Booster vaccine for classical swine fever

Abstract

To provide a technique for granting or enhancing immunity against Classical Swine Fever Virus (CSFV) in pigs.SOLUTION: A CSFV E2 subunit vaccine is administered by injection to a pig already vaccinated with a CSFV live vaccine, thereby enhancing immunity against CSFV in the pig.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a booster vaccine for classical swine fever (CSF) and its use.

Background Art

[0002] Classical swine fever (CSF) is a disease of pigs and wild boars caused by infection with the CSF virus (CSFV). CSF is characterized by strong infectivity and high lethality, and is designated as a livestock infectious disease in the Livestock Infectious Diseases Control Law. Therefore, in farms where CSF occurs, it is stipulated that epidemic prevention measures for CSF should be taken for breeding pigs and the like.

[0003] In Japan, the occurrence of CSF was first confirmed in 1887. Since then, since the development of a live vaccine for CSF in 1969, the occurrence of CSF has decreased sharply, and no occurrence of CSF has been confirmed since 1992. However, in 2018, the occurrence of CSF was confirmed at a pig farm in Gifu Prefecture for the first time in 26 years in the country, and since then, the occurrence of CSF has been expanding.

[0004] The epidemic prevention measures for CSF are mainly based on the early detection of the occurrence of CSF and the disposal of pigs infected with CSFV. However, in recent years, the infection of CSFV has been expanding in wild boars. Therefore, when it is difficult to prevent the infection of CSFV to pigs even by improving sanitary management, in areas where the risk of infection of CSFV to pigs is high, pigs are inoculated with the CSFV GPE-strain, which is a live attenuated vaccine for CSF, to prevent the occurrence of CSF.

[0005] In fact, at each farm in the country, the appropriate age for inoculation with a live vaccine for CSF is determined with the goal of conferring a neutralizing antibody or an ELISA antibody positive rate of 80% on piglets.

[0006] In recent years, it has been found that in sows vaccinated with live CSF vaccine in the presence of maternally-derived antibodies against CSFV (sows from the second generation onwards), there is a greater variation in antibody titers against CSFV compared to sows vaccinated with live CSF vaccine in a state of no immunity against CSFV (sows of the first generation), and the proportion of individuals with low antibody titers against CSFV is also large. Since sows of the first generation have an antibody titer distribution showing an obvious peak, it was easy to determine the appropriate age for vaccination with live CSF vaccine in their piglets. In contrast, in piglets derived from sows from the second generation onwards, due to the variation in maternally-derived antibody titers, it has become a problem that the appropriate age for vaccination with live CSF vaccine has a range.

[0007] Actually, in pig farms vaccinated with live CSF vaccine, the continuous occurrence of CSF mainly in weaned piglets before and after vaccination with live CSF vaccine and in fattening pigs with a short number of days elapsed after vaccination has become a major threat to pig farmers (Non-Patent Document 1).

[0008] On the other hand, overseas, as a tool for pig fever eradication, in addition to live vaccines, a recombinant CSFV E2 subunit vaccine has been put into practical use. The recombinant CSFV E2 subunit vaccine is disclosed, for example, in Patent Documents 1 to 2.

[0009] In view of the threat of CSF that causes significant economic losses to fields dependent on commercial pig farming and its products, the development of vaccines and vaccination programs that can effectively prevent CSFV infection and the occurrence of CSF is eagerly desired as various options for epidemic prevention in Japan.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0011] [Non-Patent Document 1] Enlarged Swine Fever Epidemiology Investigation Team, Recommendations Based on the 17th Enlarged Swine Fever Epidemiology Investigation Team Review Meeting, August 3, 2022 [Summary of the Invention] [Problems to be Solved by the Invention]

[0012] An object of the present invention is to provide a technique for conferring or enhancing immunity against CSFV in pigs. [Means for Solving the Problems]

[0013] The present inventors have found that by inoculating a CSFV E2 subunit vaccine into pigs that have already been inoculated with a live CSF vaccine by injection, immunity against CSFV can be successfully enhanced in the pigs. In addition, the present inventors have found that when a pig in which immunity against CSFV has been enhanced as described above is a sow, immunity against CSFV can be successfully conferred on piglets by transfer antibodies from the sow. Based on these findings, the present inventors have completed the present invention.

[0014] That is, the present invention can be exemplified as follows. [1] A method for enhancing immunity against CSFV in pigs, comprising: inoculating a booster vaccine against CSFV into the pigs by injection, wherein the pigs have been inoculated with a live vaccine against CSFV, and the booster vaccine is a CSFV E2 subunit vaccine. [2] The method according to [1], wherein the neutralizing antibody titer against CSFV after inoculation with the booster vaccine is 4 times or more the neutralizing antibody titer against CSFV at the time of inoculation with the booster vaccine. [3] The method according to [2], wherein the neutralizing antibody titer against CSFV after the booster vaccination is a value at any time point between 14 days and 42 days after the booster vaccination. [4] The method according to [2], wherein the neutralizing antibody titer against CSFV after the booster vaccination is a value at 28 days after the booster vaccination. [5] The method according to any one of [1] to [4], wherein the coefficient of variation of the neutralizing antibody titer against CSFV after the booster vaccination is 0.3 or less. [6] The method according to [5], wherein the coefficient of variation of the neutralizing antibody titer against CSFV after the booster vaccination is a value at any time point between 14 days and 42 days after the booster vaccination. [7] The method according to [5], wherein the coefficient of variation of the neutralizing antibody titer against CSFV after the booster vaccination is a value at 28 days after the booster vaccination. [8] The method according to any one of [1] to [7], wherein the E2 subunit is a polypeptide comprising the amino acid sequence of the E2 subunit found in a CSFV field epidemic strain or a variant sequence thereof. [9] The method according to any one of [1] to [8], wherein the live vaccine is a CSFV GPE - strain.

[10] The method according to any one of [1] to [9], wherein the pig is a breeding pig.

[0015]

[11] The method according to

[10] , wherein the breeding pig is a breeding pig of the second generation or later.

[12] The method according to

[10] or

[11] , wherein the breeding pig is a sow.

[13] The method according to any one of [1] to

[12] , wherein the inoculation of the live vaccine is carried out once or more than once.

[14] The method according to any one of [1] to

[13] , wherein the booster vaccination is carried out after 4 months from the first vaccination of the live vaccine.

[15] The method according to any one of

[12] to

[14] , wherein the booster vaccination is carried out until 1 week before the mother pig gives birth to piglets.

[16] A method for conferring immunity against CSFV to piglets, comprising enhancing the immunity against CSFV in the mother pig by the method according to any one of

[12] to

[15] , wherein the mother pig is a pig that nurses colostrum to the piglets.

[17] A method for suppressing the variation in the transfer antibody titer against CSFV in piglets, comprising enhancing the immunity against CSFV in the mother pig by the method according to any one of

[12] to

[15] , wherein the mother pig is a pig that nurses colostrum to the piglets.

[18] Furthermore, the method according to

[16] or

[17] , comprising nursing colostrum from the mother pig to the piglets.

[19] A booster vaccine against CSFV, wherein the booster vaccine is a CSFV E2 subunit vaccine, A booster vaccine for inoculating a pig that has been inoculated with a live vaccine against CSFV by injection.

Advantages of the Invention

[0016] According to the present invention, immunity against CSFV can be conferred or enhanced in pigs.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0018] <1>Vaccine The vaccine described in this specification is a booster vaccine for classical swine fever (CSF). Hereinafter, this vaccine is also simply referred to as the "booster vaccine".

[0019] "Classical swine fever (CSF)" means a disease of pigs caused by infection with classical swine fever virus (CSFV).

[0020] "CSF booster vaccine" means a vaccine capable of enhancing immunity against CSFV in pigs that already have immunity against CSFV. Specifically, "CSF booster vaccine" may mean a vaccine that can enhance immunity against CSFV in pigs by inoculating pigs that already have immunity against CSFV. That is, by using the booster vaccine, specifically, by inoculating a pig that already has immunity against CSFV with the booster vaccine, the immunity against CSFV in the pig can be enhanced, that is, the effect of enhancing the immunity against CSFV in the pig can be obtained. This effect is also referred to as the "booster (effect)".

