African swine fever vaccine-producing recombinant eri silkworm and use thereof

Abstract

The purpose of the present invention is to provide a method for producing an ASF antigen protein, said method having low endotoxin contamination and reduced workload in comparison with conventional methods. This purpose is achieved by a method using a recombinant eri silkworm that expresses an African swine fever virus antigen protein, in particular a recombinant eri silkworm that expresses an African swine fever virus antigen protein such as EP402R, i177L, P30, P54, pp62 and P72.

Description

African swine fever vaccine-producing recombinant Eri silkworms and their applications

[0001] The present invention relates to a recombinant Eri silkworm obtained by recombining Eri silkworms so as to express an African swine fever virus antigen protein.

[0002] African Swine Fever (ASF) is a fatal infectious disease of pigs caused by the African Swine Fever Virus (ASFV). ASF is currently designated as a notifiable disease that must be reported to the World Organization for Animal Health (WOAH). Since the outbreak of ASF, the economic losses suffered by the global pig farming industry have been immeasurable. Pigs infected with the ASF virus can die relatively quickly after infection, so they are preventatively culled to prevent the spread of infection.

[0003] An African swine fever (ASF) vaccine against ASFV is an optimal strategy for preventing or controlling ASF outbreaks. ASFV is a large double-stranded DNA virus belonging to the Asfarviridae family. ASF vaccines include inactivated ASFV vaccines that use inactivated ASFV as an antigen. However, inactivated ASFV vaccines have low immune protection potential, and there are insufficient cell lines for efficient ASFV replication in vitro. Therefore, there is a need for the development of vaccines other than those using inactivated ASFV.

[0004] Other vaccine strategies besides inactivated ASFV vaccines include subunit vaccines that utilize immunogenic African swine fever virus antigen proteins (ASF antigen proteins) expressed by the virus, nucleic acid vaccines containing ASF antigen genes that encode ASF antigen proteins, and vector vaccines engineered with ASF antigen genes. Of these, subunit vaccines are considered promising because they directly administer ASF antigen proteins to pigs.

[0005] However, the development and production of an ASF antigen protein that can be used in an ASF vaccine is difficult and is still in progress. For example, known production systems for ASF antigen protein include a method that uses recombinant Escherichia coli introduced with a recombinant vector containing a gene encoding the ASF antigen protein (see, for example, Non-Patent Document 1), and a method that uses recombinant insect cells introduced with a recombinant baculovirus containing an ASF antigen gene (see, for example, Non-Patent Document 2).

[0006] On the other hand, it has been reported that when a recombinant baculovirus modified using a protein of foot-and-mouth disease virus was injected into the blood vessels of Eri silkworm larvae, and then the obtained blood components were used to immunize rabbits and guinea pigs, the blood components were evaluated to be immunogenic (see, for example, Non-Patent Document 3).

[0007] Guanglei Zhang et al., Virol J. 2022 Jan 21;19(1):16.Mallory E. Heimerman et al, J Vet Diagn Invest. 2018 May; 30(3): 406-412.Manoj Kumar et al., Virusdisease. 2016 Mar; 27(1): 84-90.

[0008] An ASF antigen protein can be obtained according to the methods described in Non-Patent Document 1 and Non-Patent Document 2. However, the method described in Non-Patent Document 1 uses recombinant Escherichia coli, and therefore has the problem of contamination of the obtained ASF antigen protein with pathogenic foreign substances such as endotoxin.

[0009] The method described in Non-Patent Document 2 utilizes recombinant insect cells, and therefore requires the cultivation of a large number of recombinant insect cells in order to obtain a large amount of ASF antigen protein, which poses a problem of heavy workload.

[0010] Non-Patent Document 3 describes a method for producing an antigen protein of FMD virus in recombinant Eri silkworm larvae, but not a method for producing an ASF antigen protein. Furthermore, even if an ASF antigen protein is expressed in Eri silkworms, it does not necessarily result in a protein that is immunogenic in pigs.

[0011] Furthermore, little is known so far about methods for producing ASF antigen proteins with less endotoxin contamination and with less operational burden than the methods described in Non-Patent Documents 1 and 2.

[0012] Therefore, the problem that the present invention aims to solve is to provide a method for producing an ASF antigen protein with less endotoxin contamination and less operational burden than the methods described in Non-Patent Documents 1 and 2.

[0013] In an effort to solve the above problems, the present inventors have conducted extensive research into the materials, conditions, and methods for producing ASF antigen proteins, and as a result have focused on a method using Eri silkworms. The present inventors have then conducted extensive and careful research into the Eri silkworm production system for ASF antigen proteins, the type of ASF antigen protein, the growth conditions of Eri silkworms, and the transduction method.

[0014] As a result, we succeeded in producing ASF antigenic proteins in Eri silkworm pupae by infecting them with recombinant baculoviruses incorporating selected ASF antigenic proteins. Surprisingly, the ASF antigenic proteins obtained from the recombinant Eri silkworm pupae were immunogenic.

[0015] However, contrary to expectations, the ASF antigen protein produced in Eri silkworm pupae was not obtained as a soluble component and was difficult to isolate. Therefore, the inventors further repeated trial and error and finally succeeded in producing a soluble ASF antigen protein in Eri silkworm pupae.

[0016] Based on the above findings and success stories, the present inventors have finally succeeded in creating a recombinant Eri silkworm that expresses an ASF antigen protein and a method for producing an ASF antigen protein using the recombinant Eri silkworm, which solves the problems of the present invention. The present invention was completed based on the findings and success stories first obtained or achieved by the present inventors.

[0017] Therefore, the present invention provides each of the following aspects. [1] A recombinant Eri silkworm expressing an African swine fever virus antigen protein. [2] The recombinant Eri silkworm according to [1], wherein the recombinant Eri silkworm is a recombinant Eri silkworm pupa. [3] The recombinant Eri silkworm according to [1] or [2], wherein the African swine fever virus antigen protein is a protein containing at least one epitope selected from the group consisting of EP402R, i177L, P30, P54, pp62, and P72. [4] The recombinant Eri silkworm according to [1] or [2], wherein the African swine fever virus antigen protein is a soluble recombinant African swine fever virus antigen protein. [5] The recombinant Eri silkworm according to [4], wherein the soluble recombinant African swine fever virus antigen protein has a histidine tag consisting of 4 to 15 histidines at the C-terminus. [6] A method for producing an African swine fever virus antigen protein, comprising a step of obtaining an African swine fever virus antigen protein by isolating the African swine fever virus antigen protein from the recombinant Eri silkworm according to [1] or [2]. [7] A composition for pigs, comprising at least one active ingredient selected from the group consisting of the recombinant Eri silkworm according to [1] or [2] and the African swine fever virus antigen protein isolated from the recombinant Eri silkworm. [8] The composition for pigs according to [7], wherein the composition for pigs is an oral composition for pigs. [9] The composition for pigs according to [7], wherein the composition for pigs is selected from the group consisting of feed, veterinary medicines, and African swine fever vaccines.

[0018] According to the present invention, by using recombinant Eri silkworms expressing an ASF antigen protein, it is possible to produce an ASF antigen protein with less endotoxin contamination and reduced operational burden. According to the present invention, the ASF antigen protein is obtained as a soluble ASF antigen protein, which makes it easy to isolate from the recombinant Eri silkworms.

[0019] According to the present invention, the ASF antigen protein obtained has reduced endotoxin contamination and is immunogenic even when administered orally, so it is expected that the onset of African swine fever can be prevented or treated relatively easily by orally administering it to pigs as feed or mixed with feed. Thus, according to the present invention, an African swine fever vaccine for oral administration can be obtained.

[0020] FIG. 1 shows the results of examining the production of antibodies specific to ASF antigen proteins in pigs by ELISA, as described in the Examples below.

