KR102365039B1 - Suspension Cell Culture Adapted Vaccine Strain Derived from Foot-and-mouth disease virus of A/ASIA/Sea-97 Lineage and Method of Preparing the Same - Google Patents

Abstract

The present invention relates to an A/ASIA/Sea-97 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 particularly, to A/ASIA/Sea-97 genotype It relates to a foot-and-mouth disease virus vaccine strain established by alternating continuous passage of foot-and-mouth disease virus in adherent cells and floating cells to establish cell adaptability, a vaccine composition comprising an inactivated virus vaccine strain thereof as an active ingredient, and a method for producing the same.

Description

A/ASIA/Sea-97 genotype foot-and-mouth disease virus vaccine strain adapted to suspended cell culture and method for preparing the same the Same}

The present invention provides a method for producing a foot-and-mouth disease virus vaccine strain, characterized in that cell adaptability is obtained by sequentially alternating successive subculture of serotype A-derived wild-type foot-and-mouth disease virus in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells. is about

Specifically, the preparation method of the present invention comprises the following steps:

(a) inoculating LFBK-adherent cells with a serotype A-derived wild-type foot-and-mouth disease virus, followed by subculture at least 5 times to obtain a virus culture containing the cell-adapted virus;

(b) inoculating the BHK-21 adherent cells with the virus culture containing the cell-adapted virus obtained in step (a) above, followed by serial passage 4 or more 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 floating cells of BHK-21 and then subcultured continuously for 6 or more times ;

(d) selecting and isolating a candidate vaccine strain virus by evaluating at least one of 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 confirming whether at least one of a heparan sulfate receptor and a JMJD6 receptor is recognized for the selected and isolated candidate vaccine strain virus.

Also, the present invention relates to an A/ASIA/Sea-97 genotype foot-and-mouth disease virus vaccine strain adapted to floating cell culture and a foot-and-mouth disease virus vaccine composition comprising the same, and more particularly, to an A/ASIA/Sea-97 genotype foot-and-mouth disease virus vaccine composition comprising the same. It relates to a vaccine composition comprising, as an active ingredient, a foot-and-mouth disease virus vaccine strain established by alternating successive subcultures in cells and floating cells, and an inactivated virus vaccine strain thereof.

Foot-and-mouth disease (FMD) is a viral vesicular disease that infects animals with split hoofs. It is characterized by rapid replication and rapid spread. Due to its economic importance, this disease is classified as an important disease by the World Organization for Animal Health (OIE), and acts as a very important factor in international trade of livestock products. Foot-and-mouth disease virus (FMDV) is a single-stranded bipolar RNA virus belonging to the genus Picoraviridae and Aphthovirus , and has seven different serotypes (serotypes: A, O, C, Asia-1, SAT-1, SAT-2, SAT-3). In order to suppress the spread of foot-and-mouth disease, the only method used is the use of an inactivated vaccine other than the killing of animals. However, foot-and-mouth disease vaccine does not provide protection against other serotypes, and appropriate vaccines must be used according to topotype and lineage.

In the case of foot-and-mouth disease virus A serotype, the antigenicity of each virus is very diverse compared to other serotypes (Non-Patent Document 1), and therefore, if the regional type or genotype is different, the defense is often not good. Recently, the spread of the A/ASIA/Sea-97 genotype in neighboring countries such as China, Vietnam, Mongolia, and Thailand is the most prominent trend. In particular, in Korea, a G1 sublineage virus of the A/ASIA/Sea-97 genotype occurred in Pocheon, Gyeonggi-do in 2010, and a G2 sublineage virus of the A/ASIA/Sea-97 genotype was found in Gyeonggi-do in 2017. It occurred in Yeoncheon. In 2018, the A/ASIA/Sea-97 genotype occurred in Gimpo, Gyeonggi-do, proving that the genotype is a virus with a very high probability of occurrence in Korea. In addition, as of 2019, outbreaks in neighboring countries are continuously being reported, so the risk of inflow from abroad is very high. Accordingly, the need for the development of A/ASIA/Sea-97 genotype vaccine strains is also increasing.

In the foot-and-mouth disease vaccine, it is common to inactivate the foot-and-mouth disease virus by mass culturing, 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. FMD virus isolated in the field (wild-type foot-and-mouth disease virus) uses integrin receptors to infect cells and animals, but BHK-21 floating cells show 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 low (Non-Patent Document 2). Therefore, in order to use it for mass vaccine production of foot-and-mouth disease virus, it is not possible to use integrin receptors through complete cellular adaptation, but instead of floating cells (or other host cells) such as heparan sulfate or Jumonji domain-containing protein 6 (JMJD6). A virus (or vaccine strain) that recognizes the receptor must be secured, and the proliferation and antigen productivity of the foot-and-mouth disease virus in floating cells must be confirmed. In addition, the vaccine containing the corresponding 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, the serotypes and genotypes of foreign epidemic strains isolated from epithelial tissues such as cattle and pigs, particularly, foot-and-mouth disease virus prevalent in Asia have been identified (Non-Patent Document 3), and foot-and-mouth disease virus Although the use of an antigen containing an amino acid of a specific structural protein derived from , for vaccine manufacture (Patent Document 1), or a recombinant virus in which a specific gene of foot-and-mouth disease virus is deleted is disclosed for vaccine manufacture (Patent Document 1) 2), it is very difficult to develop a vaccine to effectively protect against infection against all serotypes identified so far because foot-and-mouth disease viruses do not cross-protect each other depending on the host animal and each serotype (Non-Patent Document 4) -5).

The present inventors have developed A/ASIA, one of the most frequently occurring viruses in Asia, including Korea, for the past 5 years in order to generate effective protective antibodies suitable for the serotype/regional type/genotype of the foot-and-mouth disease virus epidemics that are currently prevalent in Korea. By using the A/Yeoncheon/SKR/2017 foot-and-mouth disease virus, which is the /Sea-97 genotype, we have mass-produced a foot-and-mouth disease vaccine strain, and we have been earnestly trying to develop a preventive vaccine with high defense against the foot-and-mouth disease virus. As a result, the A/ASIA/Sea-97 genotype foot-and-mouth disease virus finally selected through a number of screening processes was transformed into LFBK adherent cells (adherent porcine kidney cells), BHK-21 adherent cells (adherent baby hamster kidney-21 cells) and BHK- 21 Suspension baby hamster kidney-21 cells were sequentially inoculated and subcultured alternately at a specific number of passages to obtain an FMD vaccine with an optimized viral titer. Next, 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 titer in the inoculated animal's serum/blood remarkably increased and maintained for a long period of time, resulting in a protective effect against the foot-and-mouth disease virus. The excellence was confirmed and the present invention was completed.