[0021] The terms "immunity against CSFV" and "immunity against CSF" may be used interchangeably. The terms "antibody against CSFV" and "antibody against CSF" may be used interchangeably. The terms "CSF vaccine", "vaccine for CSF", "vaccine against CSF", "CSFV vaccine", "vaccine for CSFV", and "vaccine against CSFV" may be used interchangeably.

[0022] The booster vaccine is a CSFV E2 subunit vaccine.

[0023] "CSFV E2 subunit vaccine" means a vaccine containing the E2 subunit of CSFV as an active ingredient. The E2 subunit of CSFV is hereinafter also simply referred to as the "E2 subunit". That is, the E2 subunit functions as an active ingredient (specifically, the active ingredient of the booster vaccine). "The E2 subunit functions as an active ingredient" or "The E2 subunit functions as the active ingredient of the booster vaccine" means that a booster effect is obtained depending on the presence of the E2 subunit.

[0024] "E2 subunit of CSFV" means a polypeptide containing the amino acid sequence of the E2 subunit found in any strain of CSFV or a variant sequence thereof. The variant sequence may or may not be found in any strain of CSFV. The said sequence contained in the E2 subunit (that is, the amino acid sequence of the E2 subunit found in any strain of CSFV or a variant sequence thereof) is also referred to as the "E2 subunit sequence".

[0025] "E2 subunit of a certain CSFV strain" means a polypeptide containing the E2 subunit sequence derived from the certain CSFV strain (that is, the amino acid sequence of the E2 subunit found in the certain CSFV strain or a variant sequence thereof). The variant sequence may or may not be found in the certain CSFV strain.

[0026] The E2 subunit may be encoded by the E2 subunit gene. That is, the "E2 subunit gene" means a polynucleotide encoding the E2 subunit. The nucleotide sequence of the E2 subunit gene is not particularly limited as long as it encodes the E2 subunit. The E2 subunit gene may be, for example, a polynucleotide containing a nucleotide sequence encoding the E2 subunit found in any strain of CSFV or a variant sequence thereof. The variant sequence may or may not be found in any strain of CSFV.

[0027] The CSFV strain from which the E2 subunit sequence or the nucleotide sequence encoding it is derived is not particularly limited as long as the E2 subunit functions as an active ingredient. Examples of the CSFV strain from which the E2 subunit sequence or the nucleotide sequence encoding it is derived include CSFV wild strains and CSFV attenuated strains. . Examples of the CSFV strain from which the E2 subunit sequence or the nucleotide sequence encoding it is derived particularly include CSFV wild strains. Examples of CSFV wild strains include CSFV field epidemic strains. That is, the E2 subunit may be, for example, the E2 subunit of a CSFV field epidemic strain, and specifically, a polypeptide containing an E2 subunit sequence derived from a CSFV field epidemic strain (that is, an amino acid sequence of the E2 subunit found in a CSFV field epidemic strain or a variant sequence thereof).

[0028] The nucleotide sequence encoding the E2 subunit found in a CSFV field epidemic strain is shown as SEQ ID NO: 1, and the amino acid sequence of the same E2 subunit is shown as SEQ ID NO: 2. That is, the E2 subunit gene may be, for example, a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 1 or a variant sequence thereof. Also, the E2 subunit may be, for example, a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 or a variant sequence thereof.

[0029] The variant sequence of the nucleotide sequence for the E2 subunit gene and the variant sequence of the amino acid sequence for the E2 subunit may each be a naturally occurring variant or an artificially modified variant. The variant sequence of the nucleotide sequence for the E2 subunit gene is not particularly limited as long as the polynucleotide containing the variant sequence encodes the E2 subunit. The variant sequence of the amino acid sequence for the E2 subunit is not particularly limited as long as the polypeptide containing the variant sequence functions as an active ingredient (specifically, the active ingredient of a booster vaccine).

[0030] That the polypeptide functions as an active ingredient (specifically, the active ingredient of a booster vaccine) can be confirmed, for example, by inoculating the polypeptide into pigs (for example, pigs that have been inoculated with a live vaccine of CSF) and confirming an increase in the antibody titer against CSFV. Inoculation of the polypeptide into pigs and measurement of the antibody titer can be carried out, for example, in the same procedure as inoculation of a booster vaccine into pigs and measurement of the antibody titer in the method described in this specification below.

[0031] That the polypeptide functions as an active ingredient (specifically, the active ingredient of a booster vaccine) can be confirmed, for example, by confirming that an antigen-antibody reaction occurs between the polypeptide and an anti-E2 subunit antibody. The antigen-antibody reaction can be measured, for example, by a conventional method.

[0032] Examples of the variant sequence of the amino acid sequence for the E2 subunit include amino acid sequences in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 2). The above "one or several" may be, for example, 1 to 65, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, or 1.

[0033] As the variant sequence of the amino acid sequence for the E2 subunit, there may also be mentioned amino acid sequences having an identity of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more with respect to the entire amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 2).

[0034] As the variant sequence of the nucleotide sequence for the E2 subunit gene, there may be mentioned a nucleotide sequence that hybridizes under stringent conditions with the complementary sequence of the above nucleotide sequence (for example, the nucleotide sequence shown in SEQ ID NO: 1). "Stringent conditions" may mean conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. As stringent conditions, there may be mentioned conditions of washing in ordinary Southern hybridization, namely, 60 °C, 1×SSC, 0.1% SDS, preferably 60 °C, 0.1×SSC, 0.1% SDS, more preferably 68 °C, 0.1×SSC, 0.1% SDS, and washing once, preferably 2 to 3 times, at a salt concentration and temperature corresponding thereto.

[0035] As the variant sequence of the nucleotide sequence for the E2 subunit gene, there may also be mentioned nucleotide sequences having an identity of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more with respect to the entire nucleotide sequence (for example, the nucleotide sequence shown in SEQ ID NO: 1).

[0036] As the variant sequence of the nucleotide sequence for the E2 subunit gene, there may also be mentioned a nucleotide sequence in which any one or more codons in the above nucleotide sequence (for example, the nucleotide sequence shown in SEQ ID NO: 1) are replaced with codons equivalent thereto (that is, a variant sequence of the above nucleotide sequence due to the degeneracy of codons). The variant sequence of the nucleotide sequence may be modified, for example, so as to have optimal codons according to the codon usage frequency of the host used for heterologous expression.

[0037] The identity between amino acid sequences and the identity between nucleotide sequences can both be calculated, for example, by conventional methods. Specifically, the identity between amino acid sequences can be calculated, for example, by blastp. Specifically, the identity between nucleotide sequences can be calculated, for example, by blastn.

[0038] The E2 subunit may be a polypeptide consisting of an E2 subunit sequence, or may be a polypeptide consisting of an amino acid sequence with one or more amino acid residues added to the N-terminus and / or C-terminus of the E2 subunit sequence. The amino acid residues that can be added to the N-terminus and / or C-terminus of the E2 subunit sequence are also referred to as "added sequences". The added sequences are not particularly limited as long as the E2 subunit functions as an active ingredient (specifically, the active ingredient of a booster vaccine). The added sequences can be independently selected for the N-terminus and C-terminus of the E2 subunit sequence, respectively. Examples of added sequences include signal peptides and peptide tags. As the signal peptide, for example, one that functions in the expression host of the E2 subunit can be selected. "The signal peptide functions in the expression host of the E2 subunit" may mean that the signal peptide contributes to the transport of the E2 subunit to the endoplasmic reticulum or extracellularly in the expression host. Examples of peptide tags include His tag, FLAG tag, GST tag, Myc tag, ZZ tag, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (Thioredoxin), GFP (green fluorescent protein), HRP (horseradish peroxidase), ALP (Alkaline Phosphatase), and the Fc region of an antibody. The peptide tag may be used, for example, for the detection and purification of the expressed E2 subunit. The added sequence may be present, for example, during the expression of the E2 subunit but not present in the finally obtained E2 subunit. For example, the signal peptide may be cleaved after the expression of the E2 subunit and thus may not be present in the finally obtained E2 subunit.

