KR20190108881A - Mixed live attenuated vaccine composition for protecting porcince rotavirus infection - Google Patents

Abstract

The present invention relates to an attenuated mixed live vaccine composition for preventing porcine rotavirus (PRV) infection. More specifically, provided are: the attenuated mixed live vaccine composition for preventing PRV infection having genotype combinations of G5, G8, G9, P[7], and P[23] by using attenuated strains of PRV isolates occur frequently in Korea; and a vaccine comprising the same.

Description

Attenuated mixed vaccine composition for prevention of swine rotavirus infection {MIXED LIVE ATTENUATED VACCINE COMPOSITION FOR PROTECTING PORCINCE ROTAVIRUS INFECTION}

The present invention relates to a vaccine composition for the prevention of Porcine Rotavirus (PRV) infection.

Rotaviruses are divided into eight serogroups up to A-H depending on the reactivity of the antibody to the VP6 capsid protein. Among these, it is the serogroup A rotavirus (henceforth "rotavirus") which causes severe diarrhea in humans and animals.

Rotaviruses have 11 segmented double-stranded RNA genomes in a virus particle consisting of three capsid layers. The genotypes of the 11 genomes of rotaviruses VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP3, NSP4, NSAP5 are Gx-P [x] -Ix-Rx-Cx-Mx-Ax-Nx -Tx-Ex-Ex-Hx. 35 G genotypes for VP7 and 50 P genotypes for VP4 have been reported in the outermost layer of viral capsids acting as rotavirus neutralizing antigens, 26 I genotypes and 21 R genotypes. There were 19 C genotypes, 19 M genotypes, 30 A genotypes, 21 N genotypes, 21 T genotypes, 27 E genotypes and 21 H genotypes.

Swine rotavirus causes severe appetite, vomiting and diarrhea in piglets and, in severe cases, dies. Histopathologic features of swine small intestine infected with swine rotavirus include atrophy, fusion, and growth of the villi by villi epithelial cell detachment.

Swine rotavirus infections are believed to occur in all countries where pigs are raised, causing significant economic damage. Argentina, 3.3%, Germany 4%, Canada 9.2%, Thailand 22.3%, Brazil 35.3% were reported.

Swine rotavirus vaccines are commercially available in Korea, and swine rotavirus infections are a problem despite the administration of swine rotavirus vaccines in pig farms. In other words, the results of swine rotavirus infection in swine diarrhea in domestic breeding were found to be 38.3%, similar to the incidence in Brazil.

The development of vaccines for the prevention of rotavirus infection is determined by the developmental distribution of genotypes of VP7 and VP4 in the outermost layer of the capsid, which act as neutralizing antigens of rotavirus. At least eleven G genotypes (G1-G6, G8-G12) have been reported for the VP7 protein of porcine rotavirus, but globally in the order of G5 (45.8%), G3 (11.2%), and G4 (9.6%). It is a bunch. However, the G genotypes that differ by region and by continent are different. For example, in the US and Canada, G5 is 71.43%, G4 is 8.19%, and G3 is 3.57%, whereas in Asia, G5 is 29.41%, G3 is 25.49%, G9 is 11.37%, and G8 is 7.84. Occurs in% order.

At least 19 P genotypes for the VP4 protein of porcine rotavirus (P [1] -P [13], P [19], P [22], P [23], P [26], P [27], P [32]) has been reported, but the genotypes that are reported worldwide are P [7] (47.4%), P [6] (15.9%) and P [13] (3.2%). P genotypes for VP4 proteins differ by region and continent. In the United States and Canada, P [7] is overwhelmingly 77.2%, but in Europe, P [6] is more common than P [7]. .

Porcine rotaviruses are changing G and P genotypes with age. In Canada, only the G5 and P [7] genotypes were reported between 1982 and 1984, but in 2005-2006, the G genotype was G4, G5, G2 and G9, and the P genotype was P [. 6], P [13], P [27] in order. In Thailand, the G4 and G3 and P [19] genotypes were predominantly reported between 2000 and 2001, but between 2006 and 2008. Genotypes G9 and G3 were P genotypes and P [23] genotypes predominantly changed.

