IgA response was evaluated, antibody production was also observed in serum and intestinal lavage fluid. When compared with the injectable vaccine, an antibody production reaction at a level comparable to that of the injectable vaccine was observed even without the combined administration of an adjuvant. When the antibody production response was evaluated by dividing the number of administrations into a group with five administrations per week and a group with two administrations per week, an enhanced antibody production response was observed in the group with five administrations per week.

Claims

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. A baculovirus-infected cell that has 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 of claim 1 and / or the cell of claim 2.

4. The oral vaccine composition of claim 3 further comprising a solution comprising an adjuvant.

5. The oral vaccine composition according to claim 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 according to claim 1 and / or the cell according to claim 2.

7. A method for producing oral pupae includes 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 a baculovirus-infectable cell with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced, and freeze-drying the infected cell.

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 antigenic 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. 10. The method according to claim 7 or 9, wherein the insect is a silkworm.

12. The method according to claim 8 or 10, wherein the cells are derived from Butterfly Moth, Anemone persica, Bombyx mori, Mamestra brassicae or Arctosporum frugiperda.

13. A method for inducing immunity against an antigen protein in a subject by administering to the subject the pupa described in claim 1, the cell described in claim 2, the oral vaccine composition described in any one of claims 3 to 5, or the food described in claim 6.

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