[0021] Each aspect of the present invention will be described in detail below, but the present invention is not limited to the details of these items and can take various forms as long as the object of the present invention is achieved.

[0022] Unless otherwise specified, each term in this specification is used in the meaning commonly used by those skilled in the art of biotechnology, veterinary medicine, pharmacology, etc., and should not be construed as having an unduly limiting meaning. Furthermore, the speculations and theories made in this specification are based on the inventors' knowledge and experience to date, and therefore the present invention is not limited solely to such speculations and theories.

[0023] "Comprising" means that elements other than those explicitly stated as included may be added (same meaning as "comprising at least"), but also encompasses "consisting of" and "consisting essentially of." That is, "comprising" can mean including the explicitly stated elements and any one or more elements, consisting of the explicitly stated elements, or consisting essentially of the explicitly stated elements. Elements include components, steps, conditions, parameters, and other limitations. The term "and / or" means any one of the associated listed items, or any or all combinations of two or more. The term "to" in a numerical range includes both the preceding and following numerical values, and also includes ranges excluding either of the included limits. For example, "0% to 100%" means 0% or more, 100% or less, and 0% or more and 100% or less. "About" means an amount within ±10% of the quantity following the term. For example, "about 100" means 100±10%, i.e., 90 to 110. "Exceeding" and "less than" refer to the lower and upper limits, respectively, without including the numerical value before them. For example, "exceeding 1" means a numerical value greater than 1, and "less than 100" means a numerical value less than 100. The number of digits in an integer value matches the number of significant figures. For example, 1 has one significant digit, and 10 has two significant digits. Furthermore, for decimal values, the number of digits after the decimal point matches the number of significant digits. For example, 0.1 has one significant digit, and 0.10 has two significant digits.

[0024] A "protein" refers to a polymer of amino acid residues and is also called a polypeptide. An amino acid sequence is the sequence information representing the linkage state of amino acids in a protein from the N-terminus to the C-terminus. Proteins are not limited by the number of amino acid residues and include peptides, oligopeptides, dimers, multimers, etc. A protein may be a full-length protein or a fragment thereof. A "gene" refers to a polymer of nucleotide residues and is also called a nucleic acid molecule or DNA. A nucleotide sequence is the sequence information representing the linkage state from the 5' end to the 3' end of a gene. Protein "expression" refers to the production of a protein having an amino acid sequence encoded by the nucleotide sequence of a gene (a protein encoded by a gene) with its original structure and properties through transcription, translation, etc. A "wild-type gene" refers to a gene inherently possessed by a wild-type organism or virus. A "wild-type protein" refers to a protein encoded by a wild-type gene. Note that, in this specification, genome and chromosome are synonymous. A "foreign gene" refers to a gene not inherently possessed in the genomic DNA of the organism into which it is introduced, and is also called a heterologous gene. A protein encoded by a foreign gene is called a "foreign protein." While wild-type organisms do not express foreign proteins, recombinant organisms do. The opposite of a "wild-type gene" is a "foreign gene." "Recombinant" refers to modifying an organism so that it expresses a protein encoded by a foreign gene within the organism, for example by integrating a foreign gene into the organism's genomic DNA or by introducing a self-replicating unit containing the foreign gene into the organism's cells. A "recombinant" organism refers to an organism that has been artificially engineered to express a foreign gene. Recombinant organisms are also called recombinants. Organisms that have been infected with a recombinant baculovirus and come to express a foreign protein (infected organisms) are also called recombinant organisms. A "wild-type" organism refers to an organism that exists in nature, can be isolated from nature, and has not been artificially engineered to express a gene. The "immunogenicity" of a protein refers to the property of stimulating the immune system of an organism into which the protein has been introduced to elicit a specific humoral and / or cellular response, and is synonymous with antigenicity."Isolation" of a protein means separating the protein from the protein source. An isolated protein contains, for example, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of components of the protein source that are different from the protein. An isolated protein is usually obtained by recovering the protein from a protein source (e.g., an organism) and then purifying it by ion exchange chromatography, affinity chromatography, sedimentation through a concentration or density gradient, or the like.

[0025] (Recombinant Eri Silkworm) One aspect of the present invention is a recombinant Eri silkworm that expresses an African swine fever virus antigen protein.

[0026] The Eri silkworm, scientific name Samia cynthia ricini, is a lepidopteran insect (silkworm) belonging to the Saturniidae family, and eats feed containing at least one species selected from the group consisting of cassava (scientific name: Manihot esculenta) and castor bean (scientific name: Ricinus communis). The Eri silkworm is considered to be a type of wild silkworm, unlike the silkworm (Bombyx mori) belonging to the Bombycidae family, which feeds on mulberry leaves.

[0027] Eri silkworms mainly take the following forms: "egg (embryo)" (from immediately after egg-laying until immediately before hatching), "larva" (from immediately after hatching until immediately before the completion of cocoon formation (divided into 1st to 5th instars)), "pupa" (from immediately before the completion of cocoon formation until immediately before adulthood), and "adult (moth)" (from immediately after adulthood until death).

[0028] The Eri silkworms used for recombinant Eri silkworms may be any form of Eri silkworm, but for example, Eri silkworm pupae and larvae are preferred because they are easy to recombine and raise, and Eri silkworm pupae are more preferred because they allow for easy protein production.

[0029] Eri silkworm larvae can be reared, for example, by referring to JP 2017-131169 A (particularly, paragraphs

[0018] to

[0025] ). Eri silkworm pupae may be reared, for example, under conditions of a temperature of 24°C to 26°C and a humidity of 40% to 60%.

[0030] The recombinant Eri silkworm is recombinantly engineered to express an African swine fever virus antigen protein, which is a foreign protein. The recombinant Eri silkworm harbors a gene (African swine fever virus antigen gene) encoding the African swine fever virus antigen protein. In the recombinant Eri silkworm, the African swine fever virus antigen gene may be inserted into intracellular genomic DNA or mitochondrial DNA, or may be present so as to be autonomously expressed within the cell, such as in the cytoplasm or nucleus. In the recombinant Eri silkworm, specifically within the cells of the recombinant Eri silkworm, the African swine fever virus antigen gene is transcribed and translated to express the African swine fever virus antigen protein.

[0031] The type of African swine fever virus antigen protein expressed in the recombinant Eri silkworm is not particularly limited, and may be one type, two or more types, or a fusion protein in which two or more types are combined. The amount of African swine fever virus antigen protein is not particularly limited, and is preferably an amount that can be confirmed, for example, by a method using immunostaining as described in the Examples below, more preferably an amount that shows immunogenicity in an organism into which it is introduced, and even more preferably an amount that shows immunogenicity in a pig into which it is introduced.

[0032] (African swine fever virus antigen protein) The African swine fever virus antigen protein is a protein that constitutes the particle structure of African swine fever virus (ASFV) and has immunogenicity.

[0033] ASFV is a double-stranded DNA virus belonging to the Asfarviridae family, and is transmitted by arthropods such as soft ticks of the genus Ornithodoros, causing African swine fever in pigs.

[0034] The ASFV may be any virus belonging to the Asfarviridae family, specifically, a virus strain belonging to the Asfivirus genus of the Asfarviridae family. Numerous isolated ASFV strains have been registered in the NCBI online database (https: / / www.ncbi.nlm.nih.gov / nuccore / ). The ASFV may be either a strain registered and classified in the database or an isolated strain that has not yet been registered. Specific examples of ASFV include, but are not limited to, the strain registered in NIBI GenBank under accession number MK128995 (version MK128995.1).

[0035] The African swine fever virus antigen protein may be either a B cell epitope or a T cell epitope as long as it has immunogenicity, but is preferably a B cell epitope in order to promote antibody production. Among the structural proteins of ASFV, those that can serve as T cell epitopes have been reported in a literature by Leana Rich De Mesa Herrera et al. (Veterinary World, 14(10):2625-2633).