According to literature that reported G and P genotype combinations for 98 strains of swine rotavirus isolated from domestic pig diarrhea, the most common G genotypes were G5 (67.3%), G8 (17.3%), and G9 (9.2%). The most common P genotype was P [7] with 92.9%, followed by P [23] and P [1] with 2.0%. In terms of G and P genotype combinations, G5P [7] is the most common (61.7%), G8P [7] is 16.3%, G9P [7] is 7.1%, GxP [7] is 5.1%, and GxP [7] is 5.1%, 3.0% for G5P [x], 2.0% for G9P [23], 1.0% for G8P [1] and GxP [1].

That is, the most common swine rotavirus G genotypes were G5, G8 and G9, and P genotypes were P [7], P [23] and P [1]. Therefore, in the domestic swine rotavirus G genotype, G8, which is known as the genotype of a cow, occurs second in Korea, and P [1], which is known as the genotype of a cow, is also generated. This means that swine rotaviruses occurring in Korea are caused by rearranged virus caused by rotavirus mixed infection of cattle and pigs.

However, the swine rotavirus vaccine currently used in Korea is an inactivated or attenuated live-dry mixed vaccine containing a mixture of swine rotavirus A1 (G5P [7] genotype combination) and pig rotavirus 10 (G9P [7] genotype combination). .

Foreign-imported vaccines also include viruses with a combination of the G5, G4, P [7] and P [6] genotypes that were prevalent in the United States in the 1990s.

Vaccine consisting of G5P [7] and G9P [7] genotype combinations produced and sold in Korea has limitations in preventing the swine rotavirus showing G8 and P [23] genotypes occurring in Korea. Able to know. In addition, the combination of G5, G4, P [7], and P [6] genotypes of foreign imported vaccines has limitations in preventing swine rotavirus infections showing G8, G9, P [23] genotypes occurring in Korea.

For this reason, swine rotavirus vaccines are sold in Korea, but since the G and P genotypes of these commercial vaccines differ from the G and P genotypes that occur in outdoor hog farms, they cannot be completely prevented. Rotavirus was a problem that is occurring at a high rate steadily occurred.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

Republic of Korea Patent No. 10-0737434

The present invention aims to develop and provide a customized vaccine consistent with the swine rotavirus G and P genotypes to effectively control swine rotavirus infection occurring in domestic pig farms.

In order to solve the above problems, the present inventors have genotyped combinations among the G and P genotype combinations of swine rotaviruses isolated from diarrhea of domestic pig house pigs so that the genotypes of swine rotaviruses occurring in domestic pig farms coincide. Using a swine rotavirus as the vaccine circumference, a live vaccine attenuated through 80 consecutive passages in MA104 cells, monkey kidney cells, using a domestically isolated swine rotavirus vaccine circumference, provides a vaccine composition suitable and effective in Korea. It has been found that this can be provided and the present invention has been completed.

One aspect of the present invention provides a vaccine composition for preventing swine rotavirus infection, comprising attenuated strain of swine rotavirus strain 174-1V, attenuated strain of K71V, and attenuated strain of PRG942V.

According to an embodiment of the present invention, the attenuated strain of swine rotavirus strain 174-1V is 174-1V-80 (Accession Number: KCTC 13429BP), and the attenuated strain of swine rotavirus strain K71V is K71V-80 (trusted Number: KCTC 13430BP), and the attenuated strain of the swine rotavirus strain PRG942V may be PRG942V-80 (Accession Number: KCTC 13431BP).

One aspect of the present invention provides a live vaccine for preventing swine rotavirus infection comprising the vaccine composition for preventing swine rotavirus infection.

According to an embodiment of the present invention, the live vaccine may further include a vaccine protector, an immune enhancer or a combination thereof.

