KR20210055668A - Suspension Cell Culture Adapted Vaccine Strain Derived from Foot-and-mouth disease virus of O/ME-SA/Ind-2001 Lineage and Method of Preparing the Same - Google Patents

Abstract

The present invention relates to an O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus vaccine strain adapted to floating cell culture, a foot-and-mouth disease virus vaccine composition comprising the same, and a method for producing the same and, more specifically, to a foot-and-mouth disease virus vaccine strain established by alternating continuous passaged culture of O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus in adherent cells and floating cells, a vaccine composition comprising an inactivated virus vaccine strain as an active ingredient, and a method for producing the same.

Description

Suspension Cell Culture Adapted Vaccine Strain Derived from Foot-and-mouth disease virus of O/ME-SA/Ind-2001 Lineage and Method of Preparing the Same}

The present invention is a method for producing a foot-and-mouth disease virus vaccine line, characterized in that cell adaptability is obtained by sequentially subculturing the O serotype-derived wild-type foot-and-mouth disease virus in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 suspended cells sequentially. It is about.

More specifically, the method for preparing a foot-and-mouth disease virus vaccine strain of the present invention includes the following steps:

(a) inoculating LFBK adherent cells with a wild-type foot-and-mouth disease virus derived from O serotype and subculturing at least five times to obtain a virus culture containing the cell-adapted virus;

(b) inoculating the virus culture containing the cell-adapted virus obtained in step (a) into the BHK-21 adherent cells and subculturing at least four times to obtain a virus culture containing the cell-adapted virus. ;

(c) obtaining a virus culture containing the cell-adapted virus by inoculating the virus culture containing the cell-adapted virus obtained in step (b) into the BHK-21 floating cells and subculturing at least six times. ;

(d) selecting and isolating candidate vaccine strain viruses by evaluating at least one of virus productivity and antigen productivity [[E]] from the virus culture containing the cell-adapted virus obtained in step (c); And

(e) selecting the final vaccine strain virus by checking whether at least one of the heparan sulfate receptor and the JMJD6 receptor is recognized for the selected and isolated candidate vaccine strain virus.

In addition, the present invention relates to an O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus vaccine line adapted for floating cell culture and a foot-and-mouth disease virus vaccine composition comprising the same, and more specifically, O/ME-SA (Middle East-South America)/Ind-2001 It relates to a vaccine composition comprising, as an active ingredient, a foot-and-mouth disease virus vaccine line and an inactivated virus vaccine line thereof established by alternating and successive subcultures in adherent cells and floating cells of the genotype foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a viral bullous disease that infects two-hoofed animals and is characterized by rapid replication and rapid transmission. Due to its economic importance, this disease is classified as an important disease by the World Animal Health Organization (OIE), and it is acting as a very important factor in the cross-border trade of livestock products. Foot-and-mouth disease virus (FMDV) is a single-stranded bipolar RNA virus that belongs to the genus Picornaviridae and Aphthovirus , and has seven different serotypes (A, O, C, Asia-1, SAT-1, SAT-2, SAT-3). In order to suppress the spread of foot-and-mouth disease, except for killing animals, the only method of using an inactivated vaccine is being used. However, since foot-and-mouth disease vaccines do not protect against other serotypes, and appropriate vaccines should be used according to the topotype and lineage, it is very important to develop vaccines in consideration of the outbreaks of neighboring countries.

In the case of foot-and-mouth disease virus O serotype, there are about 8 or more genotypes, but recently, in Asian regions such as Korea, China, Vietnam, and Myanmar, O/SEA/Mya-98, Cathay, ME-SA/PanAsia, ME-SA-Ind-2001 A genotype of foot-and-mouth disease virus is occurring. Among them, the O/ME-SA/Ind-2001 (eg, O/ME-SA/Ind-2001e) genotype is the most prevalent virus in East Asia and Southeast Asia since 2015. In particular, in Korea, the O/ME-SA/Ind 2001 genotype foot-and-mouth disease virus occurred in 2017 (the first occurrence of the genotype) and 2019, and the outbreak in neighboring countries has been continuously reported, so the risk of future outbreaks continues. . Therefore, the need to develop vaccine strains derived from the O/ME-SA/Ind 2001 genotype is also increasing.

Foot-and-mouth disease vaccine is generally produced by inactivating a foot-and-mouth disease virus by culturing it in large quantities, and then mixing the antigen with an adjuvant to prepare a vaccine. BHK-21 suspension cells are mainly used for mass culture of foot-and-mouth disease virus. Foot-and-mouth disease virus (wild-type foot-and-mouth disease virus) isolated outdoors uses integrin receptors to infect cells and animals, but BHK-21 floating cells have very high expression of integrin receptors (especially integrin alpha 5 and integrin alpha V). It is known that the virus does not proliferate well because it is poor (Non-Patent Document 1). Therefore, in order to use it for mass vaccine production of foot-and-mouth disease virus, through complete cellular adaptation, floating cells (or other host cells) such as Heparan sulfate or JMJD6 (Jumonji domain-containing protein 6), rather than integrin receptors, A virus (or vaccine strain) that recognizes the receptor must be secured, and the proliferation and antigen productivity of foot-and-mouth disease virus in floating cells must be verified. In addition, the vaccine containing the foot-and-mouth disease virus antigen must have immunogenicity capable of inducing a neutralizing antibody titer at a protective level.

According to the prior art, serotypes and genotypes of foreign epidemic strains isolated from epithelial tissues of cattle, pigs, etc., particularly in Asia, have been identified (Non-Patent Document 2), and foot-and-mouth disease virus Although it is disclosed that an antigen containing an amino acid of a specific structural protein derived from is used for vaccine production (Patent Document 1), or a recombinant virus having deleted a specific gene of foot-and-mouth disease virus is used for vaccine production (Patent Document 2), foot-and-mouth disease virus does not cross-protect each other according to the host animal and each serotype, so it is very difficult to develop a vaccine to effectively protect against infection for all serotypes identified so far (Non-Patent Document 3 -4).

In order to generate effective protective antibodies suitable for the serotype/regional/genotype of the foot-and-mouth disease virus epidemic that is currently popular in Korea, the present inventors have developed O/ME, which is one of the most frequently occurring viruses in Asia including Korea for the past 5 years. -SA/Ind-2001 Using the genotype foot-and-mouth disease virus O/Boeun/SKR/2017 foot-and-mouth disease virus, mass-produced foot-and-mouth disease vaccine strains, and thereby, have made great efforts to develop a preventive vaccine with high defense power against foot-and-mouth disease virus. As a result, the O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus finally selected through a number of screening processes were LFBK adherent cells (adherent porcine kidney cells), BHK-21 adherent cells (adherent baby hamster kidney-21 cells) and BHK-21 suspended cells (suspension baby hamster kidney-21 cells) were sequentially inoculated and subcultured alternately at a specific number of passages to harvest foot-and-mouth disease vaccine strains with optimized viral titer, and the foot-and-mouth disease vaccine strain was inactivated. Then, when the foot-and-mouth disease virus vaccine prepared by mixing with an adjuvant was inoculated into a pig or a mouse as a substitute animal, the neutralizing antibody in the serum/blood of the inoculated animal was remarkably elevated and maintained for a long period of time to protect against foot-and-mouth disease virus. It was confirmed that the effect was excellent and the present invention was completed.

Korean Patent Application Publication No. 10-2018-0133678 (December 17, 2018) Korean Patent Application Publication No. 10-2014-0083129 (July 4, 2014)

Brown F., Dev Biol Stand., 　93:85-8, 1998 Mahapatra M. et al., Vaccine, 35(51):7147-7153, 2017 Beck E. et al., EMBO J., 2(4): 555-9, 1983 Grubman MJ et al., Virology, 158(1):133-40, 1987

An object of the present invention is a new effective foot-and-mouth disease against the wild-type/outdoor-type foot-and-mouth disease virus, more specifically O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus, which is difficult to effectively defend with existing vaccines. It is to provide vaccine stocks.

Another object of the present invention is to provide a vaccine composition for preventing or treating foot-and-mouth disease virus comprising the novel foot-and-mouth disease virus vaccine line of the present invention.