[0036] The African swine fever virus antigen protein may be any protein that constitutes the ASFV particle structure, such as an envelope protein, a capsid protein, an inner lipid membrane protein, or a core-shell protein. For example, due to their high immunogenicity, EP402R, i177L, P30, P54, pp62, and P72 are preferred, EP402R, i177L, P30, and P54 are more preferred, and EP402R is even more preferred.

[0037] The African swine fever virus antigen protein may be modified at the C-terminus and / or N-terminus. For example, by tagging the C-terminus, the African swine fever virus antigen protein produced in recombinant Eri silkworms can be qualitatively or quantitatively confirmed. Examples of tags used for such tagging include peptide tags having a length of 5 to 40 amino acids, and specific examples include, but are not limited to, Flag (registered trademark) tag, HA tag, Myc tag, V5 tag, S tag, E tag, T7 tag, VSV-G tag, CBD tag, and CBP tag (each manufactured by Medical and Biological Laboratories).

[0038] Non-limiting examples of African swine fever virus antigenic proteins include EP402R, i177L, P30, P54, pp62, and P72, each of which has been modified to have a Flag tag at its C-terminus (see Table 2, which lists the nucleotide and amino acid sequences of each).

[0039] When an African swine fever virus antigen protein is expressed in a recombinant Eri silkworm, it may not be obtained as a soluble component, making isolation difficult. Therefore, the African swine fever virus antigen protein is preferably a soluble African swine fever virus antigen protein that has been modified so as to be expressed in a soluble state in the recombinant Eri silkworm.

[0040] The soluble African swine fever virus antigen protein may be any protein that can be isolated as a more soluble component in the recombinant Eri silkworm compared to the African swine fever virus antigen protein before being subjected to solubility modification. Examples of such a protein include African swine fever virus antigen protein modified at its N-terminus and / or C-terminus by adding a tag, a signal sequence, a soluble moiety, an intracellular component-binding moiety, or the like.

[0041] Examples of soluble African swine fever virus antigen proteins include, but are not limited to, African swine fever virus antigen proteins modified by the addition of a histidine tag to the C-terminus and / or N-terminus, African swine fever virus antigen proteins modified by the addition of a signal sequence to the C-terminus and / or N-terminus, African swine fever virus antigen proteins modified by the addition of a ZZ domain to the C-terminus and / or N-terminus, African swine fever virus antigen proteins modified to exclude the transmembrane region and / or intramembrane domain, and African swine fever virus antigen proteins modified by a combination of these.

[0042] The histidine (His) tag is composed of multiple histidines. The number of histidines in the His tag may be any number that confers the African swine fever virus antigen protein with the property of being able to exist as a soluble component in the recombinant Eri silkworm, but is, for example, preferably 3 to 20, more preferably 4 to 15, and even more preferably 5 to 10.

[0043] A signal sequence is a peptide having a structure that directs the transport and localization of a protein biosynthesized within a cell. The signal sequence is preferably one suitable for protein expression in Eri silkworms, and more preferably one derived from Eri silkworms or baculovirus proteins. However, the signal sequence may also be a signal sequence derived from another organism or an artificially designed signal sequence. Specific examples of signal sequences include the GP64 signal sequence and the chitinase signal sequence. When expressing an African swine fever virus antigen protein in Eri silkworms using a recombinant baculovirus in which a gene encoding an African swine fever virus antigen protein is incorporated into a baculovirus, the signal sequence is preferably the GP64 signal sequence. Specific examples of GP64 signal sequences include the sequence set forth in SEQ ID NO: 29.

[0044] The ZZ domain is the IgG binding domain of Protein A. A specific example of a ZZ domain is one having the sequence set forth in SEQ ID NO:30.

[0045] When modifying the African swine fever virus antigen protein by adding a peptide to the N-terminus and / or C-terminus, the African swine fever virus antigen protein and the added peptide may be linked via a linker. The linker is not particularly limited, but for example, a linker having a length of 2 to 50 amino acids is preferred, a linker having a length of 3 to 25 amino acids is more preferred, and a linker having a length of 3 to 20 amino acids is even more preferred. Specific examples of linkers include a GPGP linker (SEQ ID NO: 34), a GGGSAWSHPQFEK linker (SEQ ID NO: 31), a (GGGGGS) x 3 linker (SEQ ID NO: 35), and a glycine x 8 linker (SEQ ID NO: 36).

[0046] The African swine fever virus antigen protein may structurally contain a transmembrane region and / or an intramembrane domain in addition to the extramembrane domain. Such an African swine fever virus antigen protein remains within the membrane or intracellularly, resulting in reduced solubility. Therefore, the soluble African swine fever virus antigen protein is preferably an African swine fever virus antigen protein from which the transmembrane region and / or the intramembrane domain have been removed, and more preferably is composed mainly of the extramembrane domain.

[0047] The soluble African swine fever virus antigen protein may be a combination of the above-mentioned modifications. For example, the soluble African swine fever virus antigen protein may be modified by adding a His tag, a signal sequence, and / or a ZZ domain to the N-terminus and / or C-terminus of the African swine fever virus antigen protein with or without a linker, and / or by excluding the transmembrane region and / or the intramembrane domain.

[0048] Specific examples of soluble African swine fever virus antigen proteins include P30-cHis, P54-cHis, pp62-cHis, EP402R-ZZ, EP402R-cHis, i177L-cyto-ZZ, ZZ domain-P30-Linker-P54, and GP64SS-ZZ domain-P30-Linker-P54, which are described in the Examples below (see Table 2, which lists the respective nucleotide and amino acid sequences).

[0049] The African swine fever virus antigen protein contains a portion of an ASFV structural protein, which may consist of the amino acid sequence of a wild-type ASFV structural protein (wild-type protein), or may consist of an amino acid sequence of the wild-type protein with one or more amino acid deletions, substitutions, or additions.

[0050] The range of "1 to several" in "deletion, substitution, or addition of one to several amino acids" in an amino acid sequence is not particularly limited, but for example, if 100 amino acids in the amino acid sequence are considered to be one unit, the number of amino acids per unit is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and even more preferably 1, 2, 3, 4, or 5. "Amino acid deletion" means the absence or loss of an amino acid residue in a sequence, "amino acid substitution" means that an amino acid residue in a sequence has been replaced with another amino acid residue, and "amino acid addition" means that a new amino acid residue has been added to the sequence, so as to be inserted.

[0051] Specific examples of "deletion, substitution, or addition of one or more amino acids" include replacement of one or more amino acids with chemically similar amino acids. Examples include the replacement of a hydrophobic amino acid with another hydrophobic amino acid, or the replacement of a polar amino acid with another polar amino acid with the same charge. Such chemically similar amino acids are known for each amino acid in the art. Specific examples include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged basic amino acids include arginine, histidine, and lysine. Negatively charged acidic amino acids include aspartic acid and glutamic acid.

[0052] Examples of amino acid sequences having one or more amino acid deletions, substitutions, additions, etc. in the amino acid sequence of a wild-type protein include amino acid sequences that have a certain level of sequence identity to the amino acid sequence of the wild-type protein, such as amino acid sequences that have a sequence identity to the amino acid sequence of the wild-type protein of preferably 70% or more or 75% or more, more preferably 80% or more or 85% or more, even more preferably 86% or more, 87% or more, 88% or more, or 89% or more, and even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The upper limit of the sequence identity is not particularly limited, and is typically 100%.

[0053] The African swine fever virus antigen gene may be a nucleotide sequence encoding the amino acid sequence of a wild-type protein (wild-type gene), or may be a nucleotide sequence having one or more nucleotide deletions, substitutions, additions, etc. in the nucleotide sequence of the wild-type gene.