According to one embodiment of the invention, the vaccine protection agent may be a lactose phosphate glutamate gelatin mixture.

Another aspect of the present invention provides an inactivated vaccine for preventing swine rotavirus infection, comprising the vaccine composition for preventing swine rotavirus infection.

According to one embodiment of the invention, the inactivation may be achieved by adding formalin to the viral strain.

One aspect of the present invention provides a method of inducing a protective immune response against swine rotavirus infection in a pig comprising administering to the pig an immunologically effective amount of the live or inactivated vaccine.

Another aspect of the invention comprises the steps of administering the live vaccine or the inactivated vaccine to sows in late pregnancy; And it provides a method of inducing a protective immune response to swine rotavirus infection in pigs comprising the step of feeding the sows colostrum delivered from the sows.

The vaccine according to the present invention can prevent infection from each porcine rotavirus with genotype combinations of the rotavirus G and P genotypes G5, G8, G9, P [7], P [23].

The vaccine according to the present invention can produce antibodies against each virus in feces and blood when inoculated in pigs, especially piglets, and can prevent diarrhea upon infection with the above-described genotype virus. In addition, the vaccine according to the present invention can alleviate bowel-related lesions when infected with a virus showing the above genotype when orally inoculated in piglets, and can reduce the discharge of the virus through feces when infected with the above-described genotype virus. have.

In the vaccine according to the present invention, newborn piglets born after intramuscular injection in pregnant pigs can see the above effects through colostrum from the mother pig.

FIG. 1 shows the passages of the PRG942 strains (A), 174-1 strains (B) and K71 strains (C) which are the pig rotavirus vaccine strains (initial isolate strains (174-1, PRG942, K71), 20 passages (174). -1V-20, PRG942V-20, K71V-20), 40th Fleet (174-1V-40, PRG942V-40, K71V-40), 60th Fleet (174-1V-60, PRG942V-60, K71V -60) and 80's passages (174-1V-80, PRG942V-80, K71V-80)].
FIG. 2 shows the passages of the PRG942 strains (A), 174-1 strains (B), and K71 strains (C), the primary isolates (174-1, PRG942, K71), the 20th passage (174) -1V-20, PRG942V-20, K71V-20), 40th Fleet (174-1V-40, PRG942V-40, K71V-40), 60th Fleet (174-1V-60, PRG942V-60, K71V -60), the results of electron microscopic observation of the 80s passage (174-1V-80, PRG942V-80, K71V-80).
FIG. 3 shows each of the passages of the PRG942 strain (A), 174-1 strain (B) and K71 strain (C), which are the pig rotavirus vaccine strains (initiated isolates (174-1, PRG942, K71), 20 passages (174). -1V-20, PRG942V-20, K71V-20), 40th Fleet (174-1V-40, PRG942V-40, K71V-40), 60th Fleet (174-1V-60, PRG942V-60, K71V -60), passage 80 (174-1V-80, PRG942V-80, K71V-80)] shows confocal microscopic observations after immunofluorescence on infected MA104 cells.
Fig. 4 shows the respective subsidiary lines of the PRG942, 174-1 and K71 strains of the pig rotavirus vaccine (initiated isolates (174-1, PRG942, K71), the 20th passage (174-1V-20, PRG942V-20, K71V-20). ), 40s Passage (174-1V-40, PRG942V-40, K71V-40), 60s Passage (174-1V-60, PRG942V-60, K71V-60), 80s Passage (174-1V -80, PRG942V-80, K71V-80)] shows the PAGE-pattern of virus RNA extracted from infected MA104 cells.
Fig. 5 shows each of the sub- strains of the PRG942 strains, 174-1 strains and K71 strains of the pig rotavirus vaccine strains (initial isolates (174-1, PRG942, K71), the 20th passage strain (174-1V-20, PRG942V-20, K71V) -20), 40s Passage (174-1V-40, PRG942V-40, K71V-40), 60s Passage (174-1V-60, PRG942V-60, K71V-60), 80s Passage (174 -1V-80, PRG942V-80, K71V-80)] Genetic analysis of the entire nucleotide sequence of 11 genes (VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSAP4, NSAP5) Indicates.
6 to 8 show the passages (initial isolates (174-1, PRG942, K71), PRG942 (FIG. 6), 174-1 strain (FIG. 7) and K71 strain (FIG. 8) which are the pig rotavirus vaccine strains, respectively. 20th Fleet (174-1V-20, PRG942V-20, K71V-20), 40th Fleet (174-1V-40, PRG942V-40, K71V-40), 60th Fleet (174-1V-60 , PRG942V-60, K71V-60), genotypes of the eighties (174-1V-80, PRG942V-80, K71V-80)]. ) Types and locations.
Figure 9 shows the primary pathogenic strain (174-1) of each vaccine strain 2 weeks after oral administration of pig rotavirus attenuated live vaccine strain (174-1V-80, PRG942V-80 or K71V-80) according to the present invention in piglets. , 7 days, 14 days after vaccination (day of challenge), 21 days (day 7 after challenge), 28 days before vaccination to test the blood neutralizing antibody level induced after challenge with PRG942 or K71) Serum neutralizing antibody titer is shown after taking blood from each pig (14 days of challenge) and separating serum.
Figure 10 is inoculated with live vaccine before the pig rotavirus attenuated live vaccine strain 174-1V-80 strain (a), PRG942V-80 strain (b) or K71V-80 strain (c) in piglets Serum was collected from piglets at 7 days, 14 days (day of challenge), 21 days (7 days after challenge), and 28 days (14 days of challenge), and serum was isolated from ELISA. IgM, IgG and IgA antibody titers are shown.
Figure 11 before inoculating the live vaccine before the pig rotavirus attenuated live vaccine strain 174-1V-80 strain (a), PRG942V-80 strain (b) or K71V-80 strain (c) in piglets IgM, IgG and fecal in feces confirmed by ELISA method by collecting feces of each pig on 7 days, 14 days (day of challenge), 21 days (7 days after challenge), and 28 days (14 days of challenge). IgA antibody is shown.

