JP7369343B2 - Method for producing swine infectious virus - Google Patents

Description

The present invention relates to a method for producing a virus that infects pigs, and more particularly, to a method for producing a virus that infects pigs (excluding African swine fever virus) using immortalized pig kidney macrophages. The present invention also relates to a method for producing a vaccine containing a swine infectious virus (excluding African swine fever virus) using immortalized swine kidney macrophages. Furthermore, the present invention relates to a method for detecting neutralizing antibodies against swine infectious viruses (excluding African swine fever virus) using immortalized swine kidney macrophages.

Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by the PRRS virus (PRRSV), which causes respiratory symptoms in pigs during the breeding and fattening stages, as well as death and abortion in pregnant sows. Causes reproductive problems. It has invaded many farms in Japan as well, and it is estimated that it causes severe damage to the pig farming industry of 28 billion yen annually.

Live attenuated vaccines are on the market in Japan as a vaccine against PRRSV, but they are often used with the hope of alleviating symptoms and do not provide complete protection. Against this background, there is a desire to develop a vaccine that can be expected to prevent infection.

In the production of live attenuated vaccines and inactivated vaccines, an environment in which the pathogen in question can proliferate is required, and for pathogens that proliferate in a host-dependent manner, such as viruses, cells with high virus susceptibility are easily established. Is required. PRRSV is isolated using alveolar macrophages (Non-patent Document 1), and does not infect peritoneal macrophages or peripheral blood mononuclear cells just collected from living bodies, and only alveolar macrophages show high susceptibility (Non-patent Document 1). 2). For this reason, alveolar macrophages collected from the lungs of slaughtered pigs are used to isolate and propagate viruses for research purposes.

However, alveolar-derived macrophages require time, technology, and equipment to be collected from the lungs of slaughtered pigs, and individual differences often have a large effect on the condition of the collected alveolar macrophages.

On the other hand, if PRRSV is adapted, it can proliferate in other types of cells, and to produce a live attenuated PRRSV vaccine that can be used in Japan, primate cells (MA104, Marc145) or PRRSV host receptor (CD163) are used. Rodent cells with forced expression are used.

However, Marc145 has the disadvantage that it is difficult to isolate and propagate PRRSV field strains. Regarding two types of cell lines derived from porcine alveolar macrophages, there is a report that it is difficult to culture cells for the ZMAC1 strain, and that virus infection is denied for the 3D4/31 strain (Non-Patent Document 3).

Thus, there is a need for immortalized pig macrophage-derived cells that have high infection susceptibility to PRRSV and are capable of proliferating, but sufficient cells have not yet been found. Furthermore, in addition to PRRSV, there are many swine disease-related viruses for which it is difficult to isolate and propagate field strains, and there is a need for cell lines that can be expected to efficiently isolate and propagate field strains of multiple virus species. However, such cells have never existed before.

Collins JE. et al., J. Vet. Diagn. Invest. , April 1992, 4;2:117-126 Duan X. et al., Arch. Virol. , 1997, 142;12:2483-2497. Weingartl HM. et al., J. Virol. Methods. , July 2002, 104;2:203 Takenouchi T. et al., Front Vet Sci. , August 2017, 21;4:132.

The present invention has been made in view of the above-mentioned problems of the prior art, and has discovered immortalized pig-derived macrophage cells that are highly susceptible to viruses that infect pigs, such as PRRSV, and uses the cells. The present invention aims to provide a method for propagating the virus.

In order to achieve the above object, the present inventors have conducted extensive research and conducted infection tests on various cells using various swine-infecting virus strains.

As a result, first, the immortalized porcine kidney-derived macrophage (IPKM, see Non-Patent Document 4), which the present inventor had previously developed, is susceptible to various PRRSV strains, and is susceptible to these virus strains. It has become clear that it is possible to proliferate.

Macrophages exist in various locations in the body, and each has slightly different properties. Furthermore, as mentioned above, PRRSV was previously believed to be highly host- and cell-tropic, infecting only alveolar macrophages of pigs. Therefore, it was completely unexpected and surprising that kidney macrophages could be infected by PRRSV.

In addition, it has been difficult to isolate and propagate strains in the field, and the infection of macrophages was unknown: swine epidemic diarrhea virus, swine infectious gastroenteritis virus, swine parvovirus, Japanese encephalitis virus, Aujesky virus, swine circovirus. It was revealed that both virus types 2 can infect and multiply in immortalized pig kidney macrophages.

Thus, we have discovered that immortalized pig kidney macrophage-derived cells have high infection susceptibility to various types of pig-infecting viruses and are capable of propagating the viruses, and have completed the present invention. It's arrived.

That is, the present invention relates to a method for producing a pig infectious virus or vaccine using immortalized pig kidney macrophages. Furthermore, the present invention relates to a method for detecting neutralizing antibodies against a swine infectious virus using immortalized swine kidney macrophages, specifically as follows.
<1> Infecting pigs, including the step of bringing immortalized pig kidney macrophages into contact with a virus that infects pigs (excluding African swine fever virus) and multiplying the virus in the immortalized pig kidney macrophages. Virus production method.
<2> A step of bringing the immortalized pig kidney macrophage into contact with a virus that infects pigs (excluding African swine fever virus), and multiplying the virus in the immortalized pig kidney macrophage;
A method for producing a vaccine containing a virus that infects pigs, comprising a step of isolating a grown virus that infects pigs, and a step of mixing the isolated virus that infects pigs with a pharmacologically acceptable carrier or medium. .
<3> The immortalized pig kidney macrophage is an immortalized macrophage obtained by introducing a lentivirus encoding SV40 large T antigen and pig-derived telomerase reverse transcriptase into a pig-derived kidney macrophage. <1> Or the manufacturing method described in <2>.
<4> Immortalized pig kidney macrophages infected with viruses that infect pigs (excluding African swine fever virus).
<5> Pig-derived kidney macrophages are infected with viruses that infect pigs (excluding African swine fever virus) by introducing a lentivirus encoding SV40 large T antigen and pig-derived telomerase reverse transcriptase. immortalized pig kidney macrophages.
<6> A method for detecting neutralizing antibodies against a virus that infects pigs, which includes the following steps: Immortalized pig kidney macrophages and a virus that infects pigs in the presence of a biological sample isolated from a test pig. (however, excluding African swine fever virus) and multiplying the virus in the immortalized pig kidney macrophages;
a step of detecting the number of the proliferated viruses, and when the number of viruses detected in the step is smaller than the number of viruses proliferated in the immortalized pig kidney macrophages in the absence of the biological sample, the biological sample determining that the sample contains neutralizing antibodies against the virus;

According to the present invention, by using immortalized pig kidney macrophages that have high infection susceptibility to viruses that infect pigs, it becomes possible to propagate various pig-infecting virus strains, and develop vaccines against the viruses. Manufacturing becomes possible. Furthermore, by using immortalized pig kidney macrophages, it is possible to determine the presence or absence of neutralizing antibodies against the pig infection virus.