[0054] The range of "1 to several" in "deletion, substitution, or addition of one to several nucleotides" in a nucleotide sequence is not particularly limited, but for example, if 100 nucleotides in a nucleotide sequence are considered to be one unit, the number of nucleotides per unit is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and even more preferably 1, 2, 3, 4, or 5. "Nucleotide deletion" means that a nucleotide in a sequence is missing or lost, "nucleotide substitution" means that a nucleotide in a sequence is replaced with another nucleotide, and "nucleotide addition" means that a new nucleotide is added so as to be inserted.

[0055] Examples of nucleotide sequences having one or more nucleotide deletions, substitutions, additions, etc. in the nucleotide sequence of a wild-type gene include nucleotide sequences that have a certain level of sequence identity to the nucleotide sequence of the wild-type gene, such as nucleotide sequences that have a sequence identity to the nucleotide sequence of the wild-type gene of preferably 70% or more or 75% or more, more preferably 80% or more or 85% or more, even more preferably 86% or more, 87% or more, 88% or more, or 89% or more, and even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The upper limit of the sequence identity is not particularly limited, and is typically 100%.

[0056] A protein having an amino acid sequence encoded by a nucleotide sequence having one or more nucleotide deletions, substitutions, additions, etc. in the nucleotide sequence of a wild-type gene is likely to be a protein having an amino acid sequence having one or more amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the wild-type protein, but has immunogenicity equal to or greater than that of the wild-type protein.

[0057] The nucleotide sequence of a wild-type gene may be a nucleotide sequence that encodes the same amino acid sequence as that of a wild-type protein, taking advantage of the fact that there are several codons corresponding to one amino acid, but may also include a nucleotide sequence that is different from the nucleotide sequence of the wild-type gene, for example, a nucleotide sequence that has been optimized for codons, secondary structure, GC content, etc. The codon-modified nucleotide sequence is preferably a nucleotide sequence that has been codon-modified to facilitate expression in, for example, Eri silkworms.

[0058] The method for determining the sequence identity of a nucleotide sequence and an amino acid sequence is not particularly limited. For example, the sequence identity of a nucleotide sequence and an amino acid sequence can be determined using a commonly known method by aligning the nucleotide sequence of a wild-type gene and the amino acid sequence of a wild-type protein with the target nucleotide sequence and amino acid sequence, respectively, and using a program to calculate the identity between the two sequences.

[0059] As a program for calculating the identity between two nucleotide sequences and amino acid sequences, for example, the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993) is known, and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Furthermore, Gapped BLAST, a program that determines sequence identity more sensitively than BLAST, is also known (Nucleic Acids Res. 25: 3389-3402, 1997). Therefore, those skilled in the art can search for sequences that show high sequence identity to a given sequence in databases, for example, by using the programs described above, which are available, for example, on the internet website of the U.S. National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0060] While the above methods are commonly used to search databases for sequences that exhibit sequence identity, homology analysis using Genetyx Network Edition version 12.0.1 (Genetix, Inc.) can also be used to determine the sequence identity of individual sequences. This method is based on the Lipman-Pearson method (Science 227:1435-1441, 1985). When analyzing the sequence identity of nucleotide sequences, protein-coding regions (CDS or ORF) are used if possible.

[0061] The African swine fever virus antigen gene can be obtained as a nucleic acid molecule (DNA) using methods known in the art. Such methods include, for example, chemical synthesis, genetic engineering synthesis, isolation from ASFV, and a combination of these. For example, a chemically synthesized nucleic acid molecule can be obtained based on the nucleotide sequence information of the African swine fever virus antigen gene, and the nucleic acid molecule can then be subjected to polymerase chain reaction (PCR) to obtain an amplified nucleic acid molecule. The amplified nucleic acid molecule can then be optionally cloned using the Gibson Assembly system described in the Examples below to obtain an African swine fever virus antigen gene. Whether the African swine fever virus antigen gene is the desired one can be confirmed, for example, by sequencing it to confirm its nucleotide sequence.

[0062] When an African swine fever virus antigen protein is a modified wild-type protein, it has immunogenicity equal to or greater than that of the wild-type protein. That is, an African swine fever virus antigen protein obtained by modifying a wild-type protein has 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the immunogenicity of the wild-type protein. An African swine fever virus antigen protein obtained by modifying a wild-type protein may have immunogenicity greater than that of the wild-type protein.

[0063] The immunogenicity of an African swine fever virus antigen protein may be evaluated by confirming the specific immune response of an organism to which the African swine fever virus antigen protein has been administered. For example, the immunogenicity can be evaluated by confirming the production of anti-African swine fever virus antigen protein antibodies in a test animal, preferably a mammal, more preferably a mouse, rat, or pig, and even more preferably a pig, to which the African swine fever virus antigen protein has been administered, using the method described in the Examples below.

[0064] (Recombinant Baculovirus) To express an African swine fever virus antigen protein in Eri silkworms, it is preferable to use a baculovirus, i.e., Eri silkworms are recombined using a recombinant baculovirus into which an African swine fever virus antigen gene has been inserted.

[0065] The baculovirus may be either a commercially available baculovirus or a baculovirus isolated from nature. Examples of commercially available baculoviruses include, but are not limited to, the "Bac-to-Bac system" (Thermo Fisher Scientific) described in the Examples below. Examples of baculoviruses isolated from nature include, for example, baculoviruses isolated from Eri silkworms infected with a baculovirus.

[0066] Methods for inserting the African swine fever virus antigen gene into baculovirus include, for example, using the Bac-to-Bac system to insert the African swine fever virus antigen gene into a pFastBac1 transfer vector, then transforming DH10Bac Escherichia coli with the pFastBac1 transfer vector incorporating the African swine fever virus antigen gene to produce recombinant baculovirus DNA in the E. coli. Examples of methods include purifying the recombinant baculovirus DNA from the E. coli and introducing it into insect cells such as sf9 cells to produce a recombinant baculovirus, or inducing homologous recombination in insect cells such as sf9 cells to produce a recombinant baculovirus. These methods can be appropriately selected and used depending on the type of baculovirus, etc.

[0067] The African swine fever virus antigen gene to be inserted into the baculovirus may be one type alone or a combination of two or more types. For example, a recombinant baculovirus into which two African swine fever virus antigen genes have been inserted may express two African swine fever virus antigen proteins individually in Eri silkworms, or may express a fusion protein in which the two African swine fever virus antigen proteins are linked together.

[0068] (Method for producing recombinant Eri silkworms and African swine fever virus antigen protein) Recombinant Eri silkworms can be obtained by administering a recombinant baculovirus into which an African swine fever virus antigen gene has been inserted to Eri silkworms, preferably Eri silkworm pupae or larvae, more preferably Eri silkworm pupae.

[0069] The recombinant baculovirus can be administered to Eri silkworms by, for example, the method described in the Examples below. ６ pfu / mL or more, preferably 1.0 x 10 ８ The recombinant baculovirus can be administered to Eri silkworms by injecting several tens of μL to several hundreds of μL of a solution containing a recombinant baculovirus at pfu / mL or more using a syringe into Eri silkworm pupae immediately after pupation to the fifth day, preferably the first to third day after pupation, preferably about the second day after pupation, preferably into the abdomen of the Eri silkworm pupa, and then allowing the injected Eri silkworm pupa to stand at 25°C to 30°C for several days, preferably 6 to 10 days.

[0070] The African swine fever virus antigen protein to be expressed in the recombinant Eri silkworm may be one type alone or a combination of two or more types.

[0071] The production of African swine fever virus antigen protein in Eri silkworms can be confirmed by isolating the African swine fever virus antigen protein from Eri silkworms and measuring the molecular weight and immunological properties (e.g., antibody binding ability) of the obtained protein, for example, in accordance with the method described in the Examples below.