The present invention relates to a composition of a vaccine for customarily preventing swine rotavirus showing a combination of G and P genotypes of swine rotavirus in piglets in domestic pig farmers, and more specifically, G and P of domestic pig rotavirus isolates. Vaccine composition for preventing infection of porcine rotavirus showing a combination of G5, G8, G9, P [7] and P [23] genotypes based on genotypic distribution results, and pig rota comprising the same A vaccine for preventing viral infections.

Hereinafter, the present invention will be described in detail.

The present invention provides a vaccine composition for the prevention of porcine rotavirus comprising the G5, G8, G9, P [7], P [23] genotypes and / or combinations thereof.

In the present invention, the term "rotavirus" belongs to Reoviridae and encodes six structural proteins and six nonstructural proteins with 11 fragment double-stranded RNA as a genome. The two envelope capsid proteins VP4 and VP7 individually elicit attenuated antibodies to induce immunity and can be divided into two antigenic types, P (proteinase sensitivity) and G (glycoprotein). VP4 on the surface of the viral molecule plays an important role in the interaction between the virus and the cell, including invading cells and receptors that bind to the cell. Trypsin treatment of VP4 specifically divides VP5 and VP8, VP8 contains the hemagglutin of the virus, and VP5 has a hydrophobic moiety inside, allowing the virus to penetrate cells.

The term "attenuated" in the present invention means that the toxic strain is modified in such a way as to be a less toxic strain or a weaker pathogen than before the transformation, while such strain can still be replicated in the host, while clinically Significantly reduced ability to cause disease. The attenuated strains of the invention are preferably strains whose toxicity or pathogenicity has been reduced to allow for administration for vaccines, and more preferably can cause clinical disease while still allowing the strain to replicate in the host. It means to attenuate to the extent that it is absent.

The term "prevention" in the present invention is a concept including diarrhea caused by swine rotavirus infection, vomiting, viral excretion through feces, alleviation and / or prevention of lesions in the small intestine.