Pig kidney cells (PK15), green monkey kidney cells (Marc145), immortalized pig kidney macrophages (IPKM), pig alveolar macrophages (3D4/31), mouse macrophages (RAW264.7), mouse kidney macrophages (KM1), pig Fluorescent micrograph showing the results of immunofluorescent staining of the virus after 24 hours of infection with field strains 1 to 5 of reproductive and respiratory syndrome virus (PRRSV) and the vaccine strains Ingelvac and Fostera. be. In the figure, cells in which the virus has grown are detected in white. PRRSV field strain 2 and Ingelvac were infected with IPKM and Marc145, respectively, and the proteins in the lysed cells were separated by SDS-PAGE, and the results were analyzed by Western blot analysis using PRRS virus N protein antibody. This is a photo. Virus and cell combinations were performed in triplicate. In the figure, "+" indicates an infected area, and "-" indicates a non-infected area. This is a graph showing the results of inoculating Marc145 (black squares) and IPKM (hollow circles) with Ingelvac, collecting culture supernatants over time, and measuring the amount of virus in the supernatants by real-time PCR. The horizontal axis shows the elapsed time after infection, and the vertical axis shows the genome equivalent number (logarithm). A graph showing the results of inoculating PRRSV field strain 3 into Marc145 (black squares) and IPKM (hollow circles), collecting culture supernatants over time, and measuring the amount of virus in the supernatants by real-time PCR. be. The horizontal axis shows the elapsed time after infection, and the vertical axis shows the genome equivalent number (logarithm). IPKM was inoculated with a live vaccine virus solution of Aujesky virus, and the supernatant was collected 8 hours and 48 hours later ("8 hours" and "48 hours" in the figure), and after 48 hours, the cells were lysed (Figure This is a graph showing the results of real-time PCR measurement of the amount of virus contained in the supernatant and cells (middle "lysate"). The vertical axis indicates the number of cycles, and the smaller the number, the greater the amount of PCR template. A live vaccine virus solution of Japanese encephalitis virus was inoculated into IPKM, and the supernatant was collected 8 hours and 48 hours later ("8 hours" and "48 hours" in the figure), and after 48 hours, the cells were lysed (Fig. This is a graph showing the results of real-time PCR measurement of the amount of virus contained in the supernatant and cells (middle "lysate"). The vertical axis indicates the number of cycles, and the smaller the number, the greater the amount of PCR template. Porcine circovirus type 2 virus solution was inoculated into IPKM, and the supernatant was collected 8 hours and 48 hours later ("8 hours" and "48 hours" in the figure), and after 48 hours, the cells were lysed (Fig. This is a graph showing the results of real-time PCR measurement of the amount of virus contained in the supernatant and cells (middle "lysate"). The vertical axis indicates the number of cycles, and the smaller the number, the greater the amount of PCR template. Live vaccine virus solution of porcine epidemic diarrhea virus was inoculated into IPKM, supernatant was collected 8 hours and 48 hours later ("8 hours" and "48 hours" in the figure), and cells were lysed after 48 hours. (“Lysate” in the figure) is a graph showing the results of measuring the amount of virus contained in the supernatant and cells by real-time PCR. The vertical axis indicates the number of cycles, and the smaller the number, the greater the amount of PCR template. Porcine parvovirus live vaccine virus solution was inoculated into IPKM, supernatant was collected 8 hours and 48 hours later ("8 hours" and "48 hours" in the figure), and cells were lysed after 48 hours (Figure 1). This is a graph showing the results of real-time PCR measurement of the amount of virus contained in the supernatant and cells (middle "lysate"). The vertical axis indicates the number of cycles, and the smaller the number, the greater the amount of PCR template. Live vaccine virus solution of porcine infectious gastroenteritis virus was inoculated into IPKM, supernatant was collected 8 hours and 48 hours later ("8 hours" and "48 hours" in the figure), and cells were lysed after 48 hours. This is a graph showing the results of real-time PCR measurement of the amount of virus contained in the supernatant and cells ("lysate" in the figure). The vertical axis indicates the number of cycles, and the smaller the number, the greater the amount of PCR template.

As shown in the Examples below, immortalized pig kidney macrophages exhibit high infection susceptibility to various strains of viruses that infect pigs (swine infectious viruses), and are capable of propagating the viruses.

Therefore, the present invention provides a method comprising the step of bringing immortalized pig kidney macrophages into contact with a virus that infects pigs (excluding African swine fever virus), and multiplying the virus in the immortalized pig kidney macrophages. Provided is a method for producing a virus that infects. The present invention also provides immortalized pig kidney macrophages that are infected with a virus that infects pigs (excluding African swine fever virus).

(immortalized pig kidney macrophage)
In the present invention, "immortalized pig kidney macrophages", which serve as a place for the infection and proliferation of pig-infecting viruses, refer to cells that are immortalized macrophages present in the kidneys of pigs (mammalia, cetacean artiodactyla, boar family). do.

"Pig kidney macrophages" to be immortalized can be prepared by those skilled in the art according to known techniques. For example, Takenouchi T. et al., Results Immunol. , August 2014, Volume 1, Issue 4, Pages 62-67, the renal fibrous capsule is removed and the renal cortex is isolated from kidneys collected from newborn pigs. It can be prepared by isolating macrophage-like cells that loosely adhere to a monolayer cell sheet and exhibit proliferative activity, which are generated during the process of mincing and culturing the isolated renal cortex.