[0072] The African swine fever virus antigen protein can be obtained by isolating the African swine fever virus antigen protein from the recombinant Eri silkworm. Another aspect of the present invention is a method for producing an African swine fever virus antigen protein, which includes a step of isolating the African swine fever virus antigen protein from the recombinant Eri silkworm to obtain the African swine fever virus antigen protein.

[0073] Isolation of an African swine fever virus antigen protein from recombinant Eri silkworms can be carried out by the method described in the Examples below. For example, recombinant Eri silkworms are homogenized to obtain a recombinant Eri silkworm lysate, the obtained recombinant Eri silkworm lysate is then subjected to solid-liquid separation to obtain a solution, and the obtained solution is then subjected to affinity chromatography based on the tag attached to the African swine fever virus antigen protein, followed by elution, thereby isolating the African swine fever virus antigen protein from the recombinant Eri silkworms.

[0074] (Method of Using Recombinant Eri Silkworm and African Swine Fever Virus Antigen Protein) By administering the recombinant Eri silkworm and African Swine Fever Virus antigen protein to pigs, antibodies specific to the African Swine Fever Virus antigen protein can be produced in the pigs, which can result in the prevention of African Swine Fever and / or the treatment of African Swine Fever in pigs.

[0075] "Prevention" and "treatment" of African swine fever include preventing or delaying the onset of African swine fever; reducing the risk of developing African swine fever; suppressing the onset of African swine fever symptoms; alleviating and ameliorating African swine fever symptoms; preventing or delaying the worsening of African swine fever symptoms; and reversing, preventing, and delaying the progression of African swine fever symptoms.

[0076] The recombinant Eri silkworms to be administered to pigs may be live silkworms themselves, or may be live silkworms that have been subjected to processing such as drying, dissolving, crushing, pulverizing, shredding, or extraction. However, when live silkworms are used, it is preferable to subject the live silkworms to a baculovirus eradication treatment. The baculovirus eradication treatment may be carried out, for example, by adding vitamin C followed by crushing, by UV irradiation, or by a combination of these. The processing treatment may be a single type of treatment, or a combination of two or more types of treatments.

[0077] The African swine fever virus antigen protein to be administered to pigs may be isolated from recombinant Eri silkworms, or may be isolated from recombinant Eri silkworms and then subjected to processing such as drying, dissolving, crushing, pulverizing, shredding, extraction, etc. The processing may be a single type of processing or a combination of two or more types of processing.

[0078] Examples of drying treatments include freeze-drying, sun drying, hot air drying, vacuum drying, aeration drying, and reduced-pressure drying. Examples of dissolution treatments include dissolution treatments using buffers, cell lytic enzymes, surfactants, and the like. Examples of crushing treatments include crushing treatments using disrupting means such as an ultrasonic crusher, French press, Dynomill, and mortar. Examples of shredding treatments include shredding treatments using shredding means such as a slicer, cutter, and blade. Examples of extraction treatments include extraction treatments using extraction solvents such as water, warm water, and hot water; organic solvents such as methanol, ethanol, isopropanol, and acetone; and aqueous organic solvents obtained by mixing an organic solvent with water.

[0079] The processed recombinant Eri silkworm and African swine fever virus antigen proteins are preferably subjected to purification treatments such as centrifugation, filter filtration, ultrafiltration, gel filtration, separation based on differences in solubility, solvent extraction, chromatography (adsorption chromatography, hydrophobic chromatography, cation exchange chromatography, anion exchange chromatography, reverse phase chromatography, etc.), crystallization, activated carbon treatment, membrane treatment, etc.

[0080] The recombinant Eri silkworm and African swine fever virus antigen proteins may be in various forms, such as solid, liquid, gel, suspension, cream, sheet, stick, powder, granules, granules, tablet, rod, plate, block, paste, capsule, jelly, or caplet.

[0081] The recombinant Eri silkworm and the African swine fever virus antigen protein may be administered to pigs either alone or in combination. The African swine fever virus antigen protein can be said to be an isolated African swine fever virus antigen protein in the sense that it is different from the recombinant Eri silkworm.

[0082] The recombinant Eri silkworm and African swine fever virus antigen protein may be administered orally or parenterally to pigs. For example, the recombinant Eri silkworm and African swine fever virus antigen protein can be orally administered to pigs as feed or as a component of feed (internal component), or mixed with feed (external component). Furthermore, the recombinant Eri silkworm and African swine fever virus antigen protein can be parenterally administered to pigs by, for example, local injection, such as intradermal, subcutaneous, intravenous, or intramuscular, followed by percutaneous absorption, or by inhalation through mucous membranes such as the nose or pharynx.

[0083] The recombinant Eri silkworm and African swine fever virus antigen proteins can be administered orally in the form of feed, oral veterinary medicines, etc.; and can be administered parenterally in the form of parenteral veterinary medicines, skin cleansers, etc.

[0084] The recombinant Eri silkworm and African swine fever virus antigen protein can be used as a vaccine against African swine fever virus, i.e., an African swine fever vaccine. The vaccine may be a mixed cocktail vaccine containing the recombinant Eri silkworm and African swine fever virus antigen protein together with one or more other active ingredients.

[0085] When the recombinant Eri silkworm and African swine fever virus antigen protein is used as a vaccine, it is preferable to contain a component different from the recombinant Eri silkworm and African swine fever virus antigen protein, which has no activity by itself but has the effect of further enhancing the efficacy of the recombinant Eri silkworm and African swine fever virus antigen protein as a vaccine. Examples of inactive components include adjuvants and toxoids.

[0086] Non-limiting examples of adjuvants include precipitating types such as aluminum hydroxide, aluminum phosphate, and calcium phosphate, and oil-based types such as Freund's complete adjuvant and Freund's incomplete adjuvant.

[0087] The adjuvant may be administered simultaneously with the recombinant Eri silkworm and African swine fever virus antigen protein or may be administered synchronously with the recombinant Eri silkworm and African swine fever virus antigen protein, but is preferably administered simultaneously with the recombinant Eri silkworm and African swine fever virus antigen protein in order to ensure that the adjuvant's function is properly exerted. When the adjuvant is administered simultaneously with the recombinant Eri silkworm and African swine fever virus antigen protein, the adjuvant and the recombinant Eri silkworm and African swine fever virus antigen protein may be formulated as a single formulation or may be formulated separately.

[0088] The doses of the recombinant Eri silkworm and African swine fever virus antigen protein may be any amounts that can induce a specific immune response in the administered pig, but are preferably amounts that can produce antibodies specific to the African swine fever virus antigen protein (anti-African swine fever virus antigen protein antibodies) in the pig. Specific examples of the amount of African swine fever virus antigen protein per administration are preferably 0.01 mg to 1,000 mg, more preferably 0.1 mg to 100 mg, and even more preferably 1 mg to 10 mg.

[0089] The recombinant Eri silkworm and African swine fever virus antigen protein may be administered once or multiple times, but is preferably administered twice or more because a significant amount of anti-African swine fever virus antigen protein antibodies can be produced in the pig's body after the second administration. The administration interval is, for example, preferably 10 days or more, more preferably 14 days or more, even more preferably 21 days or more, and even more preferably 28 days or more after the previous administration, taking into consideration the timing at which the amount of anti-African swine fever virus antigen protein antibodies produced decreases and side effects after administration subside.

[0090] The pigs to be administered with the recombinant Eri silkworm and African swine fever virus antigen protein may be any pig that has the potential to contract African swine fever, such as livestock breeds such as Landrace, Large Yorkshire, Duroc, Berkshire, Hampshire, Middle Yorkshire, Agu, and crossbreeds of these breeds. Furthermore, Sangen pigs and Kurobuta pigs, which are branded in Japan and relatively high in value, are preferred.