In order to achieve the above object, the present invention is a wild-type foot-and-mouth disease virus, O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus, sequentially subcultured in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 suspended cells. It provides a foot-and-mouth disease vaccine line in which cell adaptability was obtained.

The present invention also provides a vaccine composition for preventing infection of foot-and-mouth disease virus, comprising the inactivated virus of the foot-and-mouth disease vaccine strain as an active ingredient.

The foot-and-mouth disease vaccine strain according to the present invention can produce foot-and-mouth disease virus antigens in large quantities, so that it can be industrially used in the production of foot-and-mouth disease vaccines, and the O serotype foot-and-mouth disease virus, especially O/ME-SA/Ind-, which is popular in Asia. It can be usefully used in the manufacture of vaccines for the defense of ME-SA regional viruses, including 2001 genotype foot-and-mouth disease virus.

1 is a wild-type foot-and-mouth disease virus O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus O/Boeun/SKR/2017 foot-and-mouth disease virus inoculation, followed by LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells. This is a graph showing the cell adaptability of the foot-and-mouth disease virus in floating cells by infecting the virus harvested by sequentially subculturing in BHK-21 adherent cells or CHO-K1 adherent cells and measuring the viral titer.
Figure 2 is a graph showing the proliferative (A) and antigen productivity (B) of the foot-and-mouth disease virus vaccine line (L5B6S9-O virus) according to the present invention.
FIG. 3 shows a photograph of an inactivated antigen produced with a foot-and-mouth disease virus vaccine line (L5B6S9-O virus) according to the present invention, confirmed by a Transmission Electron Microscope (TEM).
Figure 4 is a graph showing the degree of immunogenicity by inoculating pigs with a test vaccine containing an antigen produced with the foot-and-mouth disease virus vaccine line (L5B6S9-O virus) according to the present invention.
Figure 5 shows the same regional virus (Figure 5A) and different regional virus (Figure 5B) for the sera of pigs inoculated with a test vaccine containing an antigen produced with the foot-and-mouth disease virus vaccine line (L5B6S9-O virus) according to the present invention. The results of the neutralization test are shown in a graph.
6 is a graph showing the results of an experiment in mice to determine whether a test vaccine containing an antigen produced with the foot-and-mouth disease virus vaccine line (L5B6S9-O virus) according to the present invention protects against other field/wild type foot-and-mouth disease virus (heterologous FMDV) attacks It is represented by

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In the present specification, the term "about" used to express length, area, volume, time (period), concentration, content, etc. means that there is a tolerance of up to 10% in the corresponding value or numerical range .

In one aspect, the present invention is a novel foot-and-mouth disease vaccine strain, more specifically O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus O/Boeun/SKR/2017 foot-and-mouth disease virus, which is a wild-type/outdoor foot-and-mouth disease virus. It relates to a foot-and-mouth disease vaccine strain obtained by selection and isolation after alternately and continuously passage cultivation. The adherent cells of BHK-21 and the suspended cells of BHK-21 (baby hamster kidney cells) may be sequentially subcultured, but are not necessarily limited to the above order.

Specifically, the foot-and-mouth disease virus vaccine strain according to the present invention is completely subcultured in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 suspended cells, without being limited to the sequence of subcultures and/or the number of subcultures. It is a virus that is adapted to cells and recognizes the heparan sulfate receptor (HSR), and includes all viruses that recognize HSR having a normal function and activity and exhibit a target viral titer in a specific cell. The specific cell may be a CHO-K1 cell.

Specifically, the foot-and-mouth disease virus vaccine strain according to the present invention is completely subcultured in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 suspended cells, without being limited to the sequence of subcultures and/or the number of subcultures. As a virus that is adapted to cells and recognizes the JMJD6 receptor (Jumonji domain-containing protein 6 receptor; JMJD6R), it recognizes JMJD6R with normal function and activity, and also includes all viruses that exhibit a targeted viral titer in a specific cell. The specific cell may be a CHO-677 cell.

The continuous passage for obtaining the foot-and-mouth disease virus vaccine line according to the present invention is, as long as the foot-and-mouth disease virus vaccine line recognizing HSR and/or JMJD6R having normal function and activity is obtained, regardless of the order of passage and/or the number of passages. However, the foot-and-mouth disease virus vaccine strain according to the present invention may be obtained through alternating and continuous passage culture at least 5 times in LFBK adherent cells, at least 4 times in BHK-21 adherent cells, and at least 6 times in BHK-21 suspended cells. . Preferably, the foot-and-mouth disease virus vaccine strain according to the present invention may be obtained through alternating successive subcultures 5 times in LFBK adherent cells, 4 times in BHK-21 adherent cells, and 7 times in BHK-21 suspended cells. More preferably, the foot-and-mouth disease virus vaccine strain according to the present invention may be obtained through alternating successive subcultures 5 times in LFBK adherent cells, 6 times in BHK-21 adherent cells, and 9 times in BHK-21 suspended cells.

Foot-and-mouth disease virus vaccine according to the present invention, wild-type foot-and-mouth disease virus O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus sequentially undergoes serial passage culture in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 suspended cells. As a vaccine line, suitable cell adaptability, strictly complete cell adaptability, can be obtained. Here, the term'obtain suitable cell adaptability as a vaccine strain' or'obtain complete cell adaptability' is a wild-type virus from LFBK adherent cells, BHK-21 adherent cells, and BHK-21 suspended cells sequentially through serial subculture to BHK. -21 Means that adaptation to floating cells is complete. In other words, the term refers to various host cells, for example, LFBK adherent cells, BHK-21 adherent cells, BHK-21 floating cells, CHO adherent cells (eg, CHO-K1 adherent cells and / Or CHO-677 adherent cells), preferably in BHK-21 floating cells, recognizes receptors other than integrin receptors, such as heparan sulfate receptors and JMJD6 receptors, and can survive or proliferate normally to exhibit a predetermined viral titer. it means.

Since the foot-and-mouth disease virus vaccine line according to the present invention recognizes the heparan sulfate receptor, 1x10 ^2.0 TCID ₅₀ /mL or more, preferably 1x10 ^2.3 TCID ₅₀ /mL or more, more preferably 1x10 ^2.5 TCID _{50 in CHO-K1 adherent cells} May show viral titers above /mL.

Since the foot-and-mouth disease virus vaccine line according to the present invention recognizes the JMJD6 receptor, 1x10 ^2.0 TCID ₅₀ /mL or more, preferably 1x10 ^2.5 TCID ₅₀ /mL or more, more preferably 1x10 ^2.7 TCID ₅₀ /mL in CHO-677 adherent cells It can show the above viral titer.

The specific region virus of the present invention is a virus obtained through selection and isolation from LFBK adherent cells, BHK-21 adherent cells, and virus culture obtained through serial passage culture from BHK-21 floating cells. 1x10 ^5.5 TCID ₅₀ /mL or higher, preferably 1x10 ^6.0 TCID ₅₀ /mL or higher, most preferably 1x10 ^7.0 TCID ₅₀ /mL or higher in cells, 1x10 ^2.0 TCID ₅₀ /mL or higher in CHO-K1 adherent cells , Preferably 1×10 ^2.5 TCID ₅₀ ^{/mL or more, and preferably 1×10 2.5} TCID ₅₀ ^{/mL or more, and 1×10 2.0} TCID ₅₀ /mL or more, preferably 1×10 2.5 TCID 50 /mL or more in CHO-677 adherent cells.

Foot-and-mouth disease virus vaccine according to the present invention is a virus that has acquired complete cellular adaptability through alternating and continuous passage in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 suspended cells, and is a nucleic acid (nucleotide) represented by SEQ ID NO: 1. It may be characterized by having a nucleic acid sequence having a nucleotide sequence or substantially homologous thereto, and a polypeptide generated from the nucleic acid nucleotide sequence. In the case of nucleic acids and polypeptides of the foot-and-mouth disease vaccine strain, the term "substantial homology" refers to at least about 80% of nucleotides and amino acids, typically when the two nucleic acids and the two polypeptides or their designated sequences are optimally aligned and compared. Including at least about 85%, preferably about 90%, 91%, 92%, 93%, 94% or 95%, more preferably at least about 96%, 97%, 98%, 99% of nucleotides and amino acids. , 99.1%, 99.2%, 99.3%, 99.4% or 99.5% mean the same. The present invention includes nucleic acid sequences and polypeptide sequences having substantial homology to the specific nucleic acid sequences and amino acid sequences referred to herein.