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

Pereira Hg., Dev Biol Stand., 35:167-74, 1976 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 to provide an effective novel foot-and-mouth disease vaccine strain against the wild-type/outdoor foot-and-mouth disease virus that is currently prevalent in Korea, more specifically, the A/ASIA/Sea-97 genotype foot-and-mouth disease virus, which is difficult to effectively protect with the existing vaccine lines. is to provide

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 strain of the present invention.

In order to achieve the above object, the present invention provides cellular adaptability by sequentially subculturing a wild-type foot-and-mouth disease virus, A/ASIA/Sea-97 genotype foot-and-mouth disease, in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells. The obtained foot-and-mouth disease vaccine strain is provided.

The present invention also provides a vaccine composition for preventing infection with 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 line according to the present invention can produce a large amount of foot-and-mouth disease virus antigen, so it can be used industrially for the production of foot-and-mouth disease vaccine, and the foot-and-mouth disease virus of serotype A that is prevalent in Asia, especially the A/ASIA/Sea-97 genotype It can be usefully used in the production of vaccines for protection against foot-and-mouth disease virus.

1 is a wild-type foot-and-mouth disease virus A/ASIA/Sea-97 genotype foot-and-mouth disease virus A/Yeoncheon/SKR/2017 foot-and-mouth disease virus inoculated, followed by sequential inoculation of LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells It is a graph showing the cell adaptability in floating cells of foot-and-mouth disease virus by infecting BHK-21 adherent cells or CHO-K1 adherent cells and measuring the virus titer.
2 is a graph showing the proliferation (A) and antigen productivity (B) of the foot-and-mouth disease virus vaccine strain (L5B6S9-A virus) according to the present invention.
3 shows a photograph confirming the inactivated antigen produced with the foot-and-mouth disease virus vaccine strain (L5B6S9-A virus) according to the present invention with a transmission electron microscope (TEM).
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 strain (L5B6S9-A virus) according to the present invention.
5 shows the same regional virus (FIG. 5A) and a different regional virus (FIG. 5B) for the sera of pigs vaccinated with the test vaccine containing the antigen produced with the foot-and-mouth disease virus vaccine strain (L5B6S9-A virus) according to the present invention. The results of the neutralization test with
6 is a graph showing the results of testing in mice whether or not the test vaccine containing the antigen produced with the foot-and-mouth disease virus vaccine strain (L5B6S9-A virus) according to the present invention protects against another field/wild-type foot-and-mouth disease virus (heterologous FMDV) attack; is shown as

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

As used herein, 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 numerical value or numerical range .

In one aspect, the present invention provides a novel foot-and-mouth disease vaccine strain, more specifically, A/Yeoncheon/SKR/2017 foot-and-mouth disease virus, which is a wild-type/outdoor-type foot-and-mouth disease virus, A/Yeoncheon/SKR/2017 foot-and-mouth disease virus, alternating continuous passage between cells ( The present invention relates to a foot-and-mouth disease vaccine strain obtained by alternately and continuously passage cultivation, followed by selection and isolation, wherein the cell-to-cell alternating serial passage culture results in adhesion of LFBK (bovine calf kidney cell) adherent cells and BHK-21 (baby hamster kidney cell) cells. Cells and BHK-21 (baby hamster kidney cell) may be characterized in that the sequential subculture in floating cells, but is not necessarily limited to the above order.

Specifically, the foot-and-mouth disease virus vaccine line according to the present invention is not limited to the passage order and/or the number of passages, and is completely A virus that is adapted to cells and recognizes a heparan sulfate receptor (HSR) includes all viruses that recognize HSR with 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 line according to the present invention is not limited to the passage order and/or the number of passages, and is completely Viruses that are cellularly adapted and recognize the Jumonji domain-containing protein 6 receptor (JMJD6R), include all viruses that recognize JMJD6R with normal function and activity, and exhibit a targeted viral titer in a specific cell. The specific cell may be a CHO-677 cell.

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

The foot-and-mouth disease virus vaccine line according to the present invention was obtained by sequentially passing the A/ASIA/Sea-97 genotype foot-and-mouth disease virus, a wild-type foot-and-mouth disease virus, in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells. As such, it is possible to acquire suitable cell adaptability, strictly speaking, complete cell adaptability. Here, the term 'acquire suitable cellular adaptability as a vaccine strain' or 'acquire complete cellular adaptability' means that wild-type virus is sequentially passaged from LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells to BHK -21 It means that adaptation to floating cells is complete. In other words, the term means that the foot-and-mouth disease vaccine strain virus of the present invention is produced in 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, recognize receptors other than integrin receptors, such as heparan sulfate receptors, JMJD6 receptors, and can survive or proliferate normally and exhibit a predetermined viral titer. it means.

Since the foot-and-mouth disease virus vaccine according to the present invention recognizes the heparan sulfate 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 or more in CHO-K1 adherent cells A virus titer greater than /mL may be exhibited.

Since the foot-and-mouth disease virus vaccine 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 or more in CHO-677 adherent cells It may show more than the virus titer.

The specific region virus of the present invention is a virus obtained through selection and isolation from a virus culture obtained through continuous subculture in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells, and adheres to BHK-21. 1x10 ^5.5 TCID ₅₀ /mL or more in cells, preferably 1x10 ^6.0 TCID ₅₀ /mL or more, most preferably 1x10 ^7.0 TCID ₅₀ /mL or more, and 1x10 ^2.0 TCID ₅₀ /mL or more in CHO-K1 adherent cells , preferably exhibits a viral titer of 1x10 ^2.5 TCID ₅₀ /mL or more, and may exhibit a viral titer of 1x10 ^2.0 TCID ₅₀ /mL or more, preferably 1x10 ^2.5 TCID ₅₀ /mL or more, in CHO-677 adherent cells.