[0091] Whether or not anti-African swine fever virus antigen protein antibodies are produced in the bodies of pigs after administering the recombinant Eri silkworm and African swine fever virus antigen protein to pigs can be confirmed by the ELISA method using the blood of the administered pigs, as described in Example 4 of the Examples below.

[0092] The recombinant Eri silkworm and African swine fever virus antigen protein may be administered to pigs in combination with other ingredients. Examples of such other ingredients include excipients, stabilizers, binders, thickeners, preservatives, buffers, lubricants, oily components, humectants, cooling agents, chelating agents, pH adjusters, antioxidants, carbohydrates, fatty acids, vitamins, emulsifiers, seasonings, flavorings, coloring agents, nutrients, animal and plant ingredients such as fruit juice and eggs, and additives commonly used in the production of veterinary medicines and feed, such as medicinal ingredients. The amounts of such other ingredients are not particularly limited as long as they do not interfere with the solution of the problems of the present invention, and may be determined appropriately.

[0093] Prevention of African swine fever can be confirmed by the production of anti-African swine fever virus antigen protein antibodies in the pigs, and treatment of African swine fever can be confirmed by the alleviation, improvement, or complete recovery of African swine fever symptoms in the pigs.

[0094] (Composition for Pigs) Another aspect of the present invention is a composition for pigs, which contains a recombinant Eri silkworm and / or African swine fever virus antigen protein as an active ingredient, and optionally contains other ingredients.

[0095] The composition for pigs contains, for example, recombinant Eri silkworms and African swine fever virus antigen protein, and can be administered to pigs for use in applications where physiological effects such as anti-African swine fever virus antigen protein antibody production, African swine fever prevention, and African swine fever treatment are expected.

[0096] The porcine composition may be an oral composition for porcine administration to pigs or a parenteral composition for porcine administration to pigs, but is preferably an oral composition for porcine administration because it can be easily administered. Specific forms of the porcine composition include, but are not limited to, feed, veterinary medicines, African swine fever vaccines, etc.

[0097] The content of the recombinant Eri silkworm and African swine fever virus antigen protein in the composition for pigs is not particularly limited, but for example, the amount of the recombinant Eri silkworm and African swine fever virus antigen protein is preferably 0.001% by mass or more, more preferably 0.01% by mass to 99% by mass, and even more preferably 0.05% by mass to 50% by mass, based on the total amount of the composition, in terms of dry mass.

[0098] The method for producing the composition for pigs is not particularly limited, but examples include a method comprising mixing the recombinant Eri silkworm and African swine fever virus antigen protein with other ingredients, and molding the resulting mixture into a desired shape.

[0099] (Another aspect of the present invention) Another aspect of the present invention is a method for producing recombinant Eri silkworms, comprising the step of administering a recombinant baculovirus carrying an African swine fever virus antigen gene to Eri silkworms to obtain recombinant Eri silkworms that express an African swine fever virus antigen protein.

[0100] Another aspect of the present invention is a method for producing an African swine fever virus antigen protein, comprising the step of obtaining the above-mentioned recombinant Eri silkworm, followed by the step of isolating the African swine fever virus antigen protein from the recombinant Eri silkworm to obtain the African swine fever virus antigen protein.

[0101] Another aspect of the present invention is a method for preventing or treating African swine fever, comprising the step of administering recombinant Eri silkworms and African swine fever virus antigen proteins to pigs to prevent or treat African swine fever.

[0102] Another aspect of the present invention is a method for feeding pigs, comprising the step of maintaining or improving the health of pigs by feeding the pigs feed containing recombinant Eri silkworms and African swine fever virus antigen proteins.

[0103] In the method of each aspect of the present invention, various steps or operations can be added before, after, or between the steps described above, as long as the object of the present invention can be achieved.

[0104] The present invention will be explained in more detail below with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be achieved.

[0105] Example 1. Production of ASF antigen proteins using Eri silkworms. Several proteins expressed by African swine fever (ASF) virus were selected as candidate ASF antigen proteins. Furthermore, from among the candidate proteins, structural characteristics, expression level, ease of isolation and purification, and other factors were examined. EP402R, i177L, P30 (CP204L), P54 (E183L), pp62 (CP530R), and P72 were selected as ASF antigen proteins. The nucleotide sequences encoding these ASF antigen proteins were based on the genome information of ASFV registered in GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ) under accession numbers MK128995 and YP_009927266, respectively. DNA molecules expressing each ASF antigen protein were synthesized by standard methods. The DNA molecule is codon-optimized and has a nucleotide sequence encoding a FLAG tag (SEQ ID NO: 32) at the C-terminus.

[0106] The nucleotide sequences of the synthesized EP402R, i177L, P30, P54, pp62 and P72 are shown in SEQ ID NOs: 1 to 6. The corresponding amino acid sequences are shown in SEQ ID NOs: 7 to 12.

[0107] The resulting DNA molecules were recombined into commercially available baculoviruses ("Bac-to-Bac system"; Thermo Fisher Scientific) according to the manufacturer's instructions to obtain recombinant baculoviruses.

[0108] The recombinant baculovirus was administered to pupae of Eri silkworms (Samia cynthia ricini) to produce the ASF antigen protein in the Eri silkworms. Specifically, a highly concentrated stock solution of the recombinant baculovirus (titer 1.0 × 10) was administered to the pupae of Eri silkworms. ８ pfu / mL or more) was injected using a syringe (repeated dispenser) into the abdomen of Eri silkworm pupae on the second day after pupation, and the injected Eri silkworm pupae were then left to stand at 28°C for 7 to 8 days to produce the ASF antigen protein in the Eri silkworms. 500 Eri silkworm pupae were used for each ASF antigen protein.

[0109] ASF antigen protein produced in Eri silkworms was recovered from the silkworms using a FLAG tag. When the ASF antigen protein was expressed in insect cells (Sf9) using a recombinant baculovirus using a similar method, the ASF antigen protein was obtained as a soluble component. However, when the ASF antigen protein was expressed in Eri silkworms, although expression was confirmed, the protein was obtained as an insoluble component. Therefore, in order to obtain the ASF antigen protein as a soluble component in Eri silkworms, solubilization of the ASF antigen protein was investigated. In the solubilization study, molecular design was attempted to improve solubility while maintaining antigenicity, taking into consideration predicted three-dimensional structure, secondary structure, signal sequence, transmembrane domain, and glycosylation.

[0110] As a result of investigation, among the ASF antigen proteins, P30, P54, and pp62 were modified to have an 8xHis tag consisting of eight consecutive histidines attached to the C-terminus via a linker (GPGP), resulting in the design and synthesis of P30-cHis (SEQ ID NO: 13), P54-cHis (SEQ ID NO: 14), and pp62-cHis (SEQ ID NO: 15). The amino acid sequences of the ASF antigen proteins encoded by P30-cHis, P54-cHis, and pp62-cHis are set forth in SEQ ID NOs: 16 to 18, respectively.

[0111] For EP402R, the original signal sequence of EP402R at the N-terminus was changed to a GP64 signal sequence (SEQ ID NO: 29), and the sequence from the vicinity of the transmembrane region to the sequence of the C-terminal intramembrane (cellular) domain was substituted with a GPGP linker-8xHis tag-GPGP linker-ZZ domain or a GPGP linker-8xHis tag, resulting in the design and synthesis of EP402R-ZZ (SEQ ID NO: 19) and EP402R-cHis (SEQ ID NO: 20). The amino acid sequences of the ASF antigen proteins encoded by EP402R-ZZ and EP402R-cHis are set forth in SEQ ID NOs: 21 and 22, respectively.

[0112] EP402R-ZZ was synthesized based on ER402R with a FLAG tag added to the C-terminus, by replacing the signal sequence inherent in ER402R with a GP64 signal sequence, and then replacing the C-terminal portion of EP402R from the transmembrane region with a GPGP-8xHis tag-GPGP-ZZ domain. Genetic recombination was performed using the Gibson Assembly system (https: / / www.nebj.jp / f / 646; New England BioLabs Japan). EP402R-cHis was synthesized using a similar method.