[0039] As the E2 subunit, for example, a commercially available product may be used, or one appropriately manufactured and obtained may be used.

[0040] The method for manufacturing the E2 subunit is not particularly limited. The E2 subunit can be manufactured, for example, by expressing the gene in a host having the E2 subunit gene. Also, the E2 subunit can be manufactured, for example, by expressing the E2 subunit gene in a cell-free protein synthesis system. Further, the E2 subunit can be manufactured, for example, by separating and purifying it from CSFV.

[0041] Hereinafter, the manufacture of the E2 subunit using a host having the E2 subunit gene will be exemplified. The E2 subunit manufactured using a host having the E2 subunit gene is also referred to as a "recombinant E2 subunit".

[0042] The host is not particularly limited as long as it can express the E2 subunit that functions as an active ingredient (specifically, the active ingredient of the booster vaccine). Examples of the host include microorganisms, insect cells, plant cells, and animal cells. The cells may form an aggregate such as a tissue or an organism, for example.

[0043] Examples of the microorganisms include bacteria and yeasts. Examples of the bacteria include bacteria belonging to the family Enterobacteriaceae, coryneform bacteria, and bacteria belonging to the genus Bacillus. Examples of the bacteria belonging to the family Enterobacteriaceae include bacteria belonging to the genus Escherichia such as Escherichia coli. Examples of the coryneform bacteria include bacteria belonging to the genus Corynebacterium such as Corynebacterium glutamicum. Examples of the bacteria belonging to the genus Bacillus include Bacillus subtilis.

[0044] Examples of insects include insects of the genus Autographa such as Autographa californica, insects of the genus Trichoplusia such as Trichoplusia ni, insects of the genus Bombyx such as Bombyx mori, insects of the genus Orgyia such as Orgyia pseudotsugata, insects of the genus Lymantria such as Lymantria dispar, and insects of the genus Spodoptera such as Spodoptera exigua and Spodoptera frugiperda.

[0045] Examples of plants include plants of the Solanaceae family and plants of the Brassicaceae family. Examples of Solanaceae plants include plants of the genus Nicotiana and plants of the genus Solanum. Examples of Nicotiana plants include Nicotiana benthamiana and Nicotiana tabacum. Examples of Solanum plants include Solanum lycopersicum, Solanum melongena, and Solanum tuberosum. Examples of Brassicaceae plants include plants of the genus Arabidopsis. An example of an Arabidopsis plant is Arabidopsis thaliana.

[0046] Examples of animals include mammals. Examples of mammals include rodents and primates. Examples of rodents include hamsters, mice, rats, and guinea pigs. An example of a hamster is the Chinese hamster. Examples of primates include humans, monkeys, and chimpanzees. An example of a monkey is the African green monkey.

[0047] A host having the E2 subunit gene can be obtained, for example, by introducing the E2 subunit gene into the host.

[0048] The E2 subunit gene can be obtained, for example, by cloning from CSFV or by chemical synthesis. The obtained E2 subunit gene can be used as it is or after appropriate modification. Modification of the gene can be carried out by known methods. For example, by site-directed mutagenesis, a desired mutation can be introduced into a target site of DNA. Examples of site-directed mutagenesis methods include the method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and the method using phage (Kramer, W. and Frits, H. J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154, 367 (1987)).

[0049] The method for introducing the E2 subunit gene into the host is not particularly limited. The E2 subunit gene only needs to be retained in the host so that it can be expressed. Specifically, in the host, the E2 subunit gene only needs to be retained so that it can be expressed under the control of a promoter that functions in the host. In the host, the E2 subunit gene may be present, for example, on a vector that replicates autonomously outside the genome or may be introduced into the genome.

[0050] The promoter for expressing the E2 subunit gene is not particularly limited as long as it functions in the host. The "promoter that functions in the host" may mean a promoter having promoter activity in the host. The promoter may be a promoter derived from the host or a promoter derived from a heterologous source. The promoter may be the native promoter of the E2 subunit gene or the promoter of another gene. For example, promoters that function in bacteria of the family Enterobacteriaceae such as E. coli include the T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Pm1 promoter, PR promoter, and PL promoter. Also, for example, promoters that function in coryneform bacteria such as C. glutamicum include the lac promoter, tac promoter, trc promoter, F1 promoter, P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679 (2000)), EF-Tu promoter (Journal of Biotechnology 104 (2003) 311-323, Appl. Environ. Microbiol. 2005 Dec;71(12):8587-96.), SOD promoter, and promoters of genes such as pta, aceA, aceB, adh, amyE, and cspB. Also, for example, promoters that function in insect cells include promoters derived from baculoviruses. Examples of baculoviruses include AcMNPV, BmNPV, OpMNPV, LdMNPV, and SeMNPV. Also, for example, promoters that function in plant cells include plant-derived promoters and plant virus-derived promoters. An example of a plant-derived promoter is the ubiquitin promoter.Examples of plant viruses include Cauliflower mosaic virus (CaMV), Commelina yellow mottle virus (CoYMV), Rice tungro bacilliform virus (RTBV), Sugarcane bacilliform virus (SCBV), Soybean chlorotic mottle virus (SbCMV), Figwort mosaic virus (FMV), Carnation etched ring virus (CERV), Peanut chlorotic streak virus (PCSV), Strawberry vein banding virus (SVBV), Cacao swollen shoot virus (CSSV), and Cassava vein mosaic virus (CsVMV). Examples of promoters that function in animal cells include the SV40 promoter, EF1a promoter, RSV promoter, CMV promoter, and SRalpha promoter.

[0051] The E2 subunit gene can be introduced into a host, for example, by introducing a vector containing the gene into the host. A vector containing the E2 subunit gene is also referred to as an "expression vector for the E2 subunit gene". The expression vector for the E2 subunit gene can be constructed, for example, by ligating a DNA fragment containing the E2 subunit gene to a vector. By transforming a host with the expression vector for the E2 subunit gene, the gene can be introduced into the host. The vector may or may not be capable of autonomous replication in the host. The vector may be equipped with a marker such as a drug resistance gene. The vector may be equipped with an expression regulatory sequence such as a promoter for expressing the E2 subunit gene. The vector can be appropriately selected according to various conditions such as the type of the host. Examples of the vector include plasmid vectors and virus vectors. For example, vectors capable of autonomous replication in bacteria of the family Enterobacteriaceae such as E. coli include pUC vectors such as pUC19 and pUC18, pHSG vectors such as pHSG299, pHSG399, and pHSG398, pET vectors, pTrc99A, and pBR322. Also, for example, vectors capable of autonomous replication in coryneform bacteria such as C. glutamicum include pHM1519, pAM330, pVK4, pVK7, pVC7, and pVS7. Also, for example, vectors that can be used in insect cells include plasmid vectors such as pIZ / V5-His and virus vectors derived from baculoviruses as exemplified above. Also, for example, vectors that can be used in plant cells include plasmid vectors such as pRI vectors and pCambia vectors and virus vectors derived from plant viruses as exemplified above. Also, for example, vectors that can be used in animal cells include plasmid vectors such as pcDNA vectors, pBApo-CMV vectors, and pCI-neo (Promega), and virus vectors such as retrovirus vectors and adenovirus vectors.Depending on the type and composition of the vector, the expression vector of the E2 subunit gene may be integrated into the host genome, may replicate autonomously outside the host genome, or may be temporarily retained outside the host genome.

[0052] In addition, the E2 subunit gene can be introduced into a host, for example, by introducing a nucleic acid fragment containing the gene into the host. Examples of such nucleic acid fragments include linear DNA and linear RNA.