The wild-type or field-type foot-and-mouth disease virus used as a starting material to obtain the foot-and-mouth disease virus of the present invention may be any virus classified as a specific virus of the O/ME-SA/Ind-2001 genotype. As the genotype foot-and-mouth disease virus, for example, KVCC (Korea Veterinary Culture Collection, Korea Veterinary Genetic Resources Bank [http://kvcc.kahis.go.kr]) of the Agriculture, Forestry and Livestock Quarantine Division under the accession number KVCC-VR1700004. Foot-and-mouth disease virus can be used.

In another aspect, the present invention relates to a vaccine composition for preventing infection of foot-and-mouth disease virus, comprising the inactivated virus of the foot-and-mouth disease vaccine strain as an active ingredient.

The inactivated foot-and-mouth disease virus vaccine strain of the present invention can be obtained by inactivating the foot-and-mouth disease virus vaccine strain by a known method.

Known inactivation treatment methods include, for example, treatment with BEI (Binary Ethylenimine) used for virus inactivation, formalin, glutaaldehyde, heating, irradiation, BPL, or other inactivating agents known in the art (inactivators ) Can be used to inactivate (inactivate).

The inactivation reaction of the inactivated foot-and-mouth disease virus vaccine strain of the present invention may be preferably carried out at 26 to 37°C for 48 hours, most preferably at a temperature of 37°C for 24 hours and then first inactivated at 26°C. It can be achieved by secondary inactivation at temperature for 24 hours.

In addition to the inactivated foot-and-mouth disease virus vaccine line, the vaccine composition may further include one or more commonly used pharmaceutically and/or veterinarily acceptable carriers, diluents, antibiotics, immunomodulators, and the like.

As used herein, the term "carrier" refers to any pharmaceutically acceptable solvent (solution), dispersion medium, coating, buffer, isotonic agent, suspension, colloid for administration to a target animal (host animal). , Stabilizers, preservatives, antibiotics (antibacterial, antifungal, etc.), adsorption delaying agents, adjuvants (adjuvants), inserts, etc. In general, the use of one or more delivery vehicles for chemical compounds, in particular immunogens, is well known to those skilled in the pharmaceutical industry. Unless any conventional medium or agent is compatible with the active ingredient, its use in diagnostic, prophylactic and therapeutic compositions is contemplated. One or more supplemental active ingredient(s) may also be incorporated into or administered in conjunction with one or more of the disclosed immunogenic compositions and vaccine formulations.

The diluent may include water, saline, dextrose, ethanol, glycerol, and the like, and the isotonic agent may include sodium chloride, dextrose, mannitol, sorbitol, lactose, etc., and the stabilizer may include albumin. It may be included, but is not limited thereto.

Examples of the adjuvant (adjuvant) include mineral gels such as aluminum hydroxide gel; Surface active substances such as lysolecithin; Glycosides, such as saponin derivatives, such as Quil A or GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Alabama); Fluron polyol; Polyanion (polyanion); Non-ionic block polymers such as Pluron F-127 (BASF, USA); Peptide; Mineral oils, e.g. Montanide ISA-206 (Seppic, France), Montanide ISA-50 (Seppic, France), Cabopol, Ampigen, Ampigen Mark II (Hydronics ( Hydronic), USA), alhydrogels, oil emulsions (e.g., emulsions of mineral oil and water such as BayolF/Arlacel A), or vegetable oils, emulsions of water and emulsifiers such as lecithin; Alum; cholesterol; Freund's complete and incomplete adjuvants; Block copolymers (CytRx, Atlanta, GA); SAF-M (Chiron, Emeryville, CA); AMPHIGEN (registered trademark) adjuvant; Monophosphoryl lipid A, avridine lipid-amine adjuvant; E. coli- derived heat-labile enterotoxin (recombinant, etc.); Cholera toxin; Muramyl dipeptide; IMS1313 (Seppic, France); ISA206; Montanide 01 gel (Seppic, France); And mixtures of these adjuvants are included, but are not limited thereto, and preferably at least one adjuvant selected from the group consisting of IMS1313, Carbopol, and Montanide 01 gel. The adjuvant refers to a compound or mixture that enhances the immune response and/or promotes the rate of absorption after inoculation, and includes any absorption-promoting agent.

As used herein, the term "vaccine" is to be administered to vertebrates, preferably Artiodactyla animals such as mammals such as cattle, pigs, goats, sheep, deer, camels, giraffes, etc. It refers to a composition or formulation comprising an immunogenic composition in a possible form.

The vaccine composition according to the present invention may include other immunogenic compositions in a composition comprising a foot-and-mouth disease virus vaccine line, an immunogenic composition disclosed herein, formulated for administration to a target animal (host animal).

The "immunogenicity" of the immunogenic composition comprising the foot-and-mouth disease virus vaccine line is the ability of the foot-and-mouth disease virus to induce an immune response in the target animal (host animal) against different serotypes of foot-and-mouth disease virus, particularly O serotype foot-and-mouth disease virus ( It means the ability to act as an antigen). The immune response may be a cellular immune response primarily mediated by cytotoxic T-cells, or a humoral immune response primarily mediated by auxiliary T-cells that activate B-cells to induce antibody production.

In particular, the term "immunogenicity" refers to substances such as nucleic acids and polypeptides constituting foot-and-mouth disease virus that induce an immune response in a target animal (host animal), and is a humoral type (B cell) and/or a cellular type ( T cells) have these properties.

The immunogenic composition, and the vaccine composition comprising the same, can cause an immune response when introduced into a target animal (host animal), preferably a specific immune response against the introduced antigen(s), such an immune response is a vaccine Production of specific antibodies in cells and tissues of inoculated target animals (host animals), cytokines, and/or cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses. It is detected by activation or not, but can be easily detected using a conventional assay known to those skilled in the immunology art.

The mixing ratio of the inactivated virus and adjuvant contained in the vaccine composition may be 1 to 10: 1 to 10, preferably 1 to 5: 1 to 5, and most preferably 1 to 1, based on volume. have.

Antibiotics included in the vaccine composition may include gentamicin and methiolate, but are not limited thereto.

The vaccine composition may further include one or more immunomodulatory agents such as cytokines (eg, interleukin, interferon, etc.).

The vaccine composition is a target animal (host animal), a vaccine composition for preventing, defending, managing or treating symptoms caused by foot-and-mouth disease virus infection in mammals other than humans, which can be used as a vaccination method disclosed herein. Can be prepared in dose aliquots or multi-dose aliquots, and vaccination can be achieved through single or multiple inoculations.

The vaccine composition is preferably administered to the target animal by intramuscular or subcutaneous route, but other routes of administration, such as oral administration, nasal administration (e.g., aerosol or other needleless administration), transdermal administration, and parenteral administration ( For example, intravenous injection, intraperitoneal administration, intralymphatic administration, intradermal administration, subcutaneous administration, intraperitoneal administration, rectal administration, vaginal administration or intramuscular administration), or through a known route including a combination of these administration routes. Can be. The preferred route of administration is one or more selected from the group consisting of muscle, subcutaneous, intraperitoneal, intravenous, oral, dermal, ocular, and brain, and a more preferable route of administration is subcutaneous administration or intramuscular administration, and the most preferable route of administration is It is an intramuscular administration of the neck area.

When the vaccine composition is administered in a general injection formulation, the vaccine composition is administered to the muscle of the target animal (target animal: cow, pig, goat, sheep, deer, camel, giraffe, etc.) or to a site adjacent thereto, but the weight of the target animal And it is preferable to administer one or more times during a designated administration period in consideration of health conditions.

The vaccine composition is an immune response, infection prevention and/or foot-and-mouth disease against at least one selected from the group consisting of foot-and-mouth disease virus serotypes of O, A, C, SAT-1, SAT-2, SAT-3, and Asia-1. It may be characterized by having a therapeutic effect for viral infection, but is not limited thereto.