The foot-and-mouth disease virus vaccine line according to the present invention is a virus that has acquired complete cell adaptability through alternating successive subculture in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells, and is a nucleic acid (nucleotide) represented by SEQ ID NO: 1 It may be characterized as having a nucleic acid nucleotide sequence having a nucleotide sequence or substantially homologous thereto, and a polypeptide generated from the nucleic acid nucleotide sequence. For nucleic acids and polypeptides of the above foot-and-mouth disease vaccine strains, the term "substantial homology" means that when the two nucleic acids and the two polypeptides or their designated sequences are optimally aligned and compared, at least about 80% of the nucleotides and amino acids, usually at least about 85% of nucleotides and amino acids, preferably about 90%, 91%, 92%, 93%, 94% or 95%, more preferably at least about 96%, 97%, 98%, 99% , 99.1%, 99.2%, 99.3%, 99.4% or 99.5%. The present invention includes nucleic acid sequences and polypeptide sequences having substantial homology to specific nucleic acid sequences and amino acid sequences mentioned 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 A/ASIA/Sea-97 genotype. It may be desirable to use the G2 sublineage virus among the A/ASIA/Sea-97 genotypes. As the G2 sublineage foot-and-mouth disease virus, for example, with the accession number KVCC-VR1700006 to KVCC (Korea Veterinary Culture Collection, Korea Veterinary Gene Resources Bank [http://kvcc.kahis.go.kr]) of the Agriculture, Forestry and Livestock Quarantine Headquarters. Deposited foot-and-mouth disease virus may be used.

In another aspect, the present invention relates to a vaccine composition for preventing infection with 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 line by a known method.

As a known inactivation treatment method, for example, treatment with BEI (Binary Ethylenimine) used for virus inactivation, formalin, glutaraldehyde, heating, irradiation, BPL or other inactivating agent (inactivating agent) known in the art ) can be used to inactivate (inactivate).

The inactivation reaction of the inactivated foot-and-mouth disease virus vaccine of the present invention can be preferably carried out at 26-37 ℃ for 48 hours, and most preferably, after primary inactivation at a temperature of 37 ℃ for 24 hours, at 26 ℃ This can be accomplished by secondary inactivation at temperature for 24 hours.

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

As used herein, the term "carrier" refers to any solvent (solution), dispersion medium, coating, buffer, isotonic agent, suspension, colloid, that is pharmaceutically acceptable for administration to a subject animal (host animal). , stabilizers, preservatives, antibiotics (antibacterial agents, antifungal agents, etc.), adsorption delaying agents, adjuvants (adjuvant), inserts, and the like. The use of one or more delivery vehicles for chemical compounds in general, particularly immunogens, is well known to those skilled in the pharmaceutical arts. Insofar as any conventional medium or agent is incompatible 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 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 includes albumin 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); pluronic polyols; polyanions (polyanions); non-ionic block polymers such as Pluron F-127 (BASF, USA); peptides; Mineral oils such as Montanide ISA-206 (Seppic, France), Montanide ISA-50 (Seppic, France), Cabopol, Amphigen, Amphigen Mark II (Hydronics® Hydronic), USA), alhydrogels, oil emulsions (eg, emulsions of mineral oil and water such as BayolF/Arlacel A), or emulsions of vegetable oils, 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® adjuvants; 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, but is not limited thereto, and is 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 intended to be administered to a vertebrate, preferably an Artiodactyla animal, such as a mammal such as cattle, pigs, goats, sheep, deer, camels, giraffes, etc. 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 strain, which is 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 strain refers to the ability of the foot-and-mouth disease virus to induce an immune response in the target animal (host animal) against different serotypes of the foot-and-mouth disease virus, particularly the foot-and-mouth disease virus of serotype A ( action as an antigen). The immune response may be a cellular immune response mediated primarily by cytotoxic T-cells, or a humoral immune response mediated primarily by helper T-cells that activate B-cells to induce antibody production.

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

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, cytokines, and/or cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses in the cells and tissues of the inoculated target animal (host animal) It is detected by the presence or absence of activation, but can be easily detected using a conventional assay known to those skilled in the art of immunology.

The mixing ratio of the inactivated virus and the adjuvant included in the vaccine composition is, on a volume basis, 1-10: 1-10, preferably 1-5: 1-5, most preferably 1:1. there is.

Antibiotics included in the vaccine composition may include, but are not limited to, gentamicin and methyl oleate (Merthiolate).

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

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

The vaccine composition is preferably administered to a target animal by an intramuscular or subcutaneous route, but other routes of administration, for example, oral administration, nasal administration (eg, aerosol or other needle-free 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 a combination of these administration routes. can be A preferred route of administration is at least one selected from the group consisting of muscle, subcutaneous, intraperitoneal, intravenous, oral, dermal, eye and brain, more preferred route of administration is subcutaneous administration or intramuscular administration, and the most preferred route of administration is It is administered intramuscularly in the neck area.

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

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

In another aspect, the present invention is a complete cell adaptation through serial passage in LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells, heparan sulfate receptor (HSR) and / or JMJD6 receptor, more precisely relates to a method for producing a foot-and-mouth disease virus vaccine strain that recognizes HSR and/or JMJD6 receptors having normal function and activity.

The serial passage may be performed in any order and/or number as long as a foot-and-mouth disease virus vaccine strain that recognizes HSR having normal activity and exhibits a target virus titer in a specific cell is obtained, but LFBK adherent cells, BHK- It is preferable to proceed in the order of 21 adherent cells and BHK-21 floating cells. It is more preferable that the serial passage culture be 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 floating cells.

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

(a) LFBK adherent cells are inoculated with a wild-type foot-and-mouth disease virus, preferably A/ASIA/Sea-97 genotype foot-and-mouth disease virus, more preferably A/Yeoncheon/SKR/2017 foot-and-mouth disease virus, followed by serial passage 5 or more times. obtaining a virus culture comprising the adapted virus;

(b) After inoculating the BHK-21 adherent cells with the virus culture containing the cell-adapted virus obtained in step (a), serially passaged 4 or more times, preferably 4 to 6 times, to obtain the cell-adapted virus obtaining a virus culture comprising;

(c) After inoculating the BHK-21 floating cells with the virus culture containing the cell-adapted virus obtained in step (b), serially passaged 6 or more times, preferably 7 to 9 times, to obtain the cell-adapted virus obtaining a virus culture comprising;

(d) selecting and isolating a candidate vaccine strain virus 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 confirming whether at least one of a heparan sulfate receptor and a JMJD6 receptor is recognized for the selected and isolated candidate vaccine strain virus.