[0053] The method of introducing nucleic acids such as vectors and nucleic acid fragments into a host can be appropriately selected according to various conditions such as the type of host. Examples of methods for introducing nucleic acids into a host include the DEAE-dextran method, calcium phosphate method, lipofection method, electroporation method, and microinjection method. When the vector is a viral vector, the vector (virus) can be introduced into the host by infecting the host with the vector.

[0054] The expressed E2 subunit can be recovered, for example, as a solution containing the E2 subunit and used as an active ingredient of a booster vaccine. Examples of solutions containing the E2 subunit include a host culture solution, a host culture supernatant, and a host cell disruption extract. In addition, the expressed E2 subunit can be purified to a desired degree and used as an active ingredient of a booster vaccine, for example.

[0055] The booster vaccine may consist of the E2 subunit, or may contain components other than the E2 subunit in addition to the E2 subunit. Components other than the E2 subunit that may be contained in the booster vaccine are also referred to as "additional components". The additional components may or may not contribute to the booster effect.

[0056] Examples of additional components include components that can be normally contained in vaccines such as swine vaccines. Examples of such components include adjuvants, preservatives, and solvents.

[0057] The booster vaccine can be produced, for example, by appropriately mixing its raw materials (i.e., the E2 subunit and optionally additional components).

[0058] The content and content ratio of each component (i.e., the E2 subunit and optionally additional components) in the booster vaccine are not particularly limited as long as a booster effect can be obtained by using the booster vaccine. The content and content ratio of each component (i.e., the E2 subunit and optionally additional components) in the booster vaccine can be appropriately set according to various conditions such as the type of each component and the usage mode of the booster vaccine. For example, the content of the E2 subunit in the booster vaccine may be an amount that can obtain a booster effect when the booster vaccine is used in the manner described in the method described in this specification below.

[0059] The form of the booster vaccine is not particularly limited. The booster vaccine may be in any form such as a liquid or a powder. The booster vaccine may particularly be a liquid. The booster vaccine may be produced and provided, for example, in a form that can be used as it is, or in a form that requires preparation at the time of use. The booster vaccine may be produced and provided, for example, in a form that is diluted with a solvent at the time of use. Examples of the form that is diluted with a solvent at the time of use include concentrated forms such as dried products and concentrated solutions.

[0060] The booster vaccine can be used, for example, in the manner described in the method described in this specification below. That is, the booster vaccine may be, for example, for use in the method described in this specification below.

[0061] Specifically, the booster vaccine may be for inoculating, by injection, pigs that have been inoculated with a live vaccine against CSFV (also referred to as a "live CSFV vaccine" or a "live vaccine against CSFV").

[0062] The booster vaccine may specifically be for enhancing immunity against CSFV in pigs. More specifically, the booster vaccine may be for inoculating, by injection, pigs that have been inoculated with the live CSFV vaccine in order to enhance immunity against CSFV in pigs.

[0063] The booster vaccine may specifically be for preventing infection with CSFV in pigs, for preventing the occurrence of CSF in pigs, and / or for alleviating the symptoms during the occurrence of CSF in pigs. More specifically, the booster vaccine may be for inoculating, by injection, pigs that have been inoculated with the live CSFV vaccine in order to prevent infection with CSFV in pigs, for preventing the occurrence of CSF in pigs, and / or for alleviating the symptoms during the occurrence of CSF in pigs.

[0064] The booster vaccine may specifically be for conferring immunity against CSFV to piglets. More specifically, the booster vaccine may be for inoculating, by injection, sows that have been inoculated with the live CSFV vaccine and that nurse colostrum to the piglets in order to confer immunity against CSFV to the piglets.

[0065] The booster vaccine may specifically be for preventing infection with CSFV in piglets, for preventing the occurrence of CSF in piglets, and / or for alleviating the symptoms during the occurrence of CSF in piglets. More specifically, the booster vaccine may be for inoculating, by injection, sows that have been inoculated with the live CSFV vaccine and that nurse colostrum to the piglets in order to prevent infection with CSFV in piglets, for preventing the occurrence of CSF in piglets, and / or for alleviating the symptoms during the occurrence of CSF in piglets.

[0066] <2> Method The first aspect of the method described in this specification is a method for enhancing immunity against CSFV in pigs. Hereinafter, the same method (i.e., the first aspect of the method described in this specification) is also referred to as the "first method."

[0067] The first method includes inoculating a booster vaccine to a pig.

[0068] The pig to be inoculated with the booster vaccine is a pig that has already been inoculated with a live CSFV vaccine. The pig to be inoculated with the booster vaccine is not particularly limited other than having been inoculated with a live CSFV vaccine. That is, as the pig to be inoculated with the booster vaccine, any pig that has been inoculated with a live CSFV vaccine and desires to enhance immunity against CSVF may be selected. The pig that has been inoculated with a live CSFV vaccine may already have immunity against CSFV.

[0069] Examples of the live CSFV vaccine include attenuated strains of CSFV. Examples of the attenuated strains of CSFV include the LPC strain, C strain, GPE- strain, LOM strain, CP7_E2alf strain, Flc-LOM-BE rns strain. In particular, the GPE- strain is included as an attenuated strain of CSFV.

[0070] The number of inoculations of the live vaccine in a pig that has been inoculated with a live CSFV vaccine is not particularly limited. The number of inoculations of the live vaccine may be, for example, 1 time, or may be 2 times or more. The number of inoculations of the live vaccine may be, for example, 5 times or less, 4 times or less, 3 times or less, or 2 times or less. The number of inoculations of the live vaccine may be, for example, within a range defined by any combination of the ranges exemplified above. The number of inoculations of the live vaccine may particularly be 1 time or 2 times.

[0071] When the number of inoculations of the live vaccine in a pig that has been inoculated with a live CSFV vaccine is 2 times or more, the implementation modes of each inoculation (for example, conditions related to inoculation such as the type of live vaccine, inoculation route, inoculation amount, etc.) may or may not be the same. When the number of inoculations of the live vaccine in a pig that has been inoculated with a live CSFV vaccine is 2 times or more, the implementation modes of each inoculation may particularly be the same.

[0072] The first method may include inoculating pigs with a live CSFV vaccine before inoculating them with a booster vaccine. In this case, the pigs after inoculation with the live CSFV vaccine may be regarded as the pigs to be inoculated with the booster vaccine in the first method. That is, specifically, the first method may include inoculating pigs with a live CSFV vaccine and inoculating the pigs after inoculation with the live CSFV vaccine with the booster vaccine by injection. The pigs inoculated with the live CSFV vaccine may be pigs without a history of inoculation with the live CSFV vaccine or pigs that have been inoculated with the live CSFV vaccine one or more times.

[0073] The pigs to be inoculated with the booster vaccine may be female or male. The pigs to be inoculated with the booster vaccine may be, for example, breeding pigs. "Breeding pigs" means pigs used for breeding, and specifically, may collectively refer to female and male pigs for breeding. The breeding pigs may be multiparous or nulliparous. The purpose of breeding of the breeding pigs is not particularly limited. The purpose of breeding of the breeding pigs may be, for example, for meat use. The pigs to be inoculated with the booster vaccine (for example, breeding pigs) may specifically be breeding pigs from the second generation onwards. The pigs to be inoculated with the booster vaccine (for example, breeding pigs) may particularly be female pigs.