There are no particular restrictions on the method of immortalizing pig kidney macrophages, but it can be carried out by introducing at least one immortalizing gene. Immortalization genes include, for example, SV40 large T antigen (SV40T antigen), telomerase reverse transcriptase (TERT), Myc, and Ras, but it is preferable to introduce SV40T antigen and TERT, which improves the immortalization efficiency of pig macrophages. It is more preferable to introduce the SV40T antigen and pig-derived TERT from the viewpoint of increasing the

Introduction of an immortalizing gene can be performed by using a vector encoding the gene. Vectors may be linear or circular, and include, for example, viral vectors, plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors.

Examples of viral vectors include retrovirus vectors such as lentiviruses, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, pox virus vectors, polio virus vectors, sylvis virus vectors, and rhabdovirus vectors. Examples include viral vectors, paramyxovirus vectors, and orthomyxovirus vectors. Examples of plasmid vectors include plasmid vectors for expression in animal cells, such as pcDNA3.1, pA1-11, pXT1, pRc/CMV, pRc/RSV, and pcDNAI/Neo. Among these vectors, retrovirus vectors are preferred, and lentiviruses are more preferred, from the viewpoint of increasing the efficiency of gene introduction into pig macrophages.

In addition to the immortalizing gene, the vector of the present invention encodes expression control sequences such as promoters, enhancers, polyA addition signals, and terminators, replication origins, and proteins that bind to replication origins and control replication. Nucleotide sequence, 5' untranslated region including 5' cap structure, Shine-Dalgarno sequence, Kozak sequence, etc., 3' untranslated region including polyadenylation signal, AU-rich element, GU-rich element, etc., encoding other proteins It may also contain nucleotides etc.

By operably placing the immortalization gene downstream of the promoter, each polynucleotide can be efficiently transcribed. Examples of such "promoters" include EF1α promoter, CMV promoter, SRα promoter, SV40 early promoter, LTR promoter, RSV promoter, HSV-TK promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, metallothionein promoter, and heat shock promoter. Examples include promoters.

Examples of "nucleotides encoding other proteins" include marker genes such as reporter genes and drug resistance genes.

In addition, when introducing multiple types of immortalizing genes, these genes may be integrated into a single vector or each into separate vectors, but from the viewpoint of increasing expression efficiency, these genes may be integrated into separate vectors. It is desirable to incorporate it. Furthermore, when incorporated into a single vector, for example, by inserting IRES, 2A peptide sequences, etc. into the vector, it becomes possible to polycistronicly express multiple types of immortalizing genes.

Examples of methods for introducing the vector into cells include lipofection, microinjection, calcium phosphate, DEAE-dextran, electroporation, and particle gun methods. In addition, when the vector of the present invention is a retrovirus vector, appropriate packaging cells are selected based on the LTR sequence and packaging signal sequence possessed by the vector, and retrovirus particles are prepared using this. It's okay. Examples of packaging cells include PG13, PA317, GP+E-86, GP+envAm-12, and Psi-Crip. Furthermore, 293 cells and 293T cells, which have high transfection efficiency, can also be used as packaging cells. Further, the virus particles prepared in this manner can be introduced into cells by the Polybrene method, Protamine method, RetroNectin method, etc.

Immortalized pig kidney macrophages established by introducing an immortalization gene in this manner exhibit a proliferative property for at least 1 month or more, preferably a proliferative property for 3 months or more, and more preferably a proliferative property for 5 months or more. shows. The doubling time of immortalized pig kidney macrophages is at least 6 days, preferably 4 days, more preferably 2 days. Furthermore, the immortalized pig kidney macrophage preferably maintains the characteristics of a macrophage, for example, at least one gene among the macrophage-specific genes CD172a, CD16, Iba-1, MSR-A, MHC-II, and CD163. are expressed, preferably two genes are expressed, more preferably four genes are expressed, and particularly preferably all genes are expressed. Furthermore, the immortalized pig kidney macrophage according to the present invention retains at least one of the following macrophage functions: phagocytosis, production of inflammatory cytokines by LPS stimulation, and IL-1β maturation associated with inflammasome activity. Preferably, the second function is retained, and more preferably all functions are retained. In the present invention, Takenouchi T. et al., Front Vet Sci. , August 2017, 21;4:132. Immortalized macrophages are produced by introducing a lentivirus encoding SV40 large T antigen and pig-derived telomerase reverse transcriptase into pig-derived kidney macrophages, which are produced by the method described in (Non-Patent Document 4). It is particularly suitable for use. Note that the immortalized pig kidney macrophages obtained by the method described in Non-Patent Document 4, unlike primary cultured pig macrophages, proliferate in a densely expanded manner (in the form of a sheet). Therefore, the macrophage is useful in an infection test (CPE test) using the degeneration of infected cells as an indicator, which will be described later, since it is easy to detect changes in morphology.

(virus that infects pigs)
As shown in the Examples below, immortalized cells derived from pig kidney macrophages exhibit high infection susceptibility to various viruses that infect pigs, and can propagate the viruses. Therefore, in the present invention, the "virus that infects pigs (swine infectious virus)" may be any virus that can infect at least pigs, and includes DNA viruses (double-stranded (ds) DNA viruses, single-stranded (ss) DNA viruses, DNA viruses containing both ss and ds DNA regions), RNA viruses (single-stranded (ss) RNA viruses (positive-strand RNA viruses or negative-strand RNA viruses), double-stranded (ds) RNA viruses).

Examples of dsDNA viruses include viruses belonging to the Herpesviridae and Poxviridae families. Examples of viruses belonging to the Herpesviridae family include Aujeski's disease virus (belongs to the Herpesviridae family, Alphaherpesvirinae subfamily, and Valicerovirus genus). Viruses belonging to the Poxviridae include swinepox virus (belongs to the Poxviridae, genus Suipoxvirus), vaccinia virus (belongs to the Poxviridae, genus Orthopoxvirus), and the like.

Examples of ssDNA viruses include viruses belonging to the Circoviridae and Parvoviridae families. Examples of viruses belonging to the Circoviridae family include porcine circovirus types 1 and 2 (belonging to the Circoviridae family and the genus Circovirus). Examples of viruses belonging to the family Parvoviridae include parvoviruses (belonging to the family Parvoviridae, genus Parvovirus), and the like.