As used herein, the term "vaccine" refers to a pharmaceutical composition containing at least one immunologically active component that induces an immunological response in an animal. Suitable elements (subunit vaccines), whereby these elements destroy the entire virus or its growth culture, and then by a purification step to obtain the desired structure (s), or without limitation, bacteria, Animals requiring vaccines by synthetic procedures followed by isolation and purification by appropriate manipulation of appropriate systems, such as insects, mammals or other species, or by direct incorporation of genetic material using appropriate pharmaceutical compositions (Polynucleotide vaccination) by induction of the above synthetic process. The vaccine may comprise one or more of the above described elements or simultaneously.

The present invention provides an inactivated vaccine for preventing swine rotavirus infection, comprising the vaccine composition for preventing rotavirus infection. Inactivated vaccine means a vaccine comprising a dead virus or an active ingredient thereof, which vaccine can be prepared by methods known in the art. For example, if the virus propagates to high titers, the virus is obtained and treated with formalin, betapropriolactone (BPL), bicomponent ethyleneimine (BEI) or gamma rays, or other methods known to those skilled in the art. Can be deactivated by Preferably, it may be inactivated at a final concentration of 0.1% formalin.

The vaccine of the present invention may be in any form known in the art, such as, but not limited to, solid forms suitable for the form or suspension of solutions and injections. Such formulations may also be emulsified or encapsulated in liposomes or soluble glass, or prepared in the form of aerosols or sprays. They can also be included in transdermal patches.

The vaccine according to the present invention may include a pharmaceutically acceptable vaccine protection agent, an adjuvant, a diluent, an absorption accelerator, and the like, as necessary. Such vaccine protection agents include, but are not limited to, for example, lactose phosphate glutamate gelatin mixtures. Said adjuvant may be added to, for example, aluminum hydroxide, mineral oil or other oils or vaccines, or auxiliary molecules, such as but not limited to interferon, interleukin or growth, produced by the body after each induction by such additional components. Including, but not limited to, arguments. If the vaccine is in the form of a solution or injection, it may contain propylene glycol and an amount of sodium chloride sufficient to prevent hemolysis, if necessary (eg about 1%).

"Pharmaceutically acceptable" means non-toxic, which is physiologically acceptable and, when administered to pigs, does not inhibit the action of the active ingredient and usually does not cause gastrointestinal disorders, allergic reactions such as dizziness or the like. do.

The vaccine may be administered via a route of administration such as oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and the like, but is not limited thereto. For example, the vaccine may be administered through oral or intramuscular administration.

The present invention provides a method of inducing a protective immune response against swine rotavirus infection in a pig comprising administering to the pig an immune effective amount of a live or inactivated vaccine. The administration may be performed by methods known in the art, for example, oral or injection administration, but is not limited thereto.

The term "protection" in the present invention is evidenced by the reduction or absence of the clinical signs typically indicated by an infected host, a faster recovery time or a lower duration, or a lower viral titer in the tissue or fluid or excreta of the infected host. .

The term "effective amount" in the present invention means the amount of vaccine that can induce an immune response to reduce the frequency or severity of pandemic diarrheal virus infection in pigs, and those skilled in the art are appropriate based on the common sense. You can choose to. For example, an effective amount is 1 × 10 ⁴ to 1 × 10 ⁷ TCID50 / ml, preferably 1 × 10 ⁵ per day, for the virus strain in the case of a vaccine comprising the live vaccine composition. To 1 × 10 ⁶ TCID 50 / ml.

The present invention also provides an attenuated strain.