[0074] "The breeding pigs of the second generation and later" may mean breeding pigs that have been given transfer antibodies against CSFV from sows vaccinated with CSFV vaccine. The imparting of transfer antibodies is achieved by suckling colostrum. That is, specifically, "the breeding pigs of the second generation and later" may mean breeding pigs that have been given transfer antibodies against CSFV by suckling colostrum from sows vaccinated with CSFV vaccine. The breeding pigs of the second generation and later may or may not be born from sows vaccinated with CSFV vaccine. Typically, the breeding pigs of the second generation and later may be born from sows vaccinated with CSFV vaccine. The sow that gives birth to the breeding pigs of the second generation and later and the sow that suckles colostrum to the breeding pigs of the second generation and later may or may not be the same individual. Typically, the sow that gives birth to the breeding pigs of the second generation and later and the sow that suckles colostrum to the breeding pigs of the second generation and later may be the same individual. That is, "the breeding pigs of the second generation and later" may typically mean breeding pigs born from sows vaccinated with CSFV vaccine and given transfer antibodies from such sows. The breeding pigs of the second generation and later may be born more than 30 days after the vaccination of the CSFV vaccine in the sows vaccinated with CSFV vaccine. Also, the breeding pigs of the second generation and later may be suckled with colostrum more than 30 days after the vaccination of the CSFV vaccine in the sows vaccinated with CSFV vaccine. When the sows vaccinated with CSFV vaccine have been vaccinated with the CSFV vaccine two or more times, "after the vaccination of the CSFV vaccine" may mean after the first vaccination of the CSFV vaccine. The CSFV vaccine to be inoculated to the sows of the breeding pigs of the second generation and later is not particularly limited as long as it can induce immunity against CSFV. The CSFV vaccine to be inoculated to the sows of the breeding pigs of the second generation and later may be, for example, a live vaccine or an inactivated vaccine. Examples of the CSFV vaccine that is a live vaccine include the CSFV live vaccines as exemplified above. Examples of the CSFV vaccine that is an inactivated vaccine include the CSFV E2 subunit vaccine.In sows of breeding pigs from the second generation onwards, vaccination with a CSFV vaccine may be, for example, the first CSFV vaccine inoculation (also referred to as "initial mass vaccination") only when the breeding area of the sow is designated as an area recommended for CSFV vaccine inoculation. In addition, sows that have been vaccinated with a CSFV vaccine may have enhanced immunity against CSFV by the methods described herein (for example, the first method).

[0075] The inoculation mode of the booster vaccine (for example, conditions related to inoculation such as the inoculation dose, number of inoculations, inoculation timing, etc.) is inoculation by injection, and is not particularly limited as long as a booster effect can be obtained.

[0076] The booster vaccine may be inoculated into pigs by injection. Examples of the inoculation route of the booster vaccine include intramuscular inoculation, subcutaneous inoculation, intravenous inoculation, etc., but are not particularly limited as long as a booster effect can be obtained. The booster vaccine may be inoculated into pigs as it is, or after being appropriately prepared into a form suitable for inoculation into pigs. For example, when the booster vaccine is provided in a form that is not a liquid such as a dried product, the booster vaccine may be prepared into a liquid form with a solvent and then inoculated into pigs. The use of needleless injection is also included in inoculation by injection.

[0077] The number of inoculations of the booster vaccine may be, for example, 1 time, or may be 2 times or more. The number of inoculations of the booster vaccine may be, for example, 5 times or less, 4 times or less, 3 times or less, or 2 times or less. The number of inoculations of the booster vaccine may be, for example, within a range defined by any combination of the above-exemplified ranges. The number of inoculations of the booster vaccine may particularly be 1 time or 2 times.

[0078] When the number of inoculations of the booster vaccine is 2 times or more, the implementation mode of each inoculation (for example, conditions related to inoculation such as the composition of the booster vaccine, inoculation route, inoculation dose, etc.) may or may not be the same. When the number of inoculations of the booster vaccine is 2 times or more, the implementation mode of each inoculation may particularly be the same.

[0079] The inoculation dose of the booster vaccine may be, for example, 10 μg / dose or more, 20 μg / dose or more, 50 μg / dose or more, 100 μg / dose or more, 200 μg / dose or more, or 500 μg / dose or more when converted to the inoculation dose of the E2 subunit, and may also be 1000 μg / dose or less, 500 μg / dose or less, 200 μg / dose or less, 100 μg / dose or less, 50 μg / dose or less, or 20 μg / dose or less, and may be within the range defined by any non-contradictory combination thereof. Specifically, the inoculation dose of the booster vaccine may be, for example, 10 - 20 μg / dose, 20 - 50 μg / dose, 50 - 100 μg / dose, 100 - 200 μg / dose, 200 - 500 μg / dose, or 500 - 1000 μg / dose when converted to the inoculation dose of the E2 subunit. Specifically, the inoculation dose of the booster vaccine may be, for example, 10 - 1000 μg / dose, 20 - 500 μg / dose, or 50 - 200 μg / dose when converted to the inoculation dose of the E2 subunit.

[0080] The inoculation of the booster vaccine may be carried out, for example, after 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after the first inoculation of the live CSFV vaccine, and may also be carried out by 12 months, 9 months, 6 months, 5 months, 4 months, or 3 months after the first inoculation of the live CSFV vaccine, and may be carried out within the period defined by any non-contradictory combination thereof. In particular, the inoculation of the booster vaccine may be carried out after 4 months after the first inoculation of the live CSFV vaccine. Specifically, the inoculation of the booster vaccine may be carried out, for example, between 1 month and 2 months, 2 months and 3 months, 3 months and 4 months, 4 months and 5 months, 5 months and 6 months, 6 months and 9 months, or 9 months and 12 months after the first inoculation of the live CSFV vaccine.

[0081] The booster vaccination may be carried out, for example, when the pig to be vaccinated with the booster vaccine is a sow, up to 3 months before, 2 months before, 1 month before, 2 weeks before, or 1 week before the sow gives birth to piglets. The booster vaccination may be carried out, in particular, up to 1 week before the sow gives birth to piglets.

[0082] The booster vaccination may be carried out, for example, when the pig to be vaccinated with the booster vaccine is a sow, up to 3 months before, 2 months before, 1 month before, 2 weeks before, or 1 week before the sow nurses the piglets with colostrum. The booster vaccination may be carried out, in particular, up to 1 week before the sow nurses the piglets with colostrum.

[0083] In the first method, the immunity against CSFV in pigs may be enhanced to a desired level. The enhancement of the immunity against CSFV can be confirmed, for example, using the neutralizing antibody titer against CSFV as an indicator.

[0084] The neutralizing antibody titer against CSFV after booster vaccination may be, for example, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 7 times or more, 10 times or more, or 15 times or more the neutralizing antibody titer against CSFV at the time of booster vaccination, and may be 100 times or less, 50 times or less, 20 times or less, or 10 times or less the neutralizing antibody titer against CSFV at the time of booster vaccination, and may be within a range defined by any non - conflicting combination thereof. The neutralizing antibody titer against CSFV after booster vaccination may particularly be 4 times or more the neutralizing antibody titer against CSFV at the time of booster vaccination. Specifically, the neutralizing antibody titer against CSFV after booster vaccination may be, for example, 1.5 to 100 times, 2 to 100 times, 3 to 100 times, 4 to 100 times, or 5 to 100 times the neutralizing antibody titer against CSFV at the time of booster vaccination. The ratio of the neutralizing antibody titer against CSFV after booster vaccination to the neutralizing antibody titer against CSFV at the time of booster vaccination (= neutralizing antibody titer against CSFV after booster vaccination / neutralizing antibody titer against CSFV at the time of booster vaccination) is also referred to as the "enhancement ratio of neutralizing antibody titer".

[0085] The neutralizing antibody titer against CSFV after booster vaccination may be, for example, 0.8 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, or 2.8 or more, 3 or less, 2.8 or less, 2.6 or less, 2.4 or less, 2.2 or less, 2 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, or 1 or less, as the S / P value by the ELISA method, and may also be a range defined by any non - conflicting combination thereof. Specifically, the neutralizing antibody titer against CSFV after booster vaccination may be, for example, 0.8 - 1, 1 - 1.2, 1.2 - 1.4, 1.4 - 1.6, 1.6 - 1.8, 1.8 - 2, 2 - 2.2, 2.2 - 2.4, 2.4 - 2.6, 2.6 - 2.8, or 2.8 - 3, as the S / P value by the ELISA method. Specifically, the neutralizing antibody titer against CSFV after booster vaccination may be, for example, 0.8 - 3, 1 - 2.4, or 1 - 2, as the S / P value by the ELISA method.