[0113] For i177L, the N-terminal transmembrane domain was removed, and i177L-cyto-ZZ (SEQ ID NO: 23) was designed and synthesized by further modifying i177L by adding a GPGP linker-8xHis tag-GPGP linker-ZZ domain to the C-terminus. The amino acid sequence of the ASF antigen protein encoded by i177L-cyto-ZZ is the amino acid sequence set forth in SEQ ID NO: 24.

[0114] In addition, ZZ domain-P30-Linker-P54 (SEQ ID NO: 25) consisting of ZZ domain-P30-His linker-P54 (transmembrane region excluded) from the N-terminus to the C-terminus was designed and synthesized. Furthermore, GP64SS-ZZ domain-P30-Linker-P54 (SEQ ID NO: 26) consisting of GP64 signal sequence-ZZ domain-P30-His linker-P54 (transmembrane region excluded) from the N-terminus to the C-terminus was designed and synthesized. The amino acid sequences of the ASF antigen proteins encoded by ZZ domain-P30-Linker-P54 and GP64SS-ZZ domain-P30-Linker-P54 are set forth in SEQ ID NOs: 27 and 28, respectively.

[0115] Baculovirus was again recombined with the obtained P30-cHis, P54-cHis, pp62-cHis, EP402R-cHis, and i177L-cyto-ZZ, and the resulting recombinant baculovirus was then administered to Eri silkworm pupae to produce the ASF antigen protein in the Eri silkworm.

[0116] The ASF antigen protein produced in Eri silkworms was recovered and purified from Eri silkworms using TALON® resin with a His tag. Specifically, 15 Eri silkworm pupae infected with recombinant baculovirus were added to 100 mL of lysis buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1% CHAPS, 10% trehalose, 5 mM thiourea, 1 mM PMSF) and disrupted in a household blender to obtain a suspension. The resulting suspension was then sonicated to obtain Eri silkworm lysate. The resulting Eri silkworm lysate was centrifuged (10,000 rpm, 10 minutes, 4°C), and the supernatant was collected. The collected supernatant was sequentially filtered through a glass fiber filter (Merck Millipore) and a cellulose acetate membrane filter (0.8 μm pore size; ADVANTEC), and 50 mL of lysis buffer was added to the resulting filtrate. The resulting solution was subjected to batch treatment with 5 mL of TALON resin ("TALON Metal Affinity Resin"; TAKARA (Z5502N)) at 25°C for 1 hour to adsorb the ASF antigen protein to the resin. The adsorbed resin was then washed five times with 5 mL of lysis buffer (W1), followed by washing with 10 mL of wash buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 10% trehalose, 10 mM imidazole) (W2). After washing, the resin was eluted five times with 5 mL of elution buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 10% trehalose, 300 mM imidazole).

[0117] The production results of P30-cHis, pp62-cHis, EP402R-cHis, and i177L-cyto-ZZ are shown in Table 1. The concentration of the ASF antigen protein was determined by subjecting the ASF antigen protein and BSA to SDS-PAGE and creating a calibration curve using ImageJ based on the BSA concentration and band color intensity. As shown in Table 1, 130 mg to 230 mg of lyophilized ASF antigen protein was obtained using Eri silkworms.

[0118]

[0119] Furthermore, when EP402-cHis was used and Eri silkworm larvae were used instead of Eri silkworm pupae, the body fluid of the recovered Eri silkworm larvae was subjected to ammonium sulfate fractionation and a column packed with Ni-NTA resin (Fujifilm Wako Pure Chemical Industries, Ltd.; 141-09764) to recover and purify the ASF antigen protein, demonstrating that the ASF antigen protein can be produced in Eri silkworm larvae.

[0120] Example 2. Evaluation of immunogenicity of ASF antigen protein "TORPAC (registered trademark) Size M Capsule" (TORPAC) was filled with the lyophilized powder of the ASF antigen protein produced in Example 1. The filling amount was 0.1 mg / capsule for EP402R-cHis, 0.1 mg / capsule for P30-cHis, and 0.26 mg / capsule for pp62-cHis.

[0121] The resulting capsules filled with ASF antigen proteins were administered by probe to male C57BL / 6J-EGF mice aged 30 to 33 weeks. One capsule filled with either ASF antigen protein was administered to each mouse (Day 0). Three days after Day 0, another capsule was administered in the same manner (Day 3). Furthermore, six days after Day 0, another capsule was administered in the same manner (Day 6). Thus, each mouse received three capsules. Two mice were administered capsules for each ASF antigen protein.

[0122] Blood was collected from each mouse by intravenous injection on Day 0 before administration of the capsules, and 7 days (Day 7) and 14 days (Day 14) after the start of the test, and the ability to produce antibodies against each ASF antigen protein was evaluated by ELISA according to standard methods.

[0123] As a result, it was confirmed that antibodies against EP402R-cHis (ASF-specific serum IgG) were produced in significant amounts in the blood of mice administered EP402R-ZZ on Day 7 and Day 14 compared to Day 0. Furthermore, the amount of antibody produced was greater on Day 14 than on Day 7. Furthermore, no health abnormalities such as weight loss or diarrhea were observed in the Day 14 mice from which blood was collected.

[0124] Furthermore, the production of antibodies against the respective antigens was confirmed in mice administered with P30-cHis and pp62-cHis, although the amount of antibody production in the mice administered with P30-cHis and pp62-cHis was low.

[0125] From the above results, it was found that even when mice were used, the production of anti-ASF antigen protein antibodies could be induced by oral administration using the ASF antigen protein expressed and produced in Eri silkworms.

[0126] Example 3: Evaluation of the safety of ASF antigen protein in pigs Three-month-old three-way crossbred female pigs were orally administered feed mixed with the test substance three times: the first administration (Week 0), and two weeks (Week 2) and four weeks (Week 4) after the first administration.

[0127] The test substance used was freeze-dried lysates of Eri silkworm pupae infected with the recombinant baculovirus modified with the ASF antigen protein in Example 1. The control substance used was freeze-dried lysates of Eri silkworm pupae not infected with the baculovirus.

[0128] Test substance 1 was a mixture of EPR402R-expressing Eri silkworm pupae, P30-expressing Eri silkworm pupae, and P54-expressing Eri silkworm pupae. Test substance 2 was a mixture of i177L-expressing Eri silkworm pupae and pp62-expressing Eri silkworm pupae. Test substance 3 was a mixture of EPR402R and cholera toxin B (EPR402R + CTB)-expressing Eri silkworm pupae, P30 + CTB-expressing Eri silkworm pupae, and P54 + CTB-expressing Eri silkworm pupae. Test substance 4 was a mixture of i177L + CTB-expressing Eri silkworm pupae and pp62 + CTB-expressing Eri silkworm pupae.

[0129] The test pigs were divided into five groups of two pigs each. Pigs in Groups 1 and 3 were fed test substance 1 and test substance 3, respectively, mixed in feed at 6 g per pig. Pigs in Groups 2 and 4 were fed test substance 2 and test substance 4, respectively, mixed in feed at 4 g per pig. Pigs in Group 5 were fed the control substance mixed in feed at 2.0 g to 2.5 g per pig.

[0130] The feed used was "One Up Piglet" (Feed One Co.). The pigs were fed ad libitum. However, they were fasted from 5:00 pm on the day before the administration of the test substance. The test pigs were housed and bred in concrete-floored stalls in an open-system barn. They were allowed to drink tap water ad libitum through a waterer.

[0131] From Week 0 until the 8th week after the start of administration (Week 8), the weight, body temperature, general condition, and blood samples were taken for each test pig.