The attenuated strains of the present invention are attenuated strain 174-1V-80 (Accession No .: KCTC 13429BP) of swine rotavirus strain 174-1V, attenuated strain K71V-80 (accession number: KCTC 13430BP) and swine of pig rotavirus strain K71V. Attenuated strain PRG942V-80 (Accession No .: KCTC 13431BP) of Rotavirus strain PRG942V, which contains at least one, for example, the attenuated strain of swine Rotavirus strain 174-1V 174-1V- Mixed strain consisting of 80 (accession number: KCTC 13429BP), attenuated strain K71V-80 (accession number: KCTC 13430BP) of swine rotavirus strain K71V and attenuated strain PRG942V-80 (accession number: KCTC 13431BP) of pig rotavirus strain PRG942V Can be.

Hereinafter, one or more embodiments will be described in more detail. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example

As described in the background technology of the present invention, the inventors' prior studies have determined the G and P genotypes for the 98 strains of swine rotavirus isolated from domestic pig diarrhea feces, among which the representative genotype of 174 -1 strain (G8P [7] genotype combination), PRG942 strain (G9P [23] genotype combination), and K71 strain (G5P [7] genotype combination) were selected as vaccine vaccines, and thus the possibility of developing trivalent attenuated mixed live vaccines It was performed as follows.

Example  1. Isolate Swine Rotavirus

Swine diarrhea collected from domestic pig farms was mixed with PBS (pH 7.2) at 10% (weight percentage, weight / volume%, W / V%) and centrifuged at 3,000 xg for 30 minutes in a 4 ℃ centrifuge. . The supernatant was filtered through a 0.2 μm filter and stored at −80 ° C. to use for virus isolation.

For the isolation of porcine rotavirus, a 6-well plate containing α104, monkey kidney cells, containing 10% fetal bovine serum, 1% antibiotic (penicillin 100 units / ml, streptomycin 100 μg / ml) Monolayer culture in -MEM medium. The fecal supernatant was mixed 1: 9 with α-MEM medium containing trypsin at a concentration of 10 μg / ml and reacted at 37 ° C. for 30 minutes. The mixed solution was subjected to decimal dilution using α-MEM medium containing trypsin at a concentration of 10 μg / ml, and 300 μl was inoculated into each well of a 6-well plate in which MA104 cells were monolayer cultured. When the virus was adsorbed to MA104 cells for 1 hour after inoculation, 3 ml of the medium containing antibiotics and 1 μg / ml of trypsin was added, and then the cytopathic effect (CPE) was observed at 37 ° C. and CO ₂ cell incubator. Incubated until. After incubation, cells were completely destroyed through repeated freeze and thaw cycles, centrifuged at 3,000 xg for 30 minutes at 4 ° C, and the supernatant was collected and used for the isolation of the next single virus strain. That is, after inoculating the virus supernatant of the highest dilution factor showing the cell degeneration effect to each well of a 6-well plate in which MA104 cells were monolayer cultured in the same manner as above, the gel was overlaid and overlayed at 2-3 ° C. Incubated daily. Each plaque was then harvested and seeded in 6-well plates in which MA104 cells were monolayer cultured. This plaque assay was repeated two more times.

The plaques obtained in the third plaque assay were inoculated into 6-well plates in which MA104 cells were monolayer cultured and then cultured in a CO ₂ cell incubator at 37 ° C. until cell degeneration was observed. After repeated freezing and thawing three times, centrifuged for 30 minutes at 4 ℃, 3,000 xg. Viral genomes were extracted using RNA extraction kits in 200 μl of each supernatant.

RT-PCR was performed using various primer pairs to identify nucleotide sequences of 11 genes. Each primer pair used herein is shown in Table 1 below.

^a F: forward; R: reverse ^b Primer for 5 'RACE PCR

^c Primer for 3 'RACE PCR

After each amplification product was extracted from the gel, each nucleotide sequence was analyzed. Genetic analysis of the G and P genotypes and the remaining nine genes was performed in the DNAsis MAX DNA Basic Module. The 174-1 strains with G8P [7] genotypes, PRG942 strains with G9P [23] genotype combinations, and G5P [ 7] K71 strain with genotype combination was selected as vaccine strain and used for vaccine strain development.