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

In step (e) of the method for producing the foot-and-mouth disease virus vaccine strain according to the present invention, whether the candidate vaccine strain virus recognizes the heparan sulfate receptor can be made by measuring the viral titer in CHO-K1 adherent cells. When the virus inoculated in CHO-K1 adherent cells exhibits a virus 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 heparan sulfate receptor with

In step (e) of the method for producing the foot-and-mouth disease virus vaccine strain according to the present invention, the recognition of the JMJD6 receptor of the candidate vaccine strain virus can be achieved by measuring the viral titer in CHO-677 adherent cells. When the virus inoculated in CHO-677 adherent cells exhibits a virus 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 with

In the method for producing the foot-and-mouth disease virus vaccine strain according to the present invention, after step (e), the virus titer in at least one of BHK-21 cells, CHO-K1, and CHO-677 cells against the finally selected vaccine strain virus Measurement, sequencing of part or all of a viral gene (eg, sequencing to confirm a change in an amino acid constituting a structural protein of a virus or a nucleic acid sequence encoding the same), a test for confirming immunogenicity in a target animal, and xenogeneic At least one selected from the group consisting of tests for confirming defense against viruses may be additionally performed.

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

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

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

Pig kidney cells inoculated with A/Yeoncheon/SKR/2017 foot-and-mouth disease virus, an A/ASIA/Sea-97 genotype foot-and-mouth disease virus belonging to the A serotype foot-and-mouth disease virus isolated from the tongue tissue of a cow diagnosed as positive for foot-and-mouth disease in Yeoncheon, Gyeonggi-do in 2017 LFBK adherent cells (see Swaney LM, Vet. Microbiol ., 18:1-14, 1988, and LaRocco M. et al. , J Clin Microbiol. , 51(6):1714-20, 2013) After proliferating by passage (passaging, subculture) a certain number of times, for adaptation in BHK-21 floating cells, which are hamster kidney cells, first, BHK-21 adherent cells (BHK-21 adherent cells; for cell culture) BHK-21 cells cultured while attached to a vessel [plate]; ATCC ^® CCL-10 ^™ ), then subcultured, and then suspended in a BHK-21 suspension cell (BHK-21 suspension cell; cell culture vessel [plate]) BHK-21 cells cultured in the old state; KCTC 12945BP cell line [refer to Korean Patent No. 10-1812223]), alternating successive subculture was performed.

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

The virus inoculation amount per cell of each of LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells was 0.01 MOI (Multiplicity of infection), and the virus culture was harvested at about 16 hours after inoculation to determine the virus titer. 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℃ conditions and virus At the time of inoculation, the medium was replaced with DMEM containing 2% (v/v) FBS and subcultured under the same conditions. BHK-21 floating cells were 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) containing ProVERO-1 serum-free medium (Lonza, Switzerland) was used to suspend cells under 5% CO ₂ /37°C/110rpm conditions, and during virus inoculation, serum-free CD-BHK without additives The medium was replaced with -21 production media (Thermofisher, USA) and subcultured under the same conditions.

In addition, when converting cell types in the order of LFBK adherent cells, BHK-21 adherent cells, and BHK-21 floating cells during alternating successive subcultures, 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 lysate were removed by passing the culture through a 0.45 μm injection filter (Syringe Filter, Merck).

(2) Selection of vaccine strain viruses that have acquired complete cell adaptability

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

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

Table 1 confirms the viral titer (TCID ₅₀ /ml) of the virus harvested by alternating successive passage of the virus into cells of different combinations for establishment of a vaccine strain of wild-type foot-and-mouth disease in BHK-21 adherent cells and CHO-K1 adherent cells. (Reed: Reed LJ, Muench H. A simple method of estimating fifty percent endpoint. Am J Hyg. 27:493~497, 1938). Before passage of BHK-21 adherent cells, Chloroform was used to eliminate naive virus activity other than foot-and-mouth disease virus.

virus Alternating serial passage culture: cell line (number of passages) Inoculation MOI
(Multiplicity of infection) Virus titer in BHK-21 adherent cells
(TCID ₅₀ /ml) Whether heparan sulfate receptors are recognized by CHO-K1 adherent cells A/Yeoncheon/SKR/2017 LFBK(5), Chloroform treatment NA [not assayed] NA NA LFBK(5), Adherent BHK-21(3) 0.01 1x10 ^6.17 X LFBK(5), Adherent BHK-21(4) 0.01 1x10 ^7.50 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 ^7.50 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 ^7.50 X LFBK(5), Adherent BHK-21(4), Suspension BHK-21(6) 0.01 1x10 ^7.10 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 ^7.50 O ^***

*; **; *** : 1x10 ^2.5O TCID ₅₀ /ml in CHO-K1.

As can be seen from Table 1 above, some of the viruses harvested by performing alternating successive subcultures by varying the combination and passage number of LFBK adherent cells, BHK-21 adherent cells and BHK-21 floating cells are BHK-21 Although it exhibits good viral titer in adherent cells, it has been shown to fail to recognize the heparan sulfate receptor. Viruses that do not recognize the heparan sulfate receptor did not acquire full cellular adaptability, and therefore were excluded from the foot-and-mouth disease virus vaccine line candidates.

In addition, referring to the virus titer data in CHO-K1-adherent cells in the graph shown in FIG. 1, after passage 5 times in LFBK adherent cells (L5), and passage 4 times in BHK-21 adherent cells (B4) ), from the virus (L5B4S6-A virus) that was passaged 6 times in BHK-21 floating cells (S6), it was found that the heparan sulfate receptor was recognized, at least 4 times in BHK-21 adherent cells and at least 4 times in LFBK adherent cells. It was possible to conclude that complete cell adaptability was achieved after 5 and at least 6 consecutive passages in BHK-21 floating cells (where L denotes LFBK adherent cells and B denotes BHK-21 adherent cells). , S indicates floating cells of BHK-21, and numbers indicate the number of passages).

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

The virus culture (including L5B4S7-A virus) obtained by passage 5 times in LFBK cells, 4 times in adherent BHK-21 cells, and 7 times in BHK-21 floating cells was used in BHK-21 adherent cells to obtain a virus with a single characteristic. Viral plaques were obtained using the plaque purification method (see Polatnick J. et al. , Appl Microbiol., 12:368-73, 1964). Specifically, after undergoing the plaque purification process in BHK-21 adherent cells (single culture), and in order to obtain a level of virus capable of measuring titer, the virus passaged one more time in BHK-21 adherent cells was added to 'L5B6S7-A plaques'. called 'virus'.