[0086] The increase in the neutralizing antibody titer due to booster vaccination (i.e., the value obtained by subtracting the neutralizing antibody titer against CSFV at the time of booster vaccination from the neutralizing antibody titer against CSFV after booster vaccination) may be, for example, 0.4 or more, 0.6 or more, 0.8 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, or 2.8 or more, or may be 3 or less, 2.8 or less, 2.6 or less, 2.4 or less, 2.2 or less, 2 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, 1 or less, 0.8 or less, or 0.6 or less, as the S / P value of the ELISA method, and may also be a range defined by any non - conflicting combination thereof. Specifically, the increase in the neutralizing antibody titer due to booster vaccination may be, for example, 0.4 - 0.6, 0.6 - 0.8, 0.8 - 1, 1 - 1.2, 1.2 - 1.4, 1.4 - 1.6, 1.6 - 1.8, 1.8 - 2, 2 - 2.2, 2.2 - 2.4, 2.4 - 2.6, 2.6 - 2.8, or 2.8 - 3, as the S / P value of the ELISA method. Specifically, the increase in the neutralizing antibody titer due to booster vaccination may be, for example, 0.4 - 3, 0.6 - 2.4, or 0.8 - 2, as the S / P value of the ELISA method.

[0087] The neutralizing antibody titer against CSFV after booster vaccination may be the value at any time point after booster vaccination. The neutralizing antibody titer against CSFV after booster vaccination may be, for example, the value at any time point after 7 days, 14 days, 21 days, 28 days, or 35 days after the booster vaccination, or may be the value at any time point up to 49 days, 42 days, 35 days, 28 days, or 21 days after the booster vaccination, and may also be the value at any time point within any non - conflicting combination of these periods. In particular, the neutralizing antibody titer against CSFV after booster vaccination may be the value at any time point between 14 days and 42 days after booster vaccination. More particularly, the neutralizing antibody titer against CSFV after booster vaccination may be the value at 28 days after booster vaccination.

[0088] The method for measuring the neutralizing antibody titer against CSFV is not particularly limited as long as the neutralizing antibody titer against CSFV can be measured with the desired accuracy. The neutralizing antibody titer against CSFV can be measured, for example, by a conventional method. Examples of the method for measuring the neutralizing antibody titer include a neutralization test and an ELISA method. That is, the neutralizing antibody titer may be, for example, directly measured by a neutralization test or may be estimated by an ELISA method. For example, the neutralizing antibody titer can be estimated from the S / P value of the ELISA method (Kobayashi, Creation of a table for estimating neutralizing antibody titer from S / P value of porcine fever ELISA, Porcine Disease Association Report No. 80, 2022, 12-16). The estimation of the neutralizing antibody titer from the S / P value of the ELISA method can be carried out, for example, by ROC analysis. The measurement of the neutralizing antibody titer against CSFV by the ELISA method can be carried out, for example, using a commercially available kit for measuring the neutralizing antibody titer against CSFV. Examples of such a kit include the Porcine Fever ELISA Kit II (Nippon Gene). The measurement of the neutralizing antibody titer against CSFV by the ELISA method can be carried out, for example, as described in the examples. Note that "the enhancement ratio of the neutralizing antibody titer satisfies a certain condition" means that, unless otherwise specified, the enhancement ratio of the neutralizing antibody titer calculated by any arbitrary measurement method (which may be, for example, a neutralization test or ELISA) satisfies the certain condition. For example, "the enhancement ratio of the neutralizing antibody titer is 4-fold or more" means that the enhancement ratio of the neutralizing antibody titer calculated by any arbitrary measurement method (which may be, for example, a neutralization test or ELISA) is 4-fold or more.

[0089] In the first method, the variation in the neutralizing antibody titer against CSFV may be suppressed by inoculation with a booster vaccine. For example, the variation in the neutralizing antibody titer against CSFV may be reduced after inoculation with a booster vaccine as compared to that at the time of booster vaccine inoculation. Further, for example, the variation in the neutralizing antibody titer against CSFV may be reduced after inoculation with a booster vaccine as compared to that after inoculation with a live CSFV vaccine when the live CSFV vaccine is inoculated instead of the booster vaccine. In the first method, the variation in the neutralizing antibody titer against CSFV may be suppressed to a desired degree. Examples of the suppression of the variation in the neutralizing antibody titer against CSFV include a decrease in the coefficient of variation (= standard deviation / average value) of the neutralizing antibody titer against CSFV. The coefficient of variation of the neutralizing antibody titer against CSFV may be calculated, for example, when a plurality of individual pigs are inoculated with a booster vaccine.

[0090] The coefficient of variation of the neutralizing antibody titer against CSFV after inoculation with a booster vaccine may be, for example, 1 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.45 or less, 0.4 or less, 0.35 or less, 0.3 or less, 0.25 or less, or 0.2 or less. The coefficient of variation of the neutralizing antibody titer against CSFV after inoculation with a booster vaccine may particularly be 0.3 or less.

[0091] The coefficient of variation of the neutralizing antibody titer against CSFV after inoculation with a booster vaccine may be, for example, less than 1-fold, 0.9-fold or less, 0.8-fold or less, 0.7-fold or less, 0.6-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, or 0.2-fold or less of the coefficient of variation of the neutralizing antibody titer against CSFV at the time of booster vaccine inoculation.

[0092] The coefficient of variation of the neutralizing antibody titer against CSFV after booster vaccination may be, for example, less than 1-fold, 0.9-fold or less, 0.8-fold or less, 0.7-fold or less, 0.6-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, or 0.2-fold or less of the coefficient of variation of the neutralizing antibody titer against CSFV after inoculation with live CSFV vaccine instead of the booster vaccine.

[0093] The coefficient of variation of the neutralizing antibody titer against CSFV after booster vaccination may be a value at any time point after booster vaccination. The coefficient of variation of the neutralizing antibody titer against CSFV after booster vaccination may be, for example, a value at any time point after 7 days, 14 days, 21 days, 28 days, or 35 days after inoculation with the booster vaccine, and may also be a value at any time point up to 49 days, 42 days, 35 days, 28 days, or 21 days after inoculation with the booster vaccine, and may be a value at any time point within a non-contradictory combination of those periods. The coefficient of variation of the neutralizing antibody titer against CSFV after booster vaccination may particularly be a value at any time point between 14 days and 42 days after booster vaccination. The coefficient of variation of the neutralizing antibody titer against CSFV after booster vaccination may more particularly be a value on the 28th day after booster vaccination.

[0094] Also, "the variation in the neutralizing antibody titer against CSFV is suppressed" may mean, for example, that the neutralizing antibody titer against CSFV after booster vaccination converges within the range of the neutralizing antibody titer against CSFV after booster vaccination as exemplified above.

[0095] The second aspect of the method described in this specification is a method for preventing CSFV infection in pigs, preventing the occurrence of CSF in pigs, and / or alleviating the symptoms at the time of CSF occurrence in pigs. Hereinafter, the same method (that is, the second aspect of the method described in this specification) is also referred to as the "second method".

[0096] The second method includes enhancing the immunity against CSFV in pigs by the first method.

[0097] That is, by implementing the first method, specifically, by enhancing the immunity against CSFV by implementing the first method, the infection of pigs with CSFV may be prevented. Also, by implementing the first method, specifically, by enhancing the immunity against CSFV by implementing the first method, the occurrence of CSF in pigs may be prevented. Also, by implementing the first method, specifically, by enhancing the immunity against CSFV by implementing the first method, the symptoms at the time of the occurrence of CSF in pigs may be alleviated.

[0098] The third aspect of the method described in this specification is a method of conferring immunity against CSFV to piglets. Hereinafter, the same method (that is, the third aspect of the method described in this specification) is also referred to as the "third method".

[0099] The third method includes enhancing the immunity against CSFV in sows by the first method.

[0100] That is, by implementing the first method in sows, specifically, by enhancing the immunity against CSFV in sows by implementing the first method immunity against CSFV may be conferred to piglets.