Example  2. After Cell Culture and Virus Inoculation Subculture

For serial passage of the vaccine column to monkey kidney cells, α-MEM containing 10% fetal bovine serum and 1% antibiotic (penicillin 100 units / ml, streptomycin 100 μg / ml) was applied to MA104 cells, monkey kidney cells. Monolayer culture in medium (6-well plate).

Each of the 174-1 column, the PRG942 column, and the K71 column was added to the medium containing trypsin 10 μg / ml at a rate of 10/10 and then activated at 37 ° C. for 30 minutes. Thereafter, the cells were inoculated into each well of a 6-well plate monolayer cultured with MA104 cells from a dilution with a low dilution rate after dilution step 10 times using a medium containing 10 μg / ml of trypsin in a 96-well plate. After 1 hour of inoculation, the virus was adsorbed onto MA104 cells, and then cultured in 37 ° C. and CO ₂ cell incubator for 2 days by adding a medium containing 1 μg / ml of antibiotic and trypsin.

After incubation, cells were completely destroyed by freezing and thawing three times. Subsequently, 80 consecutive subcultures were performed by the above-described method using the supernatant of the well showing the highest dilution with rotavirus-induced cytotoxicity among each well inoculated from the low dilution of the virus to the high dilution. It was.

Each isolate was named individually according to the vaccine column and the 20, 40, 60 and 80 passages. In other words,

1) 174-1 (vaccine column), 174-1V-20 (20 passages), 174-1V-40 (40 passages), 174-1V-60 (60 passages), 174-1V-80 (80 passages);

2) PRG942 (vaccine column), PRG942V-20 (20 passages), PRG942V-40 (40 passages), PRG942V-60 (60 passages), PRG942V-80 (80 passages);

3) K71 (vaccine circumference), K71V-20 (20 passages), K71V-40 (40 passages), K71V-60 (60 passages), K71V-80 (80 passages)

Named.

Experimental Example  One. Passage  virus Titer  inspection

Each vaccine column and 20, 40, 60 and 80 passages of each attenuated strain were added to the medium containing trypsin 10 ㎍ / ml at 10/10 and then activated at 37 ℃ for 30 minutes. Then monolayer MA104 cells after 10-fold step dilution with α-MEM medium containing 1 μg / ml trypsin and 1% antibiotic (penicillin 100 units / ml, streptomycin 100 μg / ml) in 96-well plates. 100 μl was inoculated into each well of a 96-well plate incubated and incubated in 37 ° C. and CO ₂ cell incubator for 18 hours.

Each 96-well plate was washed with physiological saline (phosphate buffered saline, PBS, pH 7.2) and then fixed for 15 minutes with 80% cold acetone. After washing with PBS, the primary antibody was reacted at 37 ° C. for 1 hour with a monoclonal antibody specific for Rotavirus VP6 protein. After washing with PBS, as a secondary antibody, FITC-conjugated anti mouse IgG antibody (FITC-conjμgated anti mouse IgG antibody) was sensitized at 37 ° C for 1 hour. After washing with PBS (pH 8.0), each well was sealed with 60% glycerin and the rotavirus positive cell number was calculated using an inverted fluorescence microscope.

As a result of measuring the virus titer of each virus line passage, as shown in Table 2, the virus titer increased as the passage of each virus line was higher.

Virus strain G and P genotypes Passage 1 unit 20's 40 spaces 60 spaces 80 spaces 174-1 G8P [7] 5.9 * 10 ⁶ 6.6 * 10 ¹⁰ 7.8 * 10 ¹¹ 1.4 * 10 ¹² 1.5 * 10 ¹³ PRG942 G9P [23] 3.1 * 10 ⁶ 3.3 * 10 ⁹ 1.4 * 10 ¹¹ 8.4 * 10 ¹¹ 5.5 * 10 ¹² K71 G5P [7] 3.6 * 10 ⁹ 8.8 * 10 ¹⁰ 9.9 * 10 ¹² 1.15 * 10 ¹³ 2.5 * 10 ¹³

Experimental Example  2. Passage  Examination of cytopathic effect by virus

Each vaccine column and each attenuation strain for each of the 20, 40, 60 and 80 passages were incubated for 2 days in an 8-well chamber slide by the method described above and then fixed for 1 hour with 10% neutral formalin. Thereafter, dehydration and polymerization were carried out through various alcohols and xylene, and the cell denaturation effect caused by the virus was examined.