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

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

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

Virus titer in BHK-21 adherent cells
(TCID ₅₀ /ml) CHO-K1 adherent cells virus titer in
(TCID ₅₀ /ml) L5B6S7-A plaque virus 1x10 ^8.30 1x10 ^2.50

As can be seen from Table 2 above, the L5B6S7-A plaque-derived virus exhibited a good viral titer in CHO-K1 adherent cells, and thus it was determined that the heparan sulfate receptor was normally recognized.
(2) L5B6S9-A Confirmation of cellular adaptability and potency of viruses

In order to transform the L5B6S7-A plaque virus into a seed virus for vaccine production, the virus was produced by passage twice in BHK-21 floating cells, and finally, 'L5B6S9-A' called 'virus'.

The supernatant (L5B6S9-A virus) in BHK-21 adherent cells, CHO-K1 adherent cells, and CHO-677 (pgsD-677) adherent cells by a virus titer assay used for selection of vaccine strain viruses that have acquired cell adaptability. The measurement results are shown in Table 3 below.

Virus titer in BHK-21 adherent cells
(TCID ₅₀ /ml) CHO-K1 adherent cells virus titer in
(TCID ₅₀ /ml) Viral Titers in CHO-677 Adherent Cells
(TCID ₅₀ /ml) L5B6S9-A virus 1x10 ^7.0 1x10 ^2.70 1x10 ^2.50

As shown in Table 3 above, the L5B6S9-A virus showed a high viral titer of 1x10 ^7.0 TCID ₅₀ /ml in BHK-21 adherent cells and 1x10 ^2.7 TCID ₅₀ /ml in CHO-K1 adherent cells. and showed a virus titer of 1x10 ^2.5 TCID ₅₀ /ml in CHO-677 (pgsD-677) adherent cells, confirming that cell adaptation was completed.
(3) L5B6S9-A Confirmation of virus multiplication

The L5B6S9-A vaccine strain was inoculated into 100 ml of BHK-21 floating cell culture medium at an MOI of 0.01 to confirm the proliferation. The culture conditions were the same as those of the suspension culture during the subculture. L5B6S9-A After 8, 12, 16, and 24 hours after the foot-and-mouth disease virus inoculation, the culture supernatant was obtained and titer measurements were performed on BHK-21 adherent cells.

As a result, as shown in FIG. 2 (A), the L5B6S9-A virus exhibited the highest virus titer at the time of harvest 16 hours after inoculation, that is, an average of 1x10 ^7.15 TCID ₅₀ /ml.

(4) L5B6S9-A Confirmation of antigen productivity of virus

The L5B6S9-A virus was inoculated into 100 ml of floating cell culture medium at an MOI of 0.01 and then the virus was cultured for a specific incubation time (ie, 8 hours, 12 hours, 16 hours after inoculation). BEI (binary ethylenimine) was added to the culture to have a final concentration of 3 mM, and then reacted in a shaking incubator at 37° C. for 24 hours (first inactivation step). Then, the virus culture medium that had undergone the first inactivation step was transferred to a new container, treated with BEI at the same concentration as in 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 medium that had undergone the first inactivation step and the second inactivation step so that the final concentration of 1% (wt%) was reached. For virus concentration and purification, it was mixed with 7.5% PEG 8000, stirred at 4° C. overnight, and centrifuged with a centrifugal force of about 10,000 g for about 30 minutes, and then dissolved in Tris-NaCl [pH 7.2] buffer. The antigen obtained through the inactivation, concentration, and purification process as described above was centrifuged at a temperature of 4°C with a centrifugal force of about 110,000 g for about 180 minutes by 15-45% sucrose gradient ultracentrification method, followed by centrifugation at 259 nm The amount of antigen obtained (content of 146S antigen) was compared by quantifying it with a spectrophotometer in 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-A virus had an antigen (146S) at a level exceeding the antigen amount of 2 μg based on 1 ml of culture, which is a general antigen production standard (about 8 μg/ml of antigen produced 12 hours after inoculation). Inactivated foot-and-mouth disease virus particles) productivity was found to be very good.

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

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

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

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

The L5B6S9-A virus was finally determined as a virus vaccine strain for vaccine production, and as a result of nucleic acid sequence sequencing, it was confirmed to have the nucleic acid sequence represented by SEQ ID NO: 1. This virus vaccine was entrusted to the Korea Veterinary Culture Collection (KVCC, Korea Veterinary Resources Bank [http://kvcc.kahis.go.kr]) of the Agriculture, Forestry and Livestock Quarantine Headquarters and was registered with the accession number KVCC-VR1900045.

[Test Example 3] Immunogenicity evaluation in target animals

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

Then, in order to measure the neutralizing antibody titer of the antibody contained in the immune serum obtained from the pig inoculated with the test vaccine, sublineage A/Yeoncheon/SKR/ of foot-and-mouth disease virus serotype A, A/ASIA/Sea-97 genotype A neutralization test of foot-and-mouth disease virus in LFBK adherent cells was performed using 2017 virus (passage 6 times to LFBK adherent cells after virus isolation). The foot-and-mouth disease virus neutralization test method followed the method of the World Organization for Animal Health (OIE) Terrestrial Manual.

As a result, as shown in FIG. 4 , it was confirmed that a neutralizing antibody of 64 times (1.8Log10) or more appeared in all pigs from the time point 2 weeks after vaccination, and at the time point 4 weeks after vaccination, the test After the second vaccination (boosting), it was confirmed that the neutralizing antibody titer increased rapidly, showing a 2048-fold (3.31Log10) neutralizing antibody titer. The dotted line in FIG. 4 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.

[Test Example 4] Neutralizing antibody against wild-type virus in other regions of swine immune serum was tested

Efficacy of the L5B6S9-A virus vaccine strain according to the present invention by measuring the neutralizing antibody titer to various regional types of foot-and-mouth disease virus in the immune serum obtained in Test Example 3, that is, pig immune serum of each week O, 1, 3, and 5 was evaluated. In particular, the A/ASIA/Sea-97 genotype was used to verify the effectiveness of the test vaccine of the present invention, because the A-type foot-and-mouth disease virus has a large genetic difference even between the same regional types and does not show a significant protective effect in some cases. A neutralization test was performed for the classified A/VIT/2013 virus (Vietnam outbreak virus). A/VIT/2013 virus obtained after passage 4 times in LFBK adherent cells was used. In addition, in order to check whether it can protect against other genotypes of foot-and-mouth disease virus in other regions, a neutralization test for foot-and-mouth disease virus was performed using A22 Iraq virus (an Iraqi virus, obtained after 4 passages from LFBK adherent cells).