[0101] The immunity against CSFV in piglets may be conferred, for example, by the transfer of antibodies from sows to piglets. The transfer of antibodies to piglets is conferred by suckling from sows (especially suckling of colostrum). That is, the sow (that is, the sow whose immunity against CSFV has been enhanced by the first method) may be the sow that suckles colostrum to piglets (that is, the piglets to which immunity is to be conferred in the third method). The sow that gives birth to piglets and the sow that suckles colostrum to piglets may or may not be the same individual. The sow that gives birth to piglets and the sow that suckles colostrum to piglets may typically be the same individual.

[0102] The third method may include nursing colostrum from the mother pig to the piglet.

[0103] In the third method, the variation in the transfer antibody titer against CSFV in piglets may be suppressed. That is, in one aspect, the "method of conferring immunity against CSFV to piglets" may be read as the "method of suppressing the variation in the transfer antibody titer against CSFV in piglets".

[0104] The "transfer antibody titer against CSFV in piglets" means the neutralizing antibody titer against CSFV conferred to piglets as transfer antibodies. The transfer antibody titer against CSFV in piglets can be measured, for example, by measuring the neutralizing antibody titer against CSFV in piglets. Also, the transfer antibody titer against CSFV in piglets can be estimated, for example, from the neutralizing antibody titer against CSFV in the mother pig that nurses colostrum to the piglets. The estimation of the transfer antibody titer against CSFV in piglets from the neutralizing antibody titer against CSFV in the mother pig can be carried out, for example, as described in the examples.

[0105] Specifically, the transfer antibody titer against CSFV in piglets may be estimated to be, for example, equivalent to the neutralizing antibody titer against CSFV in the mother pig that nurses colostrum to the piglets. Therefore, regarding the suppression of the variation in the transfer antibody titer against CSFV in piglets, for example, the description regarding the suppression of the variation in the neutralizing antibody titer against CSFV in the mother pig in the first method can be applied mutatis mutandis.

[0106] The period during which the multiplication factor of the maternally-derived antibody titer against CSFV in piglets is maintained at 32-fold or more may be, for example, 30 days or more, 40 days or more, 50 days or more, or 60 days or more, assuming that the half-life of the maternally-derived antibody titer is 10.1 days. The period during which the multiplication factor of the maternally-derived antibody titer against CSFV in piglets is maintained at 100-fold or more may be, for example, 20 days or more, 30 days or more, 40 days or more, or 50 days or more, assuming that the half-life of the maternally-derived antibody titer is 10.1 days. That is, the maternally-derived antibody titer against CSFV in piglets may be a value such that the period during which the multiplication factor of the maternally-derived antibody titer against CSFV in piglets is maintained at 32-fold or more or 100-fold or more falls within the ranges exemplified above.

[0107] In addition, "the variation in the maternally-derived antibody titer against CSFV in piglets is suppressed" may mean, for example, that the maternally-derived antibody titer against CSFV in piglets converges within the range of the maternally-derived antibody titer against CSFV in piglets as exemplified above.

[0108] A fourth aspect of the method described in this specification is a method for preventing infection with CSFV in piglets, preventing the occurrence of CSF in piglets, and / or alleviating the symptoms at the time of occurrence of CSF in piglets. Hereinafter, the same method (i.e., the fourth aspect of the method described in this specification) will also be referred to as the "fourth method".

[0109] The fourth method includes conferring immunity against CSFV to piglets by the third method.

[0110] That is, by carrying out the third method, specifically, by conferring immunity against CSFV to piglets by carrying out the third method, infection with CSFV in piglets may be prevented. Further, by carrying out the third method, specifically, by conferring immunity against CSFV to piglets by carrying out the first method, the occurrence of CSF in piglets may be prevented. Further, by carrying out the first method, specifically, by conferring immunity against CSFV to piglets by carrying out the third method, the symptoms at the time of occurrence of CSF in piglets may be alleviated.

Examples

[0111] The present invention will be further specifically described below with reference to non-limiting examples.

[0112] Example 1: Preparation of booster vaccine The CSFV E2 subunit consisting of the amino acid sequence shown in SEQ ID NO: 3 was prepared by heterologous expression and purified. Using PBS as a solvent, a booster vaccine was prepared by mixing the purified CSFV E2 subunit (100 μg / dose), an oil-in-water adjuvant (25 w / v%), and thimerosal (0.01 w / v% or less). Note that amino acid numbers 20 to 350 in the amino acid sequence shown in SEQ ID NO: 3 (specifically, from "arginine (R)" which is the 20th amino acid from the N-terminus to "alanine (A)" which is the 350th amino acid from the N-terminus) are the amino acid sequence shown in SEQ ID NO: 2.

[0113] Example 2: Induction of porcine immunity by booster vaccine Fifteen fattening pigs that had been vaccinated with a live porcine vaccine at about 3 weeks of age were introduced. A test group in which 2 mL of the booster vaccine (test drug) prepared in Example 1 was administered per pig, a control group (1) in which 1 mL of a live porcine vaccine (Matsuyan Kenkyusho Co., Ltd.) was administered per pig, and a control group (2) in which 2 mL of physiological saline was administered per pig were each divided into 5 pigs, and each test substance was administered at about 150 days of age. Thereafter, the general condition was observed over time, body temperature was measured (only in the test group), the administration site was observed (only in the test group), and blood was collected.

[0114] · Observation of general condition (all pigs) In control group (1) and control group (2), no abnormalities in vitality were confirmed. In the test group, although a decrease in vitality was confirmed in 3 out of 5 pigs 4 to 6 hours after vaccine administration, all pigs had recovered the next day. Abnormalities in fecal consistency (soft stools, pasty stools) were observed sporadically in each group. Although fecal abnormalities were observed in one pig in the test group over a long period, they had been observed since before administration. In addition, one pig each in control group (1) and (2) was observed to have a slightly rapid breathing state, but both occurred sporadically.

[0115] · Rectal temperature measurement (test group only) The results of body temperature measurement in the test group are shown in Table 1. The average body temperature was 39.0 - 39.2 °C for the three days before administration, and fever was observed at 40.4 °C 4 - 6 hours after administration. The average body temperature became 39.1 °C on the day after administration, equivalent to that before administration, and then stabilized at 38.8 - 39.1 °C.

[0116]

Table 1

[0117] · Observation of the administration site (test group only) The observation results of the local administration reactions for each individual in the test group are shown in Table 2. Also, the observation of the cross-section of the administration site for each individual by autopsy (findings present) is shown in Table 3. The number of cases of manifestation for each item in the observation of local administration reactions was as follows: out of a total of 5 cases, swelling was observed in 3 cases, induration in 2 cases, and a feeling of heat in 2 cases. No other abnormalities were observed.

[0118]

Table 2 - 1

[0119]

Table 2 - 2

[0120]

Table 3 - 1

[0121]

Table 3 - 2

[0122] · Immune response Taking the day of administration of the test substance as day 0, blood was collected on days 0, 14, and 28, and the antibody titer was measured using an ELISA kit (Porcine Fever Elisa Kit II, Nippon Gene) with the serum as the specimen.

[0123] The S / P values are shown in Table 4 and Figure 1. Compared with the unvaccinated control group (control group (2)), significant differences were observed in the inactivated vaccine group (test group) 14 days and 28 days after immunization (p = 0.024, 0.024). On the other hand, no significant difference was observed between the live vaccine group (control group (1)) and the unvaccinated control group 14 days and 28 days after immunization (p = 0.14, 0.14). Also, the peak antibody titer of the inactivated vaccine group was 28 days later, and that of the live vaccine group (excluding No. 351) was 14 days later. As a result of comparing the peak increase values of each individual, a significant difference was observed between the live vaccine group and the inactivated vaccine group (p = 0.016). Scatter diagrams of the S / P values at 28 days (4 W Post-Vaccination) relative to 0 days (0 W Post-Vaccination) are shown in Figures 2 to 4. From the above, a booster effect was confirmed in the test group.