In another aspect, the present invention is fully cell-adapted through serial passage in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells, and is heparan sulfate receptor (HSR) and/or JMJD6 receptor, more precisely. Recognizing HSR and/or JMJD6 receptors having normal functions and activities, relates to a method for preparing a foot-and-mouth disease virus vaccine line.

As long as the sequence and/or the number of foot-and-mouth disease virus vaccine strains that recognize HSR with normal activity and exhibit a target viral titer in specific cells are obtained, LFBK adherent cells, BHK- It is preferable to proceed in the order of 21 adherent cells and BHK-21 floating cells. The continuous subculture is more preferably performed at least 5 times in LFBK adherent cells, at least 4 times in BHK-21 adherent cells, and at least 6 times in BHK-21 suspended cells.

For example, the manufacturing method of the foot-and-mouth disease virus vaccine strain of the present invention may include the following steps (a) to (e):

(a) LFBK adherent cells are inoculated with wild-type foot-and-mouth disease virus, preferably O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus, more preferably O/Boeun/SKR/2017 foot-and-mouth disease virus, followed by successive passage culture at least 5 times. Thereby obtaining a virus culture containing the cell-adapted virus;

(b) After inoculating the virus culture containing the cell-adapted virus obtained in step (a) onto the BHK-21 adherent cells, 4 or more times, preferably 4 to 6 times, successively passaged to obtain the cell-adapted virus. Obtaining a virus culture containing;

(c) After inoculating the virus culture containing the cell-adapted virus obtained in step (b) into the BHK-21 floating cells, the cell-adapted virus is subcultured at least 6 times, preferably 7 to 9 times. Obtaining a virus culture containing;

(d) selecting and isolating candidate vaccine strain viruses by evaluating virus productivity and antigen productivity from the virus culture containing the cell-adapted virus obtained in step (c); And

(e) selecting the final vaccine strain virus by checking whether at least one of the heparan sulfate receptor and the JMJD6 receptor is recognized for the selected and isolated candidate vaccine strain virus.

In the step (d) of the method for preparing the foot-and-mouth disease virus vaccine line according to the present invention, viral productivity is evaluated by measuring the viral titer in BHK-21 adherent cells, and viral antigen productivity is at the time when an appropriate viral titer appears (e.g., This can be accomplished by evaluating the concentration of the viral antigen (146S inactivated foot-and-mouth disease virus particles) harvested at the time when the viral titer is at its peak).

In the step (e) of the method for preparing the foot-and-mouth disease virus vaccine line according to the present invention, the recognition of the heparan sulfate receptor of the candidate vaccine line virus may be achieved by measuring the viral titer in CHO-K1 adherent cells. When the virus inoculated from CHO-K1 adherent cells ^{exhibits a viral titer of 1x10 2.0} TCID ₅₀ /mL or more, preferably 1x10 ^2.3 TCID ₅₀ /mL or more, more preferably 1x10 ^2.5 TCID ₅₀ /mL or more, normal function and activity It can be determined by recognizing the heparan sulfate receptor having

In the step (e) of the method for preparing the foot-and-mouth disease virus vaccine line according to the present invention, the recognition of the JMJD6 receptor of the candidate vaccine line virus may be achieved by measuring the viral titer in CHO-677 adherent cells. When the virus inoculated from CHO-677 adherent cells ^{exhibits a viral titer of 1x10 2.0} TCID ₅₀ /mL or more, preferably 1x10 ^2.5 TCID ₅₀ /mL or more, more preferably 1x10 ^2.7 TCID ₅₀ /mL or more, normal function and activity It can be determined by recognizing the JMJD6 receptor.

The method for preparing the foot-and-mouth disease virus vaccine line according to the present invention includes the virus titer in at least any one cell of BHK-21 cells, CHO-K1, and CHO-677 against the final selected vaccine line virus after step (e). Measurement, sequencing for part or all of the viral genes (e.g., sequencing to confirm changes in the amino acid constituting the structural protein of the virus or the nucleic acid sequence encoding the same), a test for confirming the immunogenicity in the target animal, and heterogeneity At least one selected from the group consisting of tests for confirming the defense against viruses may be additionally performed.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

[Test Example 1] Preparation of candidate vaccine strain virus through alternating and continuous cell passage culture of wild-type foot-and-mouth disease virus and selection of fully-cell adapted vaccine strain virus

(1) Preparation of candidate vaccine strain virus through alternating and continuous cell passage culture of wild-type foot-and-mouth disease virus

Pig kidney inoculated with the O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus, O/Boeun/SKR/2017 foot-and-mouth disease virus, belonging to the O serotype foot-and-mouth disease virus, isolated from blisters of cows tested positive for foot-and-mouth disease in Boeun, Chungcheongbuk-do in 2017 See LFBK adherent cells (LFBK adherent cells; Swaney LM, Vet. Microbiol ., 18:1-14, 1988, and LaRocco M. et al. , J Clin Microbiol. , 51(6):1714-20, 2013). ) In BHK-21 adherent cells (BHK-21 adherent cells; cell culture) for adaptation in BHK-21 suspended cells, which are hamster kidney cells, after proliferating by passing through (passaging, subculture) a certain number of times. BHK-21 cells cultured while attached to a container [plate] for use; ^{after subculture in ATCC ®} CCL-10 ^™ ), BHK-21 suspension cells (BHK-21 suspension cells; in a cell culture container [plate]) BHK-21 cells cultured in a suspended state; KCTC 12945BP cell line [refer to Korean Patent No. 10-1812223]) was subjected to alternating and continuous passage culture.

Specifically, as shown in Table 1 below, the O/Boeun/SKR/2017 foot-and-mouth disease virus was inoculated into LFBK adherent cells, then passaged a certain number of times, and then virus cultures were harvested from the LFBK adherent cells. Using the harvested virus culture, BHK-21 adherent cells were inoculated and subcultured a certain number of times, and then virus cultures were harvested from BHK-21 adherent cells. The harvested virus culture was used to inoculate BHK-21 suspended cells and subcultured at a certain number of times to harvest the final virus (vaccine strain candidate) culture.

Virus inoculation amount per cell of each of LFBK adherent cells, BHK-21 adherent cells, and BHK-21 suspended cells was 0.01 MOI (Multiplicity of infection), and virus titers were determined by harvesting virus cultures about 16 hours after inoculation. It was measured in BHK-21 adherent cells.

For LFBK adherent cells and BHK-21 adherent cells, Dulbecco's modified Eagle medium (DMEM) containing 10% (v/v) Fetal Bovine Serum (FBS) was used, and the _{cells were cultured under 5% CO 2} /37°C. At the time of inoculation, the medium was replaced with DMEM containing 2% (v/v) FBS and subcultured under the same conditions. BHK-21 suspended cells are dextran sulfate (Sigma-Aldrich, USA), L-glutamine (Thermo Fisher Scientific, USA), pluronic F-68 non-ionic surfactant (Thermo Fisher Scientific, USA) and HyClone cell boost 5 supplement (Fisher). Scientific, Hampton, USA) using ProVERO-1 serum-free medium (Lonza, Switzerland) in 5% CO ₂ /37°C/110 rpm conditions, and cultured cells in suspension, and serum-free CD-BHK without additives during virus inoculation. -21 The medium was replaced with production media (Thermofisher, USA) and subcultured under the same conditions.

In addition, when changing the cell type in the order of LFBK adherent cells, BHK-21 adherent cells, and BHK-21 suspended cells during alternate subculture, the supernatant obtained by centrifuging the harvested virus culture at about 3,000 rpm (supernatant ) Was used. The supernatant was prepared in a state in which impurities such as cell debris were removed by passing the culture through a 0.45 μm scanning filter (Syringe Filter, Merck).

(2) Screening of vaccine strain virus that has acquired complete cellular adaptability

In order to select a candidate vaccine strain virus with excellent cell adaptability, as described above, the combination of LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells and the number of passages were varied to perform alternating and continuous passage cultures. The titers were confirmed in BHK-21 adherent cells and CHO-K1 adherent cells.

Specifically, in order to confirm whether the virus obtained through the filtration process after alternating and continuous subculture of (1) is a foot-and-mouth disease virus that has been adapted to host cells, that is, BHK-21 floating cells, hamster kidney cells that do not have integrin receptors CHO-K1 cells (having heparan sulfate receptor, not possessing integrin receptor; ATCC ^® CCL-61 ^™ ) were inoculated with the virus at an inoculation amount of 0.01 MOI, and then it was confirmed that the virus had completed cell adaptation through viral titer measurement. _{Here, the CHO-K1 adherent cells were cultured under 5% CO 2} /37°C using RPMI 1640 medium containing 10% (v/v) FBS.

_{Table 1 shows the virus titer (TCID 50} /ml) of the virus harvested by alternating and serially subcultures with different combinations of cells to establish a vaccine line for wild-type foot-and-mouth disease virus in BHK-21 adherent cells and CHO-K1 adherent cells. (Reference: Reed LJ, Muench H. A simple method of estimating fifty percent endpoint. Am J Hyg. 27:493-497, 1938). Chloroform was used to remove non-entry virus activity other than foot-and-mouth disease virus before passage of BHK-21 adherent cells.

virus Alternate continuous passage culture: cell line (number of passages) Inoculation MOI
(Multiplicity of infection) Viral titer in BHK-21 adherent cells
(TCID ₅₀ /ml) Heparan Sulfate Receptor Recognition in CHO-K1 Adherent Cells O/Boeun/SKR/2017 LFBK(5), Chloroform treatment NA[not assayed] NA NA LFBK(5), Adherent BHK-21(3) 0.01 1x10 ^5.95 X LFBK(5), Adherent BHK-21(4) 0.01 1x10 ^6.63 X LFBK(5), Adherent BHK-21(4), Suspension BHK-21(1) 0.01 1x10 ^6.50 X LFBK(5), Adherent BHK-21(4), Suspension BHK-21(2) 0.01 1x10 ^6.50 X LFBK(5), Adherent BHK-21(4), Suspension BHK-21(3) 0.01 1x10 ^6.50 X LFBK(5), Adherent BHK-21(4), Suspension BHK-21(4) 0.01 1x10 ^7.50 X LFBK(5), Adherent BHK-21(4), Suspension BHK-21(5) 0.01 1x10 ^6.83 X LFBK(5), Adherent BHK-21(4), Suspension BHK-21(6) 0.01 1x10 ^6.30 O ^* LFBK(5), Adherent BHK-21(4), Suspension BHK-21(7) 0.01 1x10 ^6.50 O ^** LFBK(5), Adherent BHK-21(4), Suspension BHK-21(8) 0.01 1x10 ^6.50 O ^***

*; **; ^{***: 1x10 2.5O} TCID ₅₀ /ml in CHO-K1 adherent cells. As can be seen from Table 1, some of the viruses harvested by performing alternating and continuous passage culture by varying the combination of LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells and the number of passages are varied. Even if it showed good viral titer in adherent cells, it was shown that it did not recognize the heparan sulfate receptor. Viruses that do not recognize the heparan sulfate receptor did not acquire full cellular adaptability and were therefore excluded from candidates for foot-and-mouth disease virus vaccine strains.

In addition, referring to the virus titer data in CHO-K1-adhered cells in the graph shown in Fig. 1, after passage 5 times in LFBK adherent cells (L5), after passage 4 times in BHK-21 adherent cells (B4 ), since the virus (L5B4S6) subcultured 6 times in BHK-21 suspended cells (L5B4S6) has been shown to recognize the heparan sulfate receptor, at least 4 times in BHK-21 adherent cells, at least 5 times in LFBK adherent cells, And it was possible to draw a conclusion that complete cell adaptability is obtained by passing at least 6 consecutive subcultures in BHK-21 suspended cells (where L represents LFBK adherent cells, B represents BHK-21 adherent cells, S represents BHK-21 suspended cells, and numbers represent passages).

[Test Example 2] Characterization of the selected vaccine strain virus

Virus cultures (including L5B4S7-O virus) obtained by passage 5 times from LFBK cells, 4 times from adherent BHK-21 cells, and 7 times from BHK-21 floating cells were used in BHK-21 adherent cells to obtain a virus of a single characteristic. Viral plaques were obtained using a plaque purification method (see Polatnick J. et al. , Appl Microbiol., 12:368-73, 1964). Specifically, the BHK-21 adherent cells undergo a plaque purification process (once cultured), and in order to obtain a virus at a level capable of titer measurement, the virus subcultured once in BHK-21 adherent cells was added to the'L5B6S7-O plaque. It was named'virus'.

(1) Confirmation of cell adaptation of L5B6S7-O plaque virus

The titer of the virus (L5B6S7-O virus) obtained by plaque purification and subculture was confirmed 72 hours after inoculation in BHK-21 adherent cells and CHO-K1 adherent cells.

Table 2 shows the results of measuring the titers of L5B6S7-O plaque virus in BHK-21 adherent cells and CHO-K1 adherent cells.

Viral titer in BHK-21 adherent cells
(TCID ₅₀ /ml) CHO-K1 adherent cells Viral titer at
(TCID ₅₀ /ml) L5B6S7-O plaque virus 1x10 ^7.10 1x10 ^2.50

As can be seen from Table 2, the virus derived from L5B6S7-O plaque showed good viral titer in CHO-K1 adherent cells, and accordingly, it was determined that it normally recognizes the heparan sulfate receptor. (2) L5B6S9-O Confirmation of viral cell adaptability and potency

In order to transform the L5B6S7-O plaque virus into a seed virus for vaccine production, the virus was produced by subculturing it twice in BHK-21 floating cells, and finally'L5B6S9-O It was named as'virus'.

The supernatant (L5B6S9-O Virus) was measured in the viral titer in BHK-21 adherent cells, CHO-K1 adherent cells, and CHO-677 (pgsD-677) adherent cells by the viral titer assay used for selection of the vaccine strain virus that acquired cell adaptability. The measurement results are shown in Table 3 below.

Viral titer in BHK-21 adherent cells
(TCID ₅₀ /ml) CHO-K1 adherent cells Viral titer at
(TCID ₅₀ /ml) Viral titer in CHO-677 adherent cells
(TCID ₅₀ /ml) L5B6S9-O virus 1x10 ^7.00 1x10 ^2.50 1x10 ^2.50

As shown in Table 3, the L5B6S9-O virus exhibited a high viral titer of ^{1x10 7.00} TCID ₅₀ ^{/ml in BHK-21 adherent cells, and 1x10 2.50} TCID ₅₀ /ml of viral titer in CHO-K1 adherent cells. In addition, CHO-677 (pgsD-677) adherent cells showed ^{a viral titer of 1×10 2.50} TCID ₅₀ /ml, confirming that cell adaptation was complete. (3) L5B6S9-O Confirmation of viral proliferation

L5B6S9-O vaccine strain virus was inoculated into 100 ml of BHK-21 suspended cell culture medium at an MOI of 0.01 to confirm proliferation. Culture conditions were carried out in the same manner as in suspension culture during subculture. L5B6S9-O After 8, 12, 16, and 24 hours after inoculation of foot-and-mouth disease virus, a culture supernatant was obtained and titer was measured in BHK-21 adherent cells.

As a result, as shown in Fig. 2 (A), the L5B6S9-O virus exhibited the highest viral titer, that is, an average of 1×10 ^6.0 TCID ₅₀ /ml at the time of harvest 16 hours after inoculation.

(4) L5B6S9-O Confirmation of virus antigen productivity

After inoculating the L5B6S9-O virus at an MOI of 0.01 in 100 ml of the floating cell culture solution, virus culture was performed for a specific culture time (ie, 8 hours, 12 hours, 16 hours after inoculation). BEI (binary ethylenimine) was added to the culture so that the final concentration was 3mM, and then reacted in a shaking incubator at 37°C for 24 hours (first inactivation step). Then, the virus culture solution that had undergone the first inactivation step was transferred to a new container, treated with BEI at the same concentration as the first inactivation step, and reacted at a temperature of 26° C. for 24 hours (second inactivation step). Inactivation was completed by neutralizing BEI by adding 1M sodium thiosulfate to the virus culture solution that had been subjected to the first and second inactivation steps so that the concentration of 1M sodium thiosulfate became 1% (wt%). In order to concentrate and purify the virus, it was mixed with 7.5% PEG 8000, stirred overnight at 4° C., centrifuged for about 30 minutes with a centrifugal force of about 10,000 g, and dissolved in Tris-NaCl [pH 7.2] buffer. The antigen obtained through the inactivation, concentration, and purification processes as described above was centrifuged for about 180 minutes at a temperature of 4°C with a centrifugal force of about 110,000 g by a 15-45% sucrose gradient ultracentrification method, and then 259 nm. The amount of antigen obtained (content of 146S antigen) was compared by quantification with a spectrophotometer at the wavelength band of. The amount of antigen obtained was obtained by calculating the measured final antigen amount based on the virus culture medium (the amount of antigen produced per 1 ml of the virus culture medium µg).

As a result, as shown in Fig. 2 (B), the L5B6S9-O virus is at a level that exceeds 2 µg of the antigen amount based on 1 ml of the culture medium, which is a general antigen production standard (about 4.8 µg/ml of the antigen after 12 hours of inoculation). Inactivated foot-and-mouth disease virus particles) productivity was found to be very good.

(5) Confirmation of particles of L5B6S9-O virus-producing antigen (electron microscope observation)

As mentioned in Test Example 1, BEI was added to the supernatant of the L5B6S9-O virus culture to inactivate the L5B6S9-O virus, and a PEG 8000 (Polyethylene glycol 8000) solution was treated to a final concentration of 7.5% by weight (about 4 ℃, stirred for about 8 hours), centrifuged at about 10,000 g for about 30 minutes, and then the concentrated viral antigen was dissolved in Tris-NaCl [pH 7.2] buffer to obtain a virus antigen derived from L5B6S9-O virus. .

Then, the virus antigen purified by centrifuging the virus antigen derived from the L5B6S9-O virus at a temperature of about 4° C. for 180 minutes with a centrifugal force of about 110,000 g by 15 to 45% sucrose gradient ultracentrifugation method. Was obtained, and the obtained viral antigen was observed using a transmission electron microscope (TEM) to observe the particle structure of the viral antigen.

As a result, as shown in FIG. 3 below, it was observed that the inactivated antigen produced from L5B6S9-O had an intact 146S virus antigen particle structure.

The L5B6S9-O virus was finally determined as a viral vaccine strain for vaccine production, and as a result of performing nucleic acid sequence sequencing, it was confirmed to have a nucleic acid sequence represented by SEQ ID NO: 1. This virus vaccine strain was entrusted to the Korea Veterinary Culture Collection (KVCC, Korea Veterinary Genetic Resources Bank [http://kvcc.kahis.go.kr]) of the Agriculture, Forestry and Livestock Quarantine Headquarters and was registered under the accession number KVCC-VR1900044.

[Test Example 3] Evaluation of immunogenicity in target animals

To confirm the immunogenicity of the foot-and-mouth disease virus vaccine strain, 15 μg (per head) of inactivated antigen produced from the L5B6S9-O virus was mixed in the same volume with Montanide ISA 206 (Seppic, France) adjuvant, and a W/O/W emulsion ( water in oil in water emulsion) was prepared, and then 2 ml of the test vaccine was inoculated into pigs having negative antibodies to foot-and-mouth disease virus.

Then, in order to measure the neutralizing antibody titer of the antibody contained in the immune serum obtained from pigs inoculated with the test vaccine once a week for a period of 6 weeks without the second inoculation, foot-and-mouth disease virus serotype O, O//ME-SA/ Immune serum obtained from pigs inoculated with test vaccine through neutralization test of foot-and-mouth disease virus in LFBK cells using Ind-2001 genotype O/Boeun/SKR/2017 virus (passage 6 times in LFBK adherent cells after virus isolation) The immunogenicity of the foot-and-mouth disease virus antigen was confirmed by measuring the neutralizing antibody titer of the neutralizing antibody contained in. In addition, the antibody titer of the test vaccine was measured using PrioCHECK FMDV type O-SP ELISA (Thermo Fisher Scientific).

As a result, as shown in Fig. 4 (A) below, it was confirmed that neutralizing antibody titers of 45 times (1.65Log10) or more appeared in all pigs from 1 week after vaccination, and 4 weeks after vaccination. From this point, it was confirmed that the neutralizing antibody value increased sharply, showing a neutralizing antibody value of 100 times or more, and sufficient protective antibody was formed.

In addition, as shown in Fig. 4 (B) below, all pigs showed positive antibody reaction (PI [Percentage inhibition, inhibition rate] value of 50 or more) from the 4th week after inoculation of the test vaccine even in the O-type SP ELISA result. It was confirmed again that sufficient antibody formation was achieved in pig individuals with only one inoculation of. The dotted line in Fig. 4(A) shows the positive reference value (45 times) of the virus neutralizing test of the foot-and-mouth disease section of the OIE land animal manual, and the dotted line in Fig. 4(B) is the positive reference value PI value of the ELISA It shows 50.

[Test Example 4] Neutralizing antibody against field virus in other regions of pig immune serum was tested

The effectiveness of the L5B6S9-O virus vaccine strain according to the present invention by measuring the neutralizing antibody titer against various regional-type foot-and-mouth disease viruses in the immune serum obtained in Test Example 3, that is, pig immune serum for each of O, 2, 4, and 6 weeks. Was evaluated. In order to verify the effectiveness of the test vaccine of the present invention, O1 Manisa/Turkey/69 virus (ME-SA; LFBK) belonging to three regional types (ME-SA, SEA, Cathay) of O serotypes representative in Asia. Acquired after passage 4 times in cells), O/VIT/2013 virus (O/ME-SA/PanAsia; passage 5 times in ZZ-R cells (goat fetal tongue cells), then passage 6 times in LFBK cells Obtained after), O/Andong/SKR/2010 virus (O/SEA/Mya-98; obtained after 4 passages in LFBK cells) and O/Taiwan/97 virus (O Cathay; 4 passages in LFBK adherent cells) (Acquired after cultivation) was performed.

As a result, as shown in Figure 5, the immune serum obtained from 4 weeks of vaccination of pigs among the immune serum obtained in Test Example 4 is O1 Manisa/Turkey/69 virus, O/VIT/2013 virus, O/Andong/SKR /2010 virus and O/Taiwan/97 virus both showed more than 100 times the neutralizing antibody titer. The dotted line in FIG. 5 shows the positive reference value (45 times) of the virus neutralizing test of the foot-and-mouth disease section of the OIE terrestrial animal manual as in Test Example 3. From these results, it was confirmed that the vaccine strain of the present invention has a wide range of defense against allogeneic or heterogeneous regional foot-and-mouth disease virus, and thus has very high utility value as a vaccine strain.

[Test Example 5] Defense test against field-type viruses in other regions of foot-and-mouth disease virus vaccine strain (experimental animal: mouse)

7-week-old C57BL6 mice were tested with 100 μl of the O vaccine (1 μg), a mixture of 1 μg of the antigen produced by inoculating the L5B6S9-O virus of the present invention and Montanide ISA 201, and a test vaccine (O O/VIT/2013 foot-and-mouth disease virus 200 LD adapted to mice of the O/ME-SA/PanAsia genotype 10 days after vaccination of 100 μl of vaccine(0.25㎍), O vaccine(0.064㎍), O vaccine(0.016㎍)) ₅₀ were challenged and the percent survival was observed for 7 days.

As a result, as shown in Fig. 6 (A), the test vaccine containing 1 μg antigen per mouse (O vaccine (1 μg)) showed a survival rate of 100%, and the test containing 0.016 μg antigen per mouse Even the vaccine (64-fold dilution; O vaccine (0.016 μg)) was shown to maintain a survival rate of 80%.

Next, 7-week-old C57BL6 mice were inoculated with the test vaccine O vaccine (1µg) and the test vaccine O vaccine diluted 10 times (0.1µg), and 3 weeks later adapted to mice of the O/ME-SA/PanAsia genotype. The O/VIT/2013 foot-and-mouth disease virus 200 LD ₅₀ was challenged and the survival rate was observed for 7 days.

As a result, as shown in Fig. 6 (B), in the case of challenge vaccination (O/VIT/2013 foot-and-mouth disease virus 200 LD ₅₀ ) 3 weeks after vaccination in which antibody formation was complete, 1 μg and 0.1 μg of antigen per mouse It was confirmed that 100% survival rate was shown in all of the test vaccines including.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Suspension Cell Culture Adapted Vaccine Strain Derived from Foot-and-mouth disease virus of O/ME-SA/Ind-2001 Lineage and the Viral Vaccine Composition Containing the Same <130> TKPA210511-KR-D1 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 8193 <212> DNA <213> Artificial Sequence <220> <223> O/Boeun/SKR/2017 <400> 1 ttgaaagggg gcgttagggt ttcaccctag cacaccaccg gcaactcctg cgttgcactc 60 cacacttacg cccgtgtact cgcgggaacc gatggacttt cgtccaccca cctacagctg 120 gactcatggc accgcgaggc catcttagct ggattgtgcg gatgaacacc gcttgcgcac 180 ctcgcgtgac cggttaatac tcttaccact ttccgcctac ttggtcatta gcgctgtcct 240 gggcactcct gttgggggcc gttcgacgct ccacggtctc ccccgcgtaa cggactacgg 300 tgatggggcc gcttcgtgcg ggttggtcgc ttggtctgct tcggttgttg ctcgaagccc 360 acctttcacc cccccccccc ccctaagttt tccgtcgtcc ccgacgttaa aggggtgtaa 420 ccacaagctt ggaaccgtct tgcccgacgt taatgggttg taaccacgcg cttgtaccgc 480 ctttcccggc gttatcggga tgtaaccaaa agatgaacct tcacccggaa 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1380 gcacgagggt gggccacccg ctctcgtcat ttggaacatc aagcatttgc tccacaccgg 1440 aattggcact gcctcgcgac ccagcgaggt gtgcatggtc gatggcacgg acatgtgttt 1500 ggctgacttc cacgctggca tcttcctgaa agggcaagag cacgctgtgt tcgcctgcgt 1560 cacctccaac gggtggtacg cgatcgacga cgaggacttc tacccctgga cgcctgatcc 1620 gtccgacgtt ctggtgtttg tcccgtacga tcaagaacca ctcaacggag agtggaagac 1680 aaaggttcaa aagcgactca aaggagccgg gcaatccagc ccggcaactg ggtcgcagaa 1740 ccagtcaggc aacactggaa gtattatcaa caactactac atgcagcagt accagaactc 1800 tatggacaca caacttggag acaatgccac cagcggaggt tccaatgagg ggtccacaga 1860 caccacttcc acccacacaa ccaacacaca aaacaatgat tggttctcaa aactggccag 1920 ttctgctttc agcggtctgt ttggcgctct tctcgccgac aagaaaaccg aggagaccac 1980 tctcctcgag gaccgcatcc tcactacccg caacggacac acgacctcga caacccagtc 2040 gagcgtcgga gttacttacg ggtacgcaac agctgaggat tttgtgagcg ggccaaacac 2100 atctggtctc gagaccaggg ttgtgcaggc agaacggttc ttcaaaaccc atctgttcga 2160 ctgggtcacc agtgatccat tcgggcggtg ccacctgcta gaacttccaa ctgaccacaa 2220 aggggtctac ggcggcctga ctgactctta tgcttacatg agaaacggtt gggacgttga 2280 ggtcactgca gtgggaaacc agttcaacgg aggttgttta ctggtggcca tggtgccaga 2340 actttgctcc atccggagga gagagctgta ccaacttacg ctcttccctc atcagttcat 2400 caaccctcgc acgaacatga cggcgcacat cactgtgccc tttgttggcg tcaaccgcta 2460 cgaccaatac aaagtgcaca agccttggac ccttgtggtc atggtcgtgg cccctttgac 2520 tgtcaacaac gaaagtgccc cacagatcaa ggtttacgcc aacatcgccc ctaccaacgt 2580 ccacgttgcg ggtgagttcc cttccaaaga agggattttc cccgtggcgt gcagcgacgg 2640 ttacggcggt ctggtgacca ctgacccgaa aacggctgac cccgcctacg ggaaagtgtt 2700 taacccccct cgcaacatgt tgcccgggcg gttcaccaac ttccttgatg tggctgaggc 2760 gtgtcctacg tttctgcact ttgaaggtga cgtaccgtac gtgaccacga agacagattc 2820 ggacagggtg ctcgctcagt ttgacctgtc tttggcagca aaacacatgt caaacacctt 2880 cctggcaggt ctcgcccagt actacacaca gtacagcggc accatcaacc tgcacttcat 2940 gttcacaggc cccactgacg cgaaagcgcg ttacatgatt gcatatgccc cgcctggcat 3000 ggagccgcct aaaacacctg aggcggctgc tcactgcatt cacgcagagt gggacacagg 3060 gctaaactca aagttcacat tttcaatccc ttacctttcg gcggctgact acgcgtacac 3120 cgcgtctgac gctgccgaaa ccacaaatgt gcagggatgg gtgtgcttgt tccaaataac 3180 acacgggaaa gccgacggtg acgcactggt cattctggct agcgccggta aggactttga 3240 gttgcgtttg ccggttgatg cccgcacaca gaccacctcc acaggtgagt ccgctgatcc 3300 cgtgaccacc accgttgaga actacggtgg agagacacag gtccagagac gtcaacacac 3360 cgacgtttct ttcattttgg acagatttgt gaaagtaaca ccgaaagacc aaatcaatgt 3420 gttggacctg atgcaaaccc ccgctcacac tttggtaggc gcactcctcc gcaccgccac 3480 ttactacttc gcagacctag aagtggcagt gaaacacgag ggcaacctca cctgggtccc 3540 gaacggggcg cccgaggcgg cgctggacaa caccaccaac ccgacggcct accacaaggc 3600 accgctcacc cgtcttgctc tgccttacac agcaccacac cgtgttctgg ctaccgttta 3660 caacgggaac tgcaagtatg gcaagggcgc tgtgaccaac gtgaggggtg acttgcaagt 3720 ggtggctcag aaggcagcaa gaacgctgcc cacctccttt aactacggtg ccatcaaggc 3780 tacccgggtg actgaactgc tttaccgtat gaagagggcc gaaacatact gccctcggcc 3840 tctgctggcc attcacccgg aacaagccag acacaagcag aagattgtgg cacctgtgaa 3900 acagttgttg aatttcgacc tgctcaagtt ggcgggagac gttgagtcca accctgggcc 3960 cttcttcttc tccgacgtca ggtcaaactt ttccaaactt gtggaaatca tcaaccagat 4020 gcaggaagac atgtcaacaa aacacggacc cgactttaac cggttggtgt ccgcgtttga 4080 ggaattggcc actggagtga aagccatcag gaccggtctc gatgaggcca agccctggta 4140 caagctcatc aaactcctaa gccgcctgtc gtgcatggcc gctgtagcag cacggtcaaa 4200 ggacccagtc cttgtggcca tcatgctggc tgacaccggt ctcgagattc tggacagcac 4260 atttgtcgtg aagaagatct ccgactcact ctccagtctc tttcacgtgc cggcccccgc 4320 cttcagtttc ggagccccga ttctgttggc cgggttggtc aaagtcgcct cgagtttctt 4380 ccggtccacg cccgaagacc ttgagagagc agagaaacag ctcaaagcac gtgacatcaa 4440 tgacattttc gccattctta agaacggtga gtggctggtc aagttgatcc ttgctatccg 4500 cgactggatt aaagcatgga tcgcctcaga agagaagttc gtcaccatga cagacttggt 4560 gcctggcatc cttgaaaagc agcgggacct taacgacccg agcaagtaca aggaagccaa 4620 ggagtggctc gacaacgcgc gccaagcgtg tttgaagagt gggaacgtcc acattgcaaa 4680 cctctgcaaa gtgatcgccc cggcgcccag caagtcgaga cccgaacctg tagtcgtttg 4740 cctccgcggc aagtccggcc agggcaagag tttccttgcg aacgtgctcg cacaagcaat 4800 ctccactcac ttcaccggca gaaccgattc agtctggtac tgtccgcctg accccgacca 4860 cttcgacggt tataaccaac agaccgttgt cgtgatggat gatttgggcc agaaccctga 4920 cggcaaggac ttcaagtact ttgcacagat ggtctccacc acagggttca tcccgcccat 4980 ggcctcactc gaggacaaag gcaagccttt taacagtaag gtcatcatag ccaccaccaa 5040 cctgtactcg gggttcaccc cgagaacaat ggtgtgccct gatgcactga accgcaggtt 5100 tcactttgac attgatgtga gcgccaagga tgggtacaaa atcaacaaca aattggacat 5160 aaccaaagct cttgaagaca cccacaccaa cccagtggca atgtttcaat atgactgtgc 5220 ccttctcaac ggcatggccg ttgaaatgaa gagaatgcaa caagacatgt tcaagcctca 5280 accacccctc cagaacgtct accaacttgt tcaggaggtg attgaacggg tcgagctcca 5340 cgagaaagtg tcgagccacc caatcttcaa gcagatctca attccttccc aaaaatctgt 5400 gttgtacttc ctcattgaga aaggtcagca cgaagcagca attgaattct ttgaggggat 5460 ggtccacgac tccatcaagg aggagctccg acccctcatc caacagacat catttgtgaa 5520 acgcgctttc aagcgcctga aggaaaactt tgagattgtt gccctgtgtt tgactctcct 5580 ggcaaacata gtgatcatga tccgcgagac tcgcaagaga cagcagatgg tggatgatgc 5640 agtgaatgag tacattgaga aagcaaacat caccacggat gacaagactc ttgacgaggc 5700 ggaaaagaac cctctggaga tcagcggtgc cagcaccgtc ggtttcagag agagaactct 5760 cccaggacac aaggcgagtg gtgacgtgaa ctccgaaccc gccagacctg tggagggaca 5820 accacaagcc gaaggaccct acaccgggcc acttgagcgc cagaaacctc tgaaagtgcg 5880 cgccaaactg ccacaacaag aggggcctta cgctggtccg atggagagac agaaaccact 5940 gaaagtgaaa gcaaaagccc cggtcgttaa ggaaggacct tacgagggac cggtgaagaa 6000 gcctgtcgct ttgaaagtga aagcaaggaa cttgattgtc actgagagtg gtgccccacc 6060 gaccgacttg caaaagatgg ttatgggcaa cacaaagcct gttgagctca tcctcgacgg 6120 gaagacagtg gccatctgct gtgctactgg agtgtttggc actgcttacc tcgtgcctcg 6180 tcatcttttc gcagagaagt atgacaagat catgctggac ggcagagcca tgacagacag 6240 tgactacaga gtgtttgagt ttgagattaa agtaaaagga caggacatgc tctcagacgc 6300 cgcgctcatg gtgctccacc gtgggaatcg cgtgcgggac atcacgaagc acttccgtga 6360 tgttgcgagg atgaagaaag gcacccccgt cgttggcgtg atcaacaacg ctgatgttgg 6420 gagactgatt ttctctggtg aggcccttac ctacaaggac attgtagtgt gcatggatgg 6480 cgacaccatg cctggcctct ttgcctacaa ggccgccacc aaggctggtt actgtggagg 6540 agccgttctc gcaaaggacg gagctgagac tttcatcgta ggcacccact ctgcaggagg 6600 caatggagtt ggttactgct catgcgtttc caggtccatg cttctcaaga tgaaggcaca 6660 catcgaccct gaaccacacc acgaggggtt gattgttgac accagagatg tggaagaacg 6720 cgtccacgtg atgcgcaaaa ctaagcttgc acccaccgtt gcgcacggtg tgttcaaccc 6780 tgactttggc cccgctgtct tgtccaataa ggatccgcgg ttgaacgaag gtgttgtgct 6840 tgatgaagtc atcttctcca aacacaaagg ggacaccaag atgacagaag aagacaagaa 6900 gctgttccgg cgctgtgctg ctgactacgc gtcacgcctg cactccgtgc tgggtttggc 6960 aaatgcccca ttgagtatct acgaggcaat taagggcgtt gacgggctcg acgccatgga 7020 accagacact gcacctggcc tcccctgggc tctccaggga aagcgccgtg gtgccctcat 7080 tgattttgag aacggcacag tcggacccga agttgaggct gccttggaac tcatggagaa 7140 acgagagtac aagtttgttt gccagacctt cctgaaggac gaaattcgcc cgatggagaa 7200 agtacgtgcc ggcaagactc gcattgtcga tgttttgcct gttgaacaca ttctttacac 7260 cagaatgatg attggtagat tttgcgctca aatgcactca aacaacggac cgcaaattgg 7320 ctcggcggtc ggttgcaacc ctgatgttga ttggcaaaga tttggcaccc actttgccca 7380 gtacagaaat gtgtgggatg tggactattc ggcctttgat gctaaccact gcagtgacgc 7440 aatgaacatc atgtttgagg aggtgttccg cacggagttt ggtttccacc ccaacgcgga 7500 gtggattctg aagactctcg tgaacacgga gcacgcctat gagaacaaac gcatcactgt 7560 tgagggcggg atgccgtctg gctgttccgc gacaagcatt atcaacacaa ttttgaacaa 7620 catctacgtg ctctacgctt tgcgtagaca ctatgaggga gttgagctgg atacttacac 7680 catgatctcc tacggagacg acatcgtggt ggcaagtgat tatgatctgg actttgaggc 7740 tctaaagccc catttcaaat ctttgggcca aaccatcact ccagcagaca aaagtgacaa 7800 gggcttcgtc cttggacact ccataactga tgtcaccttc ctcaaaagac acttccacat 7860 ggattacgga actgggtttt ataaacctgt gatggcttcg aagactctcg aagctatcct 7920 ctcctttgca cgccgtggga ccatacagga gaagttgatc tccgtggcag gactcgcagt 7980 ccactctgga cctgatgagt accggcgtct cttcgagccc tttcagggtc tctttgagat 8040 tccaagctac agatcacttt acctgcgttg ggtgaacgcc gtgtgcggtg acgcgtaatc 8100 cctcagagaa cacattggca gaaagactct gaggcgagcg acaccgtagg agtgaaaagt 8160 ccgcaagggc ttttcccgct tcctattcca aaa 8193

Claims (6)

A method for preparing a foot-and-mouth disease virus vaccine strain, comprising the following steps:
(a) inoculating LFBK adherent cells with a wild-type foot-and-mouth disease virus derived from O serotype and subculturing at least five times to obtain a virus culture containing the cell-adapted virus;
(b) inoculating the virus culture containing the cell-adapted virus obtained in step (a) into the BHK-21 adherent cells and subculturing at least four times to obtain a virus culture containing the cell-adapted virus. ;
(c) obtaining a virus culture containing the cell-adapted virus by inoculating the virus culture containing the cell-adapted virus obtained in step (b) into the BHK-21 floating cells and subculturing at least six times. ;
(d) selecting and isolating candidate vaccine strain viruses by evaluating at least one of virus productivity and antigen productivity [[E]] from the virus culture containing the cell-adapted virus obtained in step (c); And
(e) selecting the final vaccine strain virus by checking whether at least one of the heparan sulfate receptor and the JMJD6 receptor is recognized for the selected and isolated candidate vaccine strain virus. The method of claim 1, wherein the wild-type foot-and-mouth disease virus in step (a) is an O/ME-SA/Ind-2001 genotype foot-and-mouth disease virus. The method of claim 1, wherein the step (d) undergoes a plaque purification step prior to evaluating at least one of the virus productivity and antigen productivity. The method of claim 3, wherein the plaque purification is performed using BHK-21 adherent cells. The method of claim 1, wherein in step (e), the recognition of the heparan sulfate receptor of the candidate vaccine strain virus is determined by measuring the viral titer in CHO-K1 cells, and the recognition of the JMJD6 receptor of the candidate vaccine strain virus is determined by CHO- Method for producing a foot-and-mouth disease virus vaccine line, which is determined by measuring viral titer in 677 cells. The method of claim 5, wherein when the viral titer measured in the CHO-K1 cells is 1×10 ^2.0 TCID ₅₀ /mL or more, it is determined to recognize the heparan sulfate receptor,
If the virus titer measured in the CHO-677 cells is 1x10 ^2.0 TCID ₅₀ /mL or more, it is determined to recognize the JMJD6 receptor, a method for producing a foot-and-mouth disease virus vaccine line.

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