As a result, as shown in FIG. 5 , among the immune serum obtained in Test Example 4, the immune serum obtained from the 5th week of inoculation of pigs (one week after the second inoculation) was both A/VIT/2013 virus and A22 Iraq virus. It exhibited more than 500-fold neutralizing antibody titer against 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 land animal manual, as in Test Example 3. From these results, it was confirmed that the vaccine strain of the present invention has a very high utility value as a vaccine strain because the range of protection against the homologous or heterogeneous regional foot-and-mouth disease virus is wide.

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

7-week-old C57BL6 mice were tested with a test vaccine (1 μg of the antigen produced by inoculating the L5B6S9-A virus of the present invention and 100 μl of the YC vaccine (1 μg), which is a mixture of Montanide ISA 201, and the test vaccine (YC) diluted 4-fold. 100 μl of vaccine (0.25 μg), YC vaccine (0.064 μg), YC vaccine (0.016 μg)) 10 days after inoculation, A Malaysia 97 foot-and-mouth disease virus 200 LD ₅₀ adapted to A/ASIA/Sea-97 genotype mice was administered. After challenge inoculation, the survival rate (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 (YC vaccine (1 µg)) showed a 100% survival rate, and the test containing 0.064 µg antigen per mouse Even the vaccine (8-fold dilution; YC vaccine (0.064㎍)) was found to maintain a survival rate of 60%.