Examples of ssRNA(+) viruses include viruses belonging to the Coronaviridae, Arteriviridae, Flaviviridae, Picornaviridae, Caliciviridae, and Hepeviridae. Viruses belonging to the Coronaviridae family include porcine epidemic diarrhea virus (TGE) (family Coronaviridae, genus Alphacoronavirus) and porcine infectious gastroenteritis virus (PED) (family Coronaviridae, genus Alphacoronavirus). ) etc. Examples of viruses belonging to the Arteriviridae include porcine reproductive and respiratory syndrome virus (which belongs to the Arteriviridae, Porartevirus genus (former name: Arterivirus genus)), and the like. Examples of viruses belonging to the Flaviviridae family include Japanese encephalitis virus (belongs to the Flaviviridae family, genus Flavivirus), swine fever virus (belongs to the Flaviviridae family, genus Pestivirus), and the like. Viruses belonging to the Picornaviridae family include encephalomyocarditis virus (family Picornaviridae, genus Cardiovirus), foot-and-mouth disease virus (family Picornaviridae, genus Aphthovirus), and swine vesicular disease virus (Picornaviridae). swine tesiovirus (family Picornaviridae, genus Tesiovirus), porcine sapelovirus (family Picornaviridae, genus Sapelovirus), swine enterovirus B (family Picornaviridae, genus Enterovirus) ), etc. Viruses belonging to the family Caliciviridae include porcine vesicular vesicle virus (belongs to the family Caliciviridae, genus Vesivirus), and the like. Examples of viruses belonging to the Hepeviridae family include hepatitis E virus (belongs to the Hepeviridae family and Hepevirus genus).

Examples of ssRNA(-) viruses include viruses belonging to the families Paramyxoviridae, Orthomyxoviridae, and Rhabdoviridae. Viruses belonging to the family Paramyxoviridae include Nipah virus (family Paramyxoviridae, genus Henipavirus), Lindell virus (family Paramyxoviridae, genus Morbillivirus), and the like. Viruses belonging to the Orthomyxoviridae family include swine influenza virus and avian influenza virus. Viruses belonging to the Rhabdoviridae family include vesicular stomatitis virus (belongs to the Rhabdoviridae family, genus Vesiculovirus), rabies virus (belongs to the Rhabdoviridae family, genus Lyssavirus), and the like.

As in the above example, as long as the virus is capable of infecting pigs, it is not limited to the infection symptoms (e.g., gastrointestinal symptoms, respiratory symptoms) and the route of infection (e.g., mucous membranes). do not have.

Furthermore, "African swine fever virus" refers to a virus (Asfarviridae Asfivirus) that has double-stranded DNA in its genome that belongs to the Asfarviridae, Asfivirus genus.

(Method for producing swine infectious virus)
The method for producing a swine infectious virus (propagation method, amplification method) of the present invention involves contacting immortalized swine kidney macrophages with a swine infectious virus (excluding African swine fever virus), and transmitting the virus to the immortalized swine kidney macrophage. A method comprising proliferating in kidney macrophages.

The swine infectious virus that is brought into contact with the immortalized swine kidney macrophages is as described above, but it may be not only the isolated virus itself, but also a sample that may contain the virus. Examples of such "samples" include pig-derived tissues, cells, cultures thereof, washings, or extracts, or samples from pig breeding environments (breeding facilities, etc.), washings, or cultures thereof.

"Contacting" is usually performed by adding porcine infectious virus to the medium in which immortalized porcine kidney macrophages are cultured. Such a "medium" is not particularly limited as long as it can maintain immortalized pig kidney macrophages, and can be prepared by appropriately adding well-known and commonly used medium additives based on a known basal medium. "Basal medium" includes DMEM medium, DMEM medium (high glucose), DMEM medium (low glucose), RPMI 160 medium, RPMI 1640 medium, Ham's F12 medium, KSOM medium, Eagle MEM medium, Glasgow MEM medium, αMEM medium, Examples include Ham's medium, Fisher's medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc optional improved medium, IMDM medium, Medium 199 medium, and mixed media of any of these. Examples of "medium additives" include antibiotics (penicillin, streptomycin, gentamicin, etc.), antifungal drugs, functional proteins (insulin, transferrin, lactoferrin, etc.), reducing agents (2-mercaptoethanol, monothioglycerol, catalase, etc.). , superoxide dismutase, N-acetylcysteine, etc.), lipids other than fatty acids (cholesterol, etc.), amino acids (alanine, L-glutamine, non-essential amino acids, etc.), peptides (glutathione, reduced glutathione, etc.), nucleotides, etc. (nucleosides, cytidine, adenosine 5'-monophosphate, hypoxanthine, thymidine, etc.), metal salts (iron(III) nitrate, iron(II) sulfate, copper sulfate, zinc sulfate, etc.), inorganic salts (sodium, potassium, calcium, magnesium, etc.) , phosphorus, chlorine, etc.), carbon sources (glucose, galactose, fructose, sucrose, etc.), vitamins, inorganic compounds (selenite), organic compounds (para-aminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putrescine) , pyruvate, lactic acid, triiodothyronine, etc.), buffer compounds (HEPES, sodium bicarbonate, etc.), pH indicators (phenol red, etc.), but are not limited to these.

"Propagation" of a swine-infecting virus can be carried out by culturing immortalized swine kidney macrophages that have come into contact with the virus and become infected. The culture temperature is not particularly limited, but is usually 30 to 40°C, preferably 37°C. The concentration of carbon dioxide in the gas that contacts the culture medium is not particularly limited, but is usually 1 to 10% by volume, preferably 2 to 5% by volume. The culture period after contact with the swine infectious virus is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, and more preferably 3 to 5 days.

Those skilled in the art can determine whether the swine infectious virus has proliferated using a known method. Such methods include, for example, a CPE test using cytopathic effect (CPE) as an indicator, as shown in the Examples below, and a method for detecting the degree of intracellular ATP depletion accompanying CPE (for example, Promega Co., Ltd.). Viral ToxGlo assay) provided by the company. Furthermore, methods for detecting genes derived from swine-infecting viruses or their expression can also be used. Here, gene expression may be at the transcription level (RNA level) or at the translation level (protein level). Examples of methods for detecting genes (genomic DNA, genomic RNA) or RNA include PCR (RT-PCR, real-time PCR, quantitative PCR), sequencing, DNA microarray analysis, Northern blotting or Southern blotting, and in situ hybridization. , dot blot, RNase protection assay, and mass spectrometry. Furthermore, gene or RNA levels can be quantitatively detected by counting the number of reads in the so-called next-generation sequencing method. In addition, methods for detecting proteins include, for example, detection using antibodies such as ELISA method, antibody array, immunoblotting, imaging cytometry, flow cytometry, radioimmunoassay, immunoprecipitation method, and immunohistochemical staining method. methods (immunological methods) and mass spectrometry.