[0132] As a result of observing the general condition, no abnormalities were observed in the general condition such as vitality, appetite, fecal properties, and respiratory condition from Week 0 to Week 8 in any of the groups.

[0133] Body temperature measurements showed that after the first administration at Week 0, all animals experienced an increase in body temperature (39.5°C or higher) between 1 and 6 days after administration, but all recovered by 7 days after administration. After the second administration (14 days after the first administration), no animals showed an increase in body temperature. After the third administration (28 days after the first administration), an increase in body temperature was observed in one animal in Group 1 7 days after administration (35 days after the first administration), one animal in Group 4 2 days after administration (30 days after the first administration), and one animal in Group 5 3 and 7 days after administration (31 and 35 days after the first administration). Body temperatures were normal on days 21, 42, 49, and 56 after the first administration.

[0134] The rise in body temperature observed after the first and third administrations was also observed in Group 5, which received the control substance, and therefore was deemed to be transient and had no effect on the test.

[0135] As a result of weight measurement, the average weight of each group increased steadily from Week 0 to Week 8, from 41.6 kg to 84.8 kg in Group 1, from 42.9 kg to 86.9 kg in Group 2, from 35.5 kg to 76.6 kg in Group 3, from 41.4 kg to 83.7 kg in Group 4, and from 33.9 kg to 76.2 kg in Group 5.

[0136] Based on the above, in this evaluation, the test substance was administered to healthy pigs, and although a transient fever was observed after administration, no other abnormalities were observed in body weight, body temperature, or general condition that would suggest an effect of the test substance, and therefore it was assessed that there were no problems with the safety of the test substance.

[0137] Example 4: Evaluation of immune response to ASF antigenic proteins in pigs ASF virus antigenic proteins mixed with adjuvant were administered to one pig, and the immune response was evaluated. Specifically, the ASF antigenic proteins EP402R, i177L, P30, and P54 were administered subcutaneously together with adjuvant in amounts adjusted to the pig's body weight. Blood samples were collected weekly from the pigs administered the ASF antigenic proteins, and the amount of antibodies produced was evaluated using the following ELISA method.

[0138] ASF antigen protein was immobilized at a concentration of 0.1 μg / well at 4°C for approximately 16 hours, followed by washing three times with phosphate-buffered saline (PBST) containing 0.1% Tween 20 (final concentration). 300 μL of 5-fold diluted Blocking One (Nacalai Tesque) was added to the wells, blocked at 37°C for 1-2 hours, and then washed three times with PBST. 100 μL of 250-fold diluted porcine serum with PBS was added to the wells, and a primary antigen-antibody reaction was carried out at 37°C for 1 hour, followed by washing three times with PBST. 100 μL of 2000-fold diluted anti-porcine antibody (HRP-labeled) with PBS was added to the wells, and a secondary antigen-antibody reaction was carried out at 37°C for 1 hour, followed by washing three times with PBST. After the reaction, 100 μL of TMB solution was added to the wells of the well plate using "EzELISA TMB" (ATTO), and the wells were subjected to a color reaction at 37°C for 30 minutes. After that, 100 μL of a reaction stop solution was added, and the absorbance at 450 nm was measured using a plate reader.

[0139] P402R, i177L, P30, and P54 were produced and isolated using recombinant Eri silkworm pupae according to the method described in Example 1. 2 mL of an ASF antigen protein solution containing a mixture of these four ASF antigen proteins was administered subcutaneously to a Crown miniature pig (female, 2 years old) in two separate doses (on days 0 and 28).

[0140] ASF antigen protein solution was prepared by mixing 0.5 mL each of EP402R solution (0.66 mg / mL), i177L solution (3.0 mg / mL), P30 solution (1.1 mg / mL), and P54 solution (1.2 mg / mL) to prepare a 2 mL solution. The content of each ASF antigen protein was determined by immunostaining with FLAG antibody.

[0141] After the first administration, blood samples were collected every week (up to week 8), and the production of antibodies specific to ASF antigen proteins was examined by ELISA. The results are shown in Figure 1. As shown in Figure 1, the production of antibodies specific to each antigen was confirmed starting 7 days after the first administration. Furthermore, after the second administration (day 28), the amount of antibodies produced increased significantly.

[0142] These results demonstrate that the ASF antigen protein produced in Eri silkworm pupae, recovered, and purified can function as an ASF vaccine by inducing the production of specific antibodies in pigs. Furthermore, the amount of antibodies produced was significantly increased after two doses, indicating that two or more doses are preferable.

[0143] Furthermore, ASF antigen proteins (EP402R: 0.33 mg, il77L: 1.5 mg, P30: 0.6 mg, P54: 0.6 mg) were co-injected into Crown miniature pigs (female, 2 years old) (first injection: subcutaneous injection into the left side of the neck, second injection: subcutaneous injection into the right side of the neck 4 weeks later). After injection, the animals were observed for injection site reactions (swelling, induration, erythema, heat sensation, edema, exudate) and general condition (body weight, body temperature, vitality, appetite, fecal properties, respiratory condition, etc.) for 8 weeks.

[0144] As a result, swelling, induration, erythema, and a feeling of heat were observed at the injection site for about 10 days after the first and second injections. However, the swelling, erythema, and feeling of heat returned to the same level as before injection 10 days after injection. The induration also improved over time to a level that was not a problem.

[0145] Furthermore, the body weight increased from 48.2 kg before the first administration to 51.4 kg, and the body temperature was normal, ranging from 38.1°C to 39.5°C. Furthermore, no abnormalities were observed in vitality, appetite, fecal properties, respiratory condition, etc. These results demonstrated that the ASF antigen protein does not pose a health hazard to pigs.

[0146] The nucleotide and amino acid sequences described herein are as set forth in Table 2.

[0147]

[0148]

[0149]

[0150] By utilizing the present invention, African swine fever virus antigen protein can be produced from recombinant Eri silkworms with reduced endotoxin contamination and reduced operational burden. The resulting recombinant Eri silkworm-derived African swine fever virus antigen protein can induce a specific immune response in pigs and can be used as an African swine fever vaccine. CROSS-REFERENCE TO RELATED APPLICATIONS

[0151] This application claims priority from Japanese Patent Application No. 2024-055319, filed March 29, 2024, the entire disclosure of which is incorporated herein by reference. In addition, the entire disclosures of all documents referenced in the detailed description of the invention of this application, including Non-Patent Documents 1 to 3, are incorporated herein by reference.

Claims

1. Transgenic Eri silkworms expressing African swine fever virus antigen proteins.

2. The recombinant Eri silkworm according to claim 1, wherein the recombinant Eri silkworm is a recombinant Eri silkworm pupa.

3. The recombinant Eri silkworm described in claim 1 or 2, wherein the African swine fever virus antigen protein is a protein containing at least one epitope selected from the group consisting of EP402R, i177L, P30, P54, pp62 and P72.

4. The recombinant Eri silkworm according to claim 1 or 2, wherein the African swine fever virus antigen protein is a soluble recombinant African swine fever virus antigen protein.

5. The recombinant Eri silkworm according to claim 4, wherein the soluble recombinant African swine fever virus antigen protein has a histidine tag consisting of 4 to 15 histidines at the C-terminus.

6. A method for producing an African swine fever virus antigen protein, comprising the step of obtaining the African swine fever virus antigen protein by isolating the African swine fever virus antigen protein from the recombinant Eri silkworm according to claim 1 or 2.

7. A composition for pigs, comprising at least one active ingredient selected from the group consisting of the recombinant Eri silkworm described in claims 1 and 2 and the African swine fever virus antigen protein isolated from said recombinant Eri silkworm.

8. The porcine composition of claim 7, wherein the porcine composition is an oral porcine composition.

9. The porcine composition of claim 7, wherein the porcine composition is selected from the group consisting of feed, veterinary medicine, and African swine fever vaccine.

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