Next, 7-week-old C57BL6 mice were inoculated with the test vaccine YC vaccine (1㎍) and the test vaccine YC vaccine (0.1㎍) diluted 10-fold, and then adapted to the A/ASIA/Sea-97 genotype mouse 3 weeks later. A Malaysia 97 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 inoculation (A Malaysia 97 foot-and-mouth disease virus 200 LD ₅₀ adapted to mice) 3 weeks after vaccination in which antibody formation was complete, 1 μg and 0.1 μg per mouse It was confirmed that all of the test vaccines containing the antigen showed 100% survival rate.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be 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 A/ASIA/Sea-97 Lineage and the Viral Vaccine Composition Containing the Same <130> TKPA210510-EN-D1 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 8189 <212> DNA <213> Artificial Sequence <220> <223> A/Yeoncheon/SKR/2017 <400> 1 ttgaaagggg gcgctagggt ctcagcccta acatgccaac gacagctccc gcgtcgcact 60 ccacacttac atctgtgtgc gtgcgggaac cgatggactt tcgttcaccc acctacagct 120 ggactcacga caccgcgtgg ccatttttgc tggtttgagc ggacgaacgt cgcttgcgcg 180 cctcgcgtga ccggctagta ctcttaacac tccgcctact tggtcgttag cgctgtcctg 240 ggcactcctg ttgggggccg ttcgacgctc tacggtttcc cctccccgca gcagtctacg 300 gtgatggggc cgcttcgtgc gagccgatcg cctggtgtgt tttggccgtc acccgaagcc 360 cacctttccc cccccccccc caagtactac cgtcgttccc gacgttaaag ggaggtaacc 420 acaagttttg caccttctcg tccgaagcaa aaggacggta accgcgagct ttaaaccgcc 480 tttcccggcg ttaacgggat gtaaccacaa gatagacctt cgcccggaag taaaacggca 540 acacacacag ttttgcccgt tttcatgaga aatgggacgt cagcgcacga aacgcgccgt 600 tgcttgaggt ggacttgtac aaacacgatc tacgcgggtt cccacaacca acacaaaccg 660 tgcaatttga aatcccgcct ggtcattcca ggtctagagg ggtgacactt tgtactgtgg 720 ttgactccac gctcggccca ctggcgagtg tgagtagtag cactgttgct tcgtagcgga 780 gcatggtggc cgcgggactc cctccttggt aacaaggacc cgcggggccg aaagccacgt 840 ctttgaccca ccatgtgtgc aaccccagca cggcaacctt actacgaaaa ccactttaaa 900 gtgacactga tactggtact caaccactgg tgacaggcta aggatgccct tcaggtaccc 960 cgaggtaaca cgcgacactc gggatctgag aaggggactg gggcttctgt aaaagcgccc 1020 agtttaaaaa gcttctatgc ctgaataggc gaccggaggc cggcgccttt ccctatctac 1080 cattacttac atgaatacaa ctgattgctt tatcgctttg tggcacgcca tcagagagat 1140 aaaagcacga ctgtttttga agacacaaga gaaaatggaa tttacacttc acaacggtga 1200 aaagaagacc ttttactcta ggcccaacag ccatgacaac tgctggctga acaccatcct 1260 ccaattgttc aggtacgttg atgaaccatt ctttgattgg gtctatgaat cacctgaaaa 1320 cctcactctt gaagcaatta ggcaattgga agagatcact ggtctcgagc tgcacgaggg 1380 tggcccaccc gctctcgtaa tttggaacat caagcacttg ctccataccg gaatcggtac 1440 cgcttcgcga cctagcgagg tgtgtatggt agatggtacg gacatgtgtt tggctgactt 1500 ccatgctggc atcttcctga aagggcagga acacgcagtg ttcgcctgcg tcacctccaa 1560 cggatggtat gcgattgacg acgaggactt ttacccctgg acaccagatc cgtctgacgt 1620 cctggtgttt gttccgtacg atcaggaacc actcaacggg gaatggaaaa cgaaggttca 1680 gagacgactc aaaggggccg ggcaatccag ccctgccact gggtcgcaga accaatcagg 1740 caacactggc agcattatta acaactacta catgcagcag taccagaact ccatggacac 1800 tcaacttgga gacaacgcta ttagcggagg ctccaatgag gggtccacgg acacaacctc 1860 gacacacaca accaacaccc aaaacaacga ctggttctca aagctggcaa gttccgcctt 1920 caccgggctt ttcggcgcac tgcttgccga caaaaagacc gaagagacaa ctcttctgga 1980 ggaccgcatc ctcaccactc gcaatggaca caccacctcc acaactcaat cgagtgtggg 2040 ggtcacctgc gggtattcaa ctggtgaaga ccacgtttct gggcctaaca catcaggttt 2100 ggagacgcgg gtggtgcagg ctgagaggtt tttcaagaag cacttgtttg actggacaac 2160 ggacaaaccc tttggtcaca ttgaaaaatt gaaacttccc actgaacaca aaggtgtcta 2220 cggacagctg gtagaatcct ttgcatacat gagaaatggc tgggacgtgg aggtgtctgc 2280 tgttggcaac cagttcaacg gcgggtgcct tctcgtggcc atgatacccg agttcaaaga 2340 gttcacccag cgtgagaaat accagctcac cttgttccca caccagttta tcagccccag 2400 aaccaacatg actgcgcaca tcacggtccc gtaccttggt gtgaacagat atgaccagta 2460 caagaaacac aaaccctgga cgttggtggt gatggtggtc tcaccactta ccactagctc 2520 cattggtgca acacagatca aggtctacgc caacatcgcc ccgacccacg tgcacgtggc 2580 cggcgagctc ccctcgaaag aggggatcgt gccagtcgct tgctcggacg ggtacggtgg 2640 cctggtgaca acagacccca aaacagctga ccctgtttac ggtatggtgt acaacccacc 2700 caggaccaat taccctgggc ggtttacaaa tttgttggat gtggcagagg cgtgccccac 2760 cttcctctgt ttcgacgacg ggaaaccgta tatcgtgaca aggacggacg agcaacgtct 2820 cttagccaag tttgacctct ctcttgctgc aaagcacatg tcaaacacct acctgtcagg 2880 gctagcacag tactacgcac agtactctgg caccatcaat ttgcacttca tgtttactgg 2940 ttccactgac tcaaaggccc gctacatggt ggcgtacgtt ccacccggag tggaaacgcc 3000 accggacacg cctgagaagg ctgcacactg catccacgca gaatgggaca cgggcctaaa 3060 ttccaaattc accttttcaa tcccgtacgt atctgctgca gactacgcgt acacagcgtc 3120 tgacgaggca gaaacgacaa acgtacaggg atgggtttgc atctaccaaa tcacccacgg 3180 gaaggccgaa caagacactc tggtcgtgtc ggttagcgcc ggcaaagact tcgagctgcg 3240 cctccccatt gacccccgtg cgcaaaccac cgccaccggg gaatcagcag accctgtcac 3300 aaccaccgtt gagaactacg gtggcgagac acaagtacag cggcgttacc acaccgacgt 3360 cggcttctta atggacaggt tcgtgcagat caagcctgag ggccccacac atgtcattga 3420 cctcatgcag acacaccaac acgggctggt gggcgccatg ttgcgcgcgg ccacctacta 3480 cttttctgat cttgagattg tggtgaacca cacgggtaac ctaacgtggg tacccaatgg 3540 agcacccgag gcagcactgc aaaacacgag caaccccact gcttaccaca aagcgccgtt 3600 cacgaggctt gcgctcccct acaccgcgcc acaccgcgtg ctggcaactg tgtacagtgg 3660 gacgagcaag tactccgcac ctcaaaaccg gcgaggtgac ttgggtcctc tcgcggcgag 3720 actcgctgca caactccctg cctccttcaa cttcggtgca attcgggcca cggagatccg 3780 cgaactcctt gtgcgcatga agcgcgccga gctctactgc cccaggccac tgttggcggt 3840 ggaggtgtcg tcgcaagaca gacacaagca gaaaatcatt gcccctgcaa aacaactcct 3900 gaactttgac ctgctcaagt tggcgggaga cgttgagtcc aaccctggac ccttcttctt 3960 ctccgacgtt aggtcaaatt tcaccaagct ggtggaaacc atcaaccaaa tgcaagagga 4020 catgtcaaca aaacacgggc ccgactttaa ccggttggtg tccgcgtttg aggaactggc 4080 cactggagtg aaagccatca ggaacggcct cgacgaggcc aagccctggt acaagctcat 4140 caagctccta agccgcttgt catgcatggc cgctgtagca gcacggtcca aggacccagt 4200 ccttgtggcc atcatgctgg ctgacaccgg tcttgagatt ctggacagca cctttgtcgt 4260 gaagaaaatc tccgactcgc tctccagtct ctttcacgtg ccggcccccg tctttagttt 4320 cggagccccg atcctactgg ctgggttggt caaggtcgcc tcgagcttct tccggtccac 4380 gcccgaagac ctcgagagag cggaaaaaca gctcaaagca cgtgacatca atgacatatt 4440 cgccattctc aagaacggcg agtggctggt caagctgatc ctagctatcc gcgactggat 4500 taaagcatgg atcgcctcag aagaaaagtt cgtcaccatg acggatttgg tgcctggcat 4560 ccttgaaaag cagcgggacc ttaacgaccc gagcaagtac aaggaagcca aggagtggct 4620 cgacaacgcg cgacaagcgt gtctgaagag cgggaacgtc cacatgcta acctctgcaa 4680 agtggtcacc ccagcaccga gcaggtcgag acccgagccc gtagtcgttt gcctccgagg 4740 taaatccggc cagggcaaga gtttccttgc gaacgtgctt gcacaagcaa tttccaccca 4800 ctacactggc agaaccgatt cagtttggta ctgtccacca gaccctgacc acttcgacgg 4860 ttacaaccag caaactgtcg tagtgatgga tgatttgggc cagaaccccg acggcaagga 4920 cttcaagtac tttgcccaaa tggtgtcaac aacggggttc atcccgccca tggcctcgct 4980 cgaagacaaa gggaagccct tcaacagtaa ggttatcatt gccaccacca acttgtactc 5040 gggtttcacc ccgaggacta tggtgtgccc ggacgcgctg aaccgaaggt ttcactttga 5100 cattgatgtg agtgccaagg atgggtataa aattaacaac aaattggaca taatcaaagc 5160 tcttgaggac acccacacca acccagtggc gatgttccaa tacgactgcg cccttctcaa 5220 cggcatggca gtcgaaatga agagaatgca acaggacatg ttcaagcccc aaccgcctct 5280 gcagaacgtg taccaactcg ttcaggaggt gattgatcgg gtggagctcc acgagaaggt 5340 gtcgagccac ccgattttca agcagatctc aattccttcc caaaagtccg tgctgtactt 5400 tctcattgag aagggccaac acgaggcagc aattgaattc tttgagggga tggtacacga 5460 ctccattaag gaggaactcc gacccctcgt ccaacagacc tcatttgtga aacgcgcttt 5520 taaacgcctg aaggaaaact ttgagatcgt tgccctatgt ttgactcttc tggcaaacat 5580 agtgatcatg atccgcgaga ctcgcaagag acaacaaatg gtggatgatg cagtgaatga 5640 gtacatcgag aaggcaaaca tcaccacaga tgacaagact cttgacgagg cggaaaagaa 5700 ccctctggag actaccggtg ccagcaccgt cggcttcaga gagagaactc tcccgggaca 5760 caggacgagc gatgacgtga actctgagcc cgccaaacct gtggaagagc aaccacaagc 5820 tgaaggaccc tacgccgggc cgcttgagca ccagaaacct ctgaaagtga gagccaagct 5880 accacagcag gaggggcctt acgctggtcc gttggagaga cagaaaccac tgaaagtgaa 5940 agtgaatgcc ccggtcgtga aggaaggacc ttacgaggga ccggtgaaga agcctgtcgc 6000 tttgaaagtg aaagctaaga acttgattgt cactgagagt ggtgcccccc cgaccgactt 6060 gcaaaagatg gtcatgggca acaccaagcc tgttgagctc atcctcgacg ggaagacagt 6120 agccatctgc tgtgctactg gagtgtttgg cactgcctac ctcgtgcctc gtcacctttt 6180 cgctgagaag tatgacaaga tcatgttgga cggcagggct atgacagaca gtgactacag 6240 agtgtttgag tttgagatta aagtaaaggg acaggacatg ctctcagacg ctgcgctcat 6300 ggtgctgcac cgtgggaacc gcgtgagaga catcacgaaa cactttcgtg atgcagcacg 6360 aatgaagaaa ggcacccccg tcgttggcgt gattaacaac gctgacgttg ggagactgat 6420 tttctctggc gaggccctca cctacaagga cattgtagtg tgcatggacg gagacaccat 6480 gccgggcctg tttgcctaca aagccgccac caaggcaggc tactgtgggg gagccgttct 6540 cgccaaggac ggagccgaca cgttcatcgt tggcactcac tccgcaggtg gcaatggagt 6600 tgggtactgc tcatgcgtat ccagatccat gcttctcaag atgaaagcac acatcgaccc 6660 cgaaccacac cacgaggggt taattgttga caccagagat gtggaagagc gcgtgcacgt 6720 catgcgcaaa accaagcttg cacccaccgt ggcacacggc gtgttcaacc ccgaatacgg 6780 ccctgctgcc ttgtccaaca aggatccacg gctgaatgaa ggggttgtcc tcgatgaggt 6840 catcttctcc aaacacaaag gagacacaaa aatgtcacca gaggacaaag cgctgttccg 6900 ccgctgtgcc gctgactacg cgtcacgctt gcacagtgcg ctgggtacgg caaacgcccc 6960 actgagcatc tacgaggcca tcaaaggtgt cgacggactc gacgccatgg aaccagacac 7020 agcgcctggt ctcccctggg ccctccaggg gaagcgccgc ggcgcgctga ttgacttcga 7080 gaacggcacg gtcggacccg aagttcaggc tgccctagag ctcatggaga aaagagaata 7140 caagtttgct tgccagacct tcctgaagga cgagattcgc ccgatggaga aagtgcgtgc 7200 cggtaagacc cgcattgtcg atgtcttgcc tgttgaacat attctttaca ccaggatgat 7260 gattggcaga ttttgtgcgc aaatgcactc aaacaacggc ccgcaaattg gatctgcggt 7320 tggctgtaac cctgatgttg attggcaaag atttggcaca catttcgccc aatacagaaa 7380 cgtgtgggat gtggactatt cggccttcga tgctaaccac tgcagtgacg caatgaacat 7440 catgtttgag gaggtgtttc gcacagactt tggcttccac ccaaacgctg agtggatact 7500 gaagaccctt gtgaacacgg aacacgccta cgagaacaaa cgcattactg ttgaaggcgg 7560 gatgccctct ggttgttccg caactagcat cattaacaca attttgaaca acatctacgt 7620 gctctacgcg ctgcgtagac actatgaggg agttgagctg gacacttaca ccatgatctc 7680 ctacggagac gacatcgtgg tggcgagtga ttatgacctg gactttgagg ctcttaagcc 7740 tcacttcaaa tctcttggcc aaaccattac tccagctgac aaaagtgaca aaggttttgt 7800 tcttggtcat tccattactg atgttacttt cctcaaaaga cacttccaca tggattatgg 7860 aactgggttt tacaaacctg tgatggcctc gaagaccctc gaggctatcc tctcctttgc 7920 acgccgtggg accatacagg agaagttgac ctccgtggca ggactcgctg tccactccgg 7980 acctgacgag taccggcgtc tctttgagcc cttccagggc ctctttgaga ttccaagcta 8040 cagatcactt tacctgcgtt gggtgaacgc cgtgtgtggc gacgcataat ccctcagatg 8100 tcacaattgg cagaaagact ctgaggcgag cgacgccata ggagtgaaaa gctcgaaagg 8160 gcttttcccg cttccttttc caaaaaaaa 8189