(Vaccine manufacturing method)
The method for producing a vaccine containing a swine infection virus of the present invention includes the steps of: bringing an immortalized swine kidney macrophage into contact with a swine infection virus, and multiplying the virus in the immortalized swine kidney macrophage;
The method comprises the steps of: isolating the propagated swine infectious virus; and mixing the isolated swine infectious virus with a pharmacologically acceptable carrier or medium.

The process of propagating the virus in the immortalized pig kidney macrophages is as described above. "Isolation" of the propagated swine infectious virus means separation, purification and/or concentration of the immortalized swine kidney macrophages and/or the cells from the culture medium. Examples of methods for isolating the virus include filtration of culture medium, disruption of cells (sonication, hypotonic solution treatment, freeze-thaw, etc.), centrifugation (ultracentrifugation, density gradient centrifugation, etc.), and concentration ( ammonium sulfate, resin columns, polyethylene glycol salting out, etc.).

The swine infectious virus isolated in this way may be used as a vaccine as it is (so-called live vaccine), may be used in an attenuated live form (so-called live attenuated virus), or can be used as a vaccine in inactivated form. May be used. Furthermore, as long as it has immunogenicity, some of these isolated swine infectious viruses (proteins, polypeptides, sugars, glycoproteins, lipids, nucleic acids, etc.) may be used as vaccines.

A live attenuated virus is a virus that has a reduced level of toxicity compared to a virus isolated from the field. Attenuated viruses can be grown by known methods, e.g., propagation in the presence of mutagens, acclimation to cultured cells by continuous (long-term) passage in vitro, conditions deviating from their natural growth environment (e.g. can be obtained by subjecting a swine infectious virus to growth under high temperature conditions). Furthermore, a live attenuated virus can also be obtained by deleting or recombining a specific gene of the virus using genome editing, gene modification technology, or the like.

Virus inactivation can also be performed by those skilled in the art using known methods. Examples of such inactivation methods include formaldehyde treatment, UV irradiation, X-ray irradiation, electron beam irradiation, gamma ray irradiation, alkylation treatment, ethylene-imine treatment, thimerosal treatment, β-propiolactone treatment, and glutaraldehyde treatment. .

Examples of the "pharmacologically acceptable carrier" to be mixed with the isolated swine infectious virus include stabilizers, excipients, preservatives, surfactants, chelating agents, and binders. Examples of the "pharmacologically acceptable medium" include water, physiological saline, phosphate buffer, and Tris-HCl buffer. As these carriers and media, those skilled in the art can select and use known carriers and media used in the field as appropriate or in combination, depending on the dosage form and method of use of the vaccine. The form of the vaccine is not particularly limited; for example, it may be in the form of a suspension or in a freeze-dried form.

From the viewpoint of enhancing the vaccine effect, an adjuvant may be further mixed. Examples of adjuvants include inorganic substances such as aluminum gel adjuvants, microorganisms or microorganism-derived substances (BCG, muramyl dipeptide, Bordetella pertussis, pertussis toxin, cholera toxin, etc.), surfactant substances (saponin, deoxycol), etc. Examples include emulsions of oily substances (mineral oil, vegetable oil, animal oil, etc.), alum, etc.

(Method for detecting neutralizing antibodies)
The method of detecting neutralizing antibodies against a virus that infects pigs according to the present invention includes:
In the presence of a biological sample isolated from a test pig, immortalized pig kidney macrophages are brought into contact with a virus that infects pigs (excluding African swine fever virus), and the virus is introduced into the immortalized pig kidney macrophages. a step of propagating
a step of detecting the number of the proliferated viruses, and when the number of viruses detected in the step is smaller than the number of viruses proliferated in the immortalized pig kidney macrophages in the absence of the biological sample, the biological sample The method includes the step of determining that the sample contains neutralizing antibodies against the virus.

The "neutralizing antibody" according to the present invention refers to an antibody that suppresses infection or proliferation of a pig-infecting virus. Such antibodies also include all classes and subclasses of immunoglobulins. Furthermore, there are no particular restrictions on the ``swine to be tested'' as long as they are pigs, regardless of their past experience with swine-infecting viruses. "Biological samples" isolated from test pigs include pig-derived samples (e.g., blood (serum, plasma, etc.), mucus (saliva, nasal secretions, milk, gastrointestinal secretions, etc.), and those purified from them. antibodies).

"Contact" is as described above. Regarding the growth conditions, the culture temperature is not particularly limited, but is usually 30 to 40°C, preferably 37°C. The concentration of carbon dioxide in the gas that contacts the culture medium is not particularly limited, but is usually 1 to 10% by volume, preferably 2 to 5% by volume. The culture period in the presence or absence of a biological sample is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, and more preferably 3 to 5 days. Further, as described above, the propagated virus can be detected by a CPE test or the like or by detecting a gene derived from a swine-infecting virus or its expression. As described above, in the detection method of the present invention, the presence or absence of neutralizing antibodies is determined using not only the virus number itself but also the gene (genome DNA amount, etc.) that reflects the virus number or its expression level as an indicator. It's okay.

EXAMPLES Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

(Example 1)
Immortalized pig kidney macrophages (IPKM) and mouse kidney macrophages (KM1) were cultured in MG medium (composition: DMEM (manufactured by Sigma), 10% fetal bovine serum (FBS), 0.1% bovine insulin (manufactured by Sigma). , 0.5% mercaptoethanol (manufactured by Wako Corporation), 1% penicillin/streptomycin (manufactured by Thermo Fisher Scientific), and 200 mg/L gentamicin (manufactured by Wako Corporation)). Pig kidney cells (PK15) were placed in MEM containing 10% FBS (manufactured by Thermo Fisher Scientific), and green monkey kidney cells (Marc145) were placed in MEM containing 5% FBS (manufactured by Thermo Fisher Scientific). and maintained each. Porcine alveolar macrophages (3D4/31) were treated with RPMI1640 manufactured by Sigma (10% FBS, 2mM glutamine, 4.5g/L glucose, 1.5g/L NaHCO ₃ , 10mM HEPES, 1mM sodium pyruvate, 0.1mM (contains non-essential amino acids). Mouse macrophages (RAW264.7) were maintained in DMEM (manufactured by Thermo Fisher Scientific) containing 10% FBS.