[0124] Also, based on the data in Table 4, the coefficient of variation of the ELISA S / P value was calculated. As a result, in the test group, the coefficient of variation of the ELISA S / P value on day 0 was approximately 0.76, and that on day 28 was approximately 0.14. The coefficient of variation of the ELISA S / P value decreased due to the inoculation of the booster vaccine (that is, the variation in the neutralizing antibody titer was suppressed). Also, in the live vaccine group (control group (1)), the coefficient of variation of the ELISA S / P value on day 28 was approximately 0.30, and the variation in the neutralizing antibody titer was suppressed in the test group compared with the live vaccine group (control group (1)).

[0125]

Table 4

[0126] · Conclusion Transient decline in vitality, fever, and swelling at the administration site were observed after administration of the test drug, but no serious adverse reactions were observed. From the results of this test, it was confirmed that the test drug has a higher antibody-inducing ability (booster effect) than the live vaccine.

[0127] Example 3: Estimation of the persistence period of maternally derived antibodies in piglets The persistence period of maternally-derived antibodies in piglets was estimated from the ELISA S / P values measured in Example 2 according to the following procedure.

[0128] ·Estimation of neutralizing antibody titer from ELISA S / P value Kobayashi (Creation of a table for estimating neutralizing antibody titer from S / P value of porcine fever ELISA, Journal of the Japanese Swine Diseases Research Association No. 80, 2022, 12-16) created estimation tables for estimating neutralizing antibody titer from the S / P value of ELISA in three methods to more simply determine the appropriate age for vaccination with porcine fever vaccine. Among these, the estimation table created by ROC analysis had a better degree of agreement with the measured values compared to other methods (simple regression analysis and non-linear regression analysis). Therefore, the present inventors estimated the neutralizing antibody titer from the ELISA S / P value measured in Example 2 using the estimation table created by ROC analysis.

[0129] ·Estimation of maternally-derived antibody titer transferred to offspring from maternal blood antibodies It is known that maternally-derived antibodies transferred to offspring reach the same or higher concentration levels as maternal serum 12-24 hours after the start of lactation (Minoru Shimizu, Yoshikatsu Kodama (1999): Immune mechanism, 69-79, Swine Diseases - Physiology, Diseases, Feeding - <4th Edition>, edited by Mamoru Kashiwazaki et al., Kindai Shuppan, Tokyo). Therefore, the present inventors assumed that the maternally-derived antibody titer transferred to offspring is at the same level as the antibody titer in maternal serum.

[0130] ·Half-life of maternally-derived antibodies It is known that the half-life of maternally-derived antibodies in pigs is 9.1 - 14.2 days (Minoru Shimizu, Yoshikatsu Kodama (1999): Immune mechanism, 69-79, Swine Diseases - Physiology, Diseases, Feeding - <4th Edition>, edited by Mamoru Kashiwazaki et al., Kindai Shuppan, Tokyo). Also, the Ministry of Agriculture, Forestry and Fisheries assumes that the half-life of maternally-derived antibodies in pigs is 10.1 days ("Regarding the immunization status etc. after vaccination of porcine fever vaccine to breeding pigs (draft)", Ministry of Agriculture, Forestry and Fisheries, Consumer Affairs and Food Safety Bureau, July 11, 2022). Therefore, the present inventors assumed that the half-life of maternally-derived antibodies in pigs is 10.1 days.

[0131] The results are shown in Table 5.

[0132] ·Defensive antibody level of maternally-derived antibodies The magnification of the transfer antibody titer required for the defense against CSF infection is set at 32-fold (Editorial Committee of Pig J. (2023): Vaccine Program to Protect Swine in Kyushu, Pig J., 2, 12-20). Therefore, when determining the period during which the estimated transfer antibody is maintained at 32-fold or more, it was 60.6 days or more in the test group, suggesting that CSF infection can be defended by transfer antibodies until the general piglet usage of the live CSF vaccine, which is "administer the first injection at 1 to 2 months of age". On the other hand, it was 18.2 days in the positive control group and 0 days in the negative control group, raising concerns about the occurrence of an immune blank period in the piglet usage of the live CSF vaccine.

[0133] There is also a theory that the magnification of the transfer antibody titer required for the defense against CSF infection is about 100-fold. Therefore, when determining the period during which the estimated transfer antibody is maintained at 100-fold or more, it was 49.9 days or more in the test group, suggesting that it can be defended by transfer antibodies until the general piglet usage of the live CSF vaccine, which is "administer the first injection at 1 to 2 months of age". On the other hand, it was 1.6 days in the positive control group and 0 days in the negative control group, raising concerns about the occurrence of an immune blank period of more than one month in the piglet usage of the live CSF vaccine.

[0134] From the above, it became clear that the method of the present invention is also effective in conferring immunity against CSFV to piglets.

[0135]

Table 5

[0136] <Explanation of the Sequence Listing> Sequence number: 1: Nucleotide sequence encoding the E2 subunit found in the CSF epidemic field strain 2: Amino acid sequence of the E2 subunit found in the CSF epidemic field strain 3: Amino acid sequence with an additional sequence added to the amino acid sequence of the E2 subunit found in the CSF epidemic field strain

Claims

1. A method for enhancing immunity against classical swine fever virus (CSFV) in pigs, comprising: inoculating the pig with a booster vaccine against CSFV by injection; wherein the pig has been inoculated with a live vaccine against CSFV; and the booster vaccine is a CSFV E2 subunit vaccine.

2. The method according to claim 1, wherein the neutralizing antibody titer against CSFV after inoculation with the booster vaccine is at least 4 times the neutralizing antibody titer against CSFV at the time of inoculation with the booster vaccine.

3. The method according to claim 2, wherein the neutralizing antibody titer against CSFV after inoculation with the booster vaccine is a value at any time point between 14 days and 42 days after inoculation with the booster vaccine.

4. The method according to claim 2, wherein the neutralizing antibody titer against CSFV after inoculation with the booster vaccine is a value at 28 days after inoculation with the booster vaccine.

5. The method according to claim 1, wherein the coefficient of variation of the neutralizing antibody titer against CSFV after inoculation with the booster vaccine is 0.3 or less.

6. The method according to claim 5, wherein the coefficient of variation of the neutralizing antibody titer against CSFV after inoculation with the booster vaccine is a value at any time point between 14 days and 42 days after inoculation with the booster vaccine.

7. The method according to claim 5, wherein the coefficient of variation of the neutralizing antibody titer against CSFV after inoculation with the booster vaccine is a value at 28 days after inoculation with the booster vaccine.

8. The method according to claim 1, wherein the E2 subunit is a polypeptide comprising the amino acid sequence of the E2 subunit found in a CSFV epidemic field strain or a variant sequence thereof.

9. The method according to claim 1, wherein the live vaccine is a CSFV GPE - strain.

10. The method according to claim 1, wherein the pig is a breeding pig.

11. The method according to claim 10, wherein the breeding pig is a breeding pig of the second generation or later.

12. The method according to claim 10, wherein the breeding pig is a sow.

13. The method according to claim 1, wherein the inoculation of the live vaccine is carried out once or more than once.

14. The method according to claim 1, wherein the booster vaccination is carried out after 4 months from the first inoculation of the live vaccine.

15. The method according to claim 12, wherein the booster vaccination is carried out up to 1 week before the sow gives birth to a piglet.

16. A method for conferring immunity against classical swine fever virus (CSFV) to piglets, comprising enhancing the immunity against CSFV in sows by the method according to claim 12, wherein the sow is a sow that suckles colostrum to the piglets.

17. A method for suppressing the variation in the transfer antibody titer against classical swine fever virus (CSFV) in piglets, comprising enhancing the immunity against CSFV in sows by the method according to claim 12, wherein the sow is a sow that suckles colostrum to the piglets.

18. The method according to claim 16 or 17, further comprising suckling colostrum from the sow to the piglets.

19. A booster vaccine against classical swine fever virus (CSFV), wherein the booster vaccine is a CSFV E2 subunit vaccine, A booster vaccine for inoculating pigs already inoculated with a live vaccine against CSFV by injection.

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