As a result, typical cytopathic effects such as dropout and circularization of MA104 cells were observed by infection of each vaccine strain by passage compared to unvaccinated MA104 cells. The cell degeneration effects produced by each passage were identical (see Figure 1).

Experimental Example  3. Passage  Virus Confocal  Photomicrograph

Each vaccine column and each attenuated strain of 20, 40, 60 and 80 passages were incubated in a CO ₂ cell incubator at 37 ° C. for 18 hours in an 8-well chamber slide by the method described above. After washing with physiological saline (pH 7.2), and fixed with 80% cold acetone for 15 minutes. After washing with PBS, the primary antibody was reacted at 37 ° C. for 1 hour with a monoclonal antibody specific for Rotavirus VP6 protein. After washing with PBS, FITC-conjugated anti-mouse IgG antibody was sensitized at 37 ° C. for 1 hour as a secondary antibody. After washing with PBS (pH 8.0), each well was dispensed with the inclusion solution containing DAPI, and positive cells were photographed with a confocal microscope.

As a result of observing the type of virus of each vaccine strain by electron microscopy, rotavirus of 60-80 nm size was confirmed, and there was no change of virus particles according to passage of vaccine strain (see FIG. 2).

Experimental Example  4. Passage  Test for Morphological Changes in Viruses

After each incubation of each vaccine column and each attenuation strain for 20, 40, 60 and 80 passages in the T-150 flask via the above-described method, three freezes and thaws were repeated, and the culture was 3,000 in a 4 ° C centrifuge. Centrifugation at xg for 30 minutes. The supernatant was then centrifuged at 141,000 × g for 10 hours in a 4 ° C. ultra-centrifuge. After removing the supernatant, the precipitate was negatively stained with 50 μl of phosphotungstic acid (PTA, pH 7.0), and then incubated for 2 minutes by placing 25 μl of each on a carbon-coated grid membrane. . After that, the excess liquid was removed from the grid, and dried and examined for viruses under an electron microscope.

As a result of immunofluorescence staining, fluorescence of rotavirus-specific antibodies was observed in the cytoplasm of all MA104 cells inoculated with each passage, and it was confirmed that there was no change in the viral characteristics according to passage of the vaccine strain. 3).

Experimental Example  5. Passage  Virus electrophoresis pattern analysis

After each incubation in the T-150 flask by the method described above, each vaccine column and each attenuation strain for each of the 20, 40, 60 and 80 passages were repeated three times of freezing and thawing, and then the culture solution was centrifuged in a 4 ° C centrifuge. Centrifugation for 30 minutes at 3,000 xg.

Viral genomes were extracted using RNA extraction kits in 200 μl of each supernatant. After each of the extracted viral genomes were electrophoresed in a polyacrylamide gel for 16 to 18 hours, the gel was silver stained, and the PAGE-pattern on the gel was photographed to determine whether there was a change in the PAGE-pattern for each passage. Inspected.

As a result of analyzing the PAGE-pattern, it was confirmed that each passage vaccine shows the 4-2-3-2 pattern, which is the PAGE pattern of rotavirus. In addition, no change in the PAGE-pattern with the vaccine column was observed even as each vaccine line increased the passage, and in particular, the sequence rearrangement of the genome was not observed. That is, it was confirmed that there was no change in the size of the viral genome due to passage (see FIG. 4).

Experimental Example  6. Passage  The entire genome of the 11 genomes of the virus and Homology  analysis

After each incubation of each vaccine column and each attenuation strain for 20, 40, 60 and 80 passages in the T-150 flask via the above-described method, three freezes and thaws were repeated