Then, IPKM, KM1, and RAW264.7 were seeded at 2x10 ⁵ cells/well each, and Marc145, PK15, and 3D4/31 were each seeded at 2x10 ⁴ cells/well in an 8-well chamber slide at 37°C. , 5% CO ₂ and saturated water vapor.

On the day after sowing, PRRSV field strain 1 (cluster II), PRRSV field strain 2 (cluster III), PRRSV field strain 3 (cluster IV), PRRSV field strain 4 (cluster IV), and PRRSV field strain, which are derived from farms and have different clusters, were collected on the day after sowing. 5 (Cluster IV) was inoculated into each of the cells. In addition, 10 μL of live attenuated vaccine virus solutions of Boehringer Ingelheim's Ingelvac (Cluster II) and Zoetis' Fostera (Cluster I) as vaccine strains were each inoculated into the cells. The cells were then cultured at 37° C. under 5% CO ₂ and saturated water vapor.

20-24 hours after virus inoculation, cells infected with the virus were fixed with 80% acetone (20% phosphate buffered saline (PBS)). Next, after washing with PBST (PBS containing 0.1% Triton-X100) and PBS and blocking with Immunoblock (manufactured by DS Pharma), anti-PRRS virus M protein antibody (manufactured by GeneTex) was used as the primary antibody. The cells were immunostained using alexa Fluor 488-labeled goat-derived anti-rabbit IgG (manufactured by Thermo Fisher Scientific) as a secondary antibody. The cells were then photographed using a fluorescence microscope (manufactured by Nikon), and cells in which virus infection and proliferation had occurred were detected as green fluorescence-positive cells by immunofluorescence staining. The results obtained are shown in FIG.

As is clear from the results shown in FIG. 1, among the infection tests using combinations of various cells and multiple PRRSV strains, positive cells indicating infection were detected for all strains only for IPKM.

On the other hand, no positive cells were detected for any strain of 3D4/31, which is registered as a porcine alveolar macrophage in a cell bank in the United States. H. M. A similar report was also made by Weingartl et al., Journal of Virological Methods, 104:203-216 (2002), stating that in the natural environment, PRRSV shows cell tropism for porcine alveolar macrophages, but not for 3D4/31. It was confirmed that PRRSV was not infectious.

Furthermore, no positive cells were detected in pig kidney cells (PK15) or two mouse macrophages (KM1, RAW264.7).

Furthermore, although not shown in the figure, all PRRSV strains infected immortalized pig blood macrophages, but the number of positive cells was lower than that of IPKM despite infecting the same number of cells with the same viral load. Ta.

Regarding Marc145, positive cells were detected in the vaccine strain Ingelbach and some field strains, but as mentioned above, IPKM showed the best infection growth among all the field strains used. Furthermore, it was revealed that PRRSV multiplies in IPKM regardless of its cluster, suggesting that infection and multiplication are possible in IPKM regardless of PRRSV classification.

(Example 2)
IPKM or Marc145 was seeded in a 24-well plate at 3 x 10 ⁵ cells/well/2 mL, and the next day, Ingelvac (live vaccine virus solution 200 μL) and field strain 2 (virus solution 100 μL) were inoculated and incubated at 37°C. , 5% CO ₂ and saturated steam for 1 hour.

One hour after virus inoculation, the cell surface was washed once with serum-free medium, 2 mL of MEM (1% FBS) was added, and cultured at 37° C., 5% CO ₂ and saturated water vapor. After 24 hours for Ingelvac and 24 hours for field strain 2, each cell was lysed with 200 μL of mammalian protein extraction buffer (manufactured by GE Healthcare) and centrifuged to remove insoluble matter.

1/4 volume of Laemmli sample buffer was added to the supernatant after centrifugation and boiled, and 5 μL for Marc145 and 25 μL for IPKM were each subjected to SDS polyacrylamide gel electrophoresis. After electrophoresis, the separated proteins were transferred to a PVDF membrane (product name: Immobilon-P, manufactured by GE Healthcare). The transferred membrane was blocked (product name: Blocking One, manufactured by Nacalai Tesque), and anti-PRRS virus N protein antibody (manufactured by GeneTex) was used as the primary antibody, and HRP-labeled anti-rabbit IgG antibody (Millipore) was used as the secondary antibody. (manufactured by Seiko Co., Ltd.). The membrane reacted with the antibody was treated with a chemiluminescent reagent (product name: ECL select, manufactured by GE Healthcare), and then analyzed using a chemiluminescent scanner (product name: C-DiGit blot scanner, manufactured by LI-COR Bioscience). , detected viral proteins in the solution of whole cells in the wells. The results obtained are shown in FIG. 2. As shown in FIG. 2, each combination of cells and virus was performed in triplicate, and the concentrations of the detected bands were almost uniform, so the reliability of the results was high.

As is clear from the results shown in FIG. 2, N protein of field strain 2 could not be detected in the cell lysate of Marc145. On the other hand, a detectable level of N protein production was observed in Marc145 24 hours after Ingelvac infection. Since Ingelvac is a virus grown in MA104, which is the parent strain of Marc145, it is natural that Ingelvac shows good proliferation in Marc145.

On the other hand, since the above immunostaining has a high detection sensitivity, although infection was recognized (see FIG. 1), Ingelvac-derived N protein could not be detected from the IPKM cell lysate. However, surprisingly, contrary to the Ingelbach infection, field strain 2 was detectable by IPKM 48 hours after virus inoculation, despite the same number of cells seeded and the same amount of virus infected. Levels of N protein production were observed.

These results revealed that IPKM has good propagation efficiency for PRRSV field strains.

(Example 3)
IPKM or Marc145 was seeded in a 6-well plate at 3 x 10 ⁵ cells/well/2 mL, and the next day, Ingelvac (live vaccine virus solution 100 μL) and field strain 3 (virus solution 100 μL) were inoculated. The mixture was allowed to stand for 1 hour at 5% CO ₂ under saturated steam. One hour after virus inoculation, the cell surface was washed three times with PBS, 2.2 mL of 1% MEM was added, and 0.2 mL of the medium was immediately collected. After adding 0.2 mL of medium at 14, 16, 20, 24, and 38.5 hours after infection and mixing well, 0.2 mL of culture supernatant was collected.

Nucleic acids were extracted from the recovered medium using a nucleic acid purification kit (product name: Maxwell (registered trademark) 16 Virus Total Nucleic Acid Purification Kit, manufactured by Promega), and mixed with M-MLV reverse transcriptase (manufactured by Promega) and random primers. A reverse transcription reaction was performed using the dT primer and dT primer. The obtained reverse transcription product was used as a template and was used as described in Sipos et al. Viral immunology 16; 335-346 (2003) and Eschbaumer et al. Canadian journal of veterinary research 79; 170-179 (2015). Using primer, SYBR (registered trademark) pre- A reaction solution of Mix ExTaqII (manufactured by Takara) was prepared according to the manual, and the PRRSV gene was detected using a CFX96 Touch Deep Well real-time PCR analysis system (manufactured by BIO-RAD). That is, the amount of progeny virus released into the medium was monitored by detecting the PRRS virus gene in the culture supernatant using real-time PCR. The results obtained are shown in FIGS. 3 and 4.

As shown in FIGS. 3 and 4, for both IPKM and Marc145, an increase in the amount of virus genes in the culture supernatant was observed over time compared to the culture medium collected immediately after infection.

In the culture supernatant of Marc145 infected with Ingelvac, genes derived from progeny viruses increased over time. As mentioned above, Ingelvac is a virus grown in the parent strain of Marc145, so it is natural that Ingelvac shows good growth in Marc145, but surprisingly, in IPKM, Ingelvac, which is similar to Marc145, It has become clear that proliferation is possible.

On the other hand, field strain 3 hardly proliferated on Marc145. However, in IPKM, field strain 3 was observed to proliferate over time.

These results revealed that IPKM allows the progeny viruses of field strains to propagate efficiently.

(Example 4)
IPKM or Marc145 was seeded in a 96-well plate at 8 x ¹⁰⁴ cells/well/0.1 mL, and the next day, 10 μL of the virus culture supernatant of 32 strains that had been isolated using the standard method was inoculated and incubated at 37°C. The mixture was left standing under 5% CO ₂ and saturated steam for 48 hours. The 32 strains include 6 strains isolated from lung emulsion, 24 strains isolated from serum, 1 strain of the vaccine strain Ingelbach, and 1 strain of the Japanese standard strain. 48 hours after virus inoculation, the presence or absence of a change in cell morphology (cytopathic effect) was determined visually. The results obtained are shown in Table 1. In Table 1, strains in which cytopathic effects were observed were counted as + (plus), and strains in which no change was observed were counted as - (minus).

As shown in Table 1, when infected with 32 strains including the vaccine strain Ingelback, IPKM had a cytopathic effect on 11 strains. On the other hand, six strains showed cytopathic effects upon infection with Marc145. More specifically, there are 5 strains (4 strains isolated from serum and 1 strain isolated from lung emulsion) that showed cytopathic effects in common between IPKM and Marc145, and only IPKM showed cytopathic effects. There were 6 strains that showed efficacy (5 strains isolated from serum, 1 strain isolated from lung emulsion), and only 1 strain, the vaccine strain, showed cytopathic effect only in Marc145. Therefore, IPKM exhibited a cytopathic effect in about twice as many strains as Marc145, demonstrating the high efficiency of IPKM in field strain isolation and proliferation.

(Example 5)
IPKM or Marc145 were seeded in 6-well plates at ^3x10 cells/well/2 mL. The next day, 100 μL of virus solution of field strain 1 (cluster II), field strain 2 (cluster III), field strain 3 (cluster IV), and field strain 4 (cluster IV), which are derived from farms and have different clusters, was added to the cells. was inoculated. In addition, 100 μL of live vaccine virus solutions of Boehringer Ingelheim's Ingelvac (Cluster II) and Zoetis' Fostera (Cluster I) as vaccine strains were each inoculated into the cells. Then, it was allowed to stand for 1 hour at 37° C. under 5% CO ₂ and saturated steam.

One hour after virus inoculation, the cell surface was washed once with serum-free medium, 2 mL of MEM (containing 1% FBS) was added, and cultured at 37° C., 5% CO ₂ and saturated steam. 3 to 4 days after infection, the culture medium from each well was collected, and 100 μL of it was added to IPKM or Marc145 that had been seeded in a 6-well plate the previous day, so that the combination of cells and virus of the collection source and the inoculation target corresponded. Inoculated.

After inoculating the recovered medium and allowing it to stand for 1 hour, the cell surface was washed once with serum-free medium, the medium was replaced with MEM (containing 1% FBS), and incubated at 37°C with 5% _CO2 for 3 to 4 days. , cultured under saturated water vapor. Then, the culture was repeated up to 10 times.

Then, nucleic acids are extracted from the culture medium collected during each passage using a nucleic acid purification kit (product name: Maxwell (registered trademark) 16 virus total nucleic acid purification kit, manufactured by Promega), and M-MLV reverse transcriptase (manufactured by Promega), a random primer, and a dT primer, a reverse transcription reaction was performed. Using the obtained reverse transcription product as a template, a reaction solution for ExTaqII (manufactured by Takara) was prepared according to the manual, and the PRRSV gene was amplified using a T100 thermal cycler (manufactured by BIO-RAD).

The obtained PCR products were subjected to electrophoresis in a 1% agarose gel in 1x Tris-borate-EDTA (TBE) buffer (buffer containing 89mM Tris, 89mM boric acid, and 2mM EDTA) and gel red (manufactured by Biotium). Stained. Next, the band derived from the PCR product was visualized under a transilluminator to determine the presence or absence of a PCR product of the desired size.

The presence or absence of amplification by PCR was detected for each of the six PRRSV strains using two primer sets. Then, the ratio (percentage) of the number of products confirmed to be amplified among the 12 types of PCR products was calculated. The results obtained are shown in Table 2.

As is clear from the results shown in Table 2, two types of PCR products were detected at all passage stages in all virus strains inoculated into IPKM. On the other hand, in Marc145 inoculated in the same manner as IPKM, PCR products were detected only in Ingelvac at all passage stages. Furthermore, when the base sequences of the PCR products derived from the two field strains detected at the 5th passage in Marc145 were decoded, a mutation in which a stop codon appeared, although not shown in the diagram, was detected.

(Example 6)
IPKM was seeded in a 6-well plate at ^3x10 cells/well/2 mL. The next day, swine epidemic diarrhea virus (Nisseiken PED live vaccine, Nisseiken), swine infectious gastroenteritis virus (swine infectious gastroenteritis live virus dried prophylactic solution, Kaketsuken), and swine parvovirus (“Kyoto Biken”) were detected. Porcine parvo live vaccine, Kyoto Biken), Japanese encephalitis virus (“Kyoto Biken” Japanese encephalitis vaccine, Kyoto Biken), Live vaccine virus solution of Auskie virus (Porciris Begonia DF・50, Intervet) or porcine circovirus 100 μL of each type 2 virus solution was inoculated and allowed to stand for 2 hours at 37° C. under 5% CO ₂ and saturated steam.

Two hours after each virus inoculation, the cell surface was washed once with serum-free medium, and 2 mL of MEM (containing 1% FBS) was added. Then, 0.2 mL of the medium was collected at 8 hours and 48 hours after infection.

Forty-eight hours after infection, cells were lysed with mammalian protein extraction buffer (manufactured by GE Healthcare). Next, nucleic acids are extracted from the obtained cell lysate and the collected medium using a nucleic acid purification kit (product name: Maxwell (registered trademark) 16 virus total nucleic acid purification kit, manufactured by Promega), and M-MLV reversal is performed. Reverse transcription reaction was performed using transcriptase (manufactured by Promega), a random primer, and a dT primer. Using the obtained reverse transcription product as a template and a primer set corresponding to each virus, a reaction solution of SYBR (registered trademark) premix ExTaqII (manufactured by Takara) was prepared according to the manual, and the mixture was transferred to the CFX96 Touch Deep Well real-time PCR analysis system. (manufactured by BIO-RAD) to detect the genes of each virus. The results obtained are shown in Figures 5 to 10.

The genes of swine epidemic diarrhea virus and swine infectious gastroenteritis virus are described by Kim et al. (2007) J. virol. Meth. The primer set described in 146(1-2) 172-177 was used for amplification. The gene of porcine parvovirus was described by Kim et al. (2003) J Vet Med Sci 65(6):741-744. The gene of Japanese encephalitis virus was amplified using the primer set described in the Pathogen Detection Manual (National Institute of Infectious Diseases), page 636. The gene of Aujesky virus was described by Belak et al. (1989) Arch Virol: 108, 279-286. In addition, the gene of porcine circovirus type 2 was developed by Takahagi Y. , et al. (2008) J. Vet. Med. Amplification was performed using the primer set described in Sci:70,603-606.

As is clear from the results shown in Figures 5 to 10, for all live vaccine viruses, the Ct value of the virus gene 48 hours after inoculation was smaller than that of the virus gene in the culture supernatant collected 8 hours after inoculation. , and an increase in the number of viruses was observed. In addition, even though the cell surface was washed 2 hours after inoculation with the virus solution, a larger amount of virus was detected in the cell lysate than in the culture supernatant, indicating that all viruses infected IPKM and multiplied. It became clear that it could be done.

As explained above, according to the present invention, by using immortalized porcine kidney macrophages that have high infection susceptibility to swine-infecting viruses, it becomes possible to propagate various swine-infecting virus strains, and It will become possible to manufacture and develop vaccines. Furthermore, by using immortalized pig kidney macrophages, it is possible to determine the presence or absence of neutralizing antibodies against the pig infection virus.

Claims (4)

A step of contacting immortalized pig kidney macrophages with a virus that infects pigs (excluding African swine fever virus), and multiplying the virus in the immortalized pig kidney macrophages,
The immortalized pig kidney macrophage is an immortalized macrophage (IPKM) obtained by introducing a lentivirus encoding SV40 large T antigen and pig-derived telomerase reverse transcriptase into a primary cultured macrophage derived from pig kidney, and
A method for producing a virus that infects pigs, wherein the virus that infects pigs (excluding African swine fever virus) is PRRS virus . contacting immortalized pig kidney macrophages with a virus that infects pigs (excluding African swine fever virus), and multiplying the virus in the immortalized pig kidney macrophages;
isolating the grown pig-infecting virus; and mixing the isolated pig-infecting virus with a pharmacologically acceptable carrier or medium,
The immortalized pig kidney macrophage is an immortalized macrophage (IPKM) obtained by introducing a lentivirus encoding SV40 large T antigen and pig-derived telomerase reverse transcriptase into a primary cultured macrophage derived from pig kidney, and
A method for producing a vaccine containing a virus that infects pigs, wherein the virus that infects pigs (excluding African swine fever virus) is PRRS virus . Immortalized pig kidney macrophages infected with a virus that infects pigs (excluding African swine fever virus) ,
The immortalized pig kidney macrophage is an immortalized macrophage (IPKM) obtained by introducing a lentivirus encoding SV40 large T antigen and pig-derived telomerase reverse transcriptase into a primary cultured macrophage derived from pig kidney, and
Immortalized pig kidney macrophages, wherein the virus that infects pigs (excluding African swine fever virus) is PRRS virus . A method for detecting neutralizing antibodies against a virus infecting pigs, the method comprising:
In the presence of a biological sample isolated from a test pig , immortalized pig kidney macrophages are brought into contact with a virus that infects pigs (excluding African swine fever virus), and the virus is introduced into the immortalized pig kidney macrophages. a step of propagating
a step of detecting the number of the proliferated viruses, and when the number of viruses detected in the step is smaller than the number of viruses proliferated in the immortalized pig kidney macrophages in the absence of the biological sample, the biological sample the step of determining that the sample contains neutralizing antibodies against the virus;
The immortalized pig kidney macrophage is an immortalized macrophage (IPKM) obtained by introducing a lentivirus encoding SV40 large T antigen and pig-derived telomerase reverse transcriptase into a primary cultured macrophage derived from pig kidney, and
The method, wherein the virus that infects pigs (excluding African swine fever virus) is a PRRS virus .