Claims (6)

A method for preparing a foot-and-mouth disease virus vaccine, comprising the steps of:
(a) inoculating LFBK-adhered cells with a serotype A-derived wild-type foot-and-mouth disease virus and then serially subcultured to obtain a virus culture containing a virus having acquired cell adaptability;
(b) BHK-21 adherent cells are inoculated with the virus culture containing the virus obtained in step (a), and then serially subcultured to obtain a virus culture containing the virus obtained in the cell adaptability step;
(c) inoculating the BHK-21 floating cells with the virus culture containing the virus obtained in step (b) above, and then serially subcultured to obtain a virus culture containing the virus obtained in the cell adaptability step;
(d) selecting and isolating a candidate vaccine strain virus by evaluating at least one of virus productivity and antigen productivity from a virus culture containing the virus obtained in step (c); and
(e) selecting the final vaccine strain virus by confirming whether at least one of a heparan sulfate receptor and a 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 A/ASIA/Sea-97 genotype foot-and-mouth disease virus. The method according to claim 1, wherein a plaque purification step is performed before evaluating at least one of the virus productivity and antigen productivity in step (d). The method according to claim 3, wherein the plaque purification is performed using BHK-21 adherent cells. According to 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 whether the candidate vaccine strain virus recognizes the JMJD6 receptor is determined by CHO- A method for producing a foot-and-mouth disease virus vaccine strain, which is determined by measuring the virus titer in 677 cells. The method of claim 5, wherein when the viral titer measured in the CHO-K1 cells is 1x10 ^2.0 TCID ₅₀ /mL or more, it is determined that the heparan sulfate receptor is recognized,
When the viral titer measured in the CHO-677 cells is 1x10 ^2.0 TCID ₅₀ /mL or more, it is determined that the JMJD6 receptor is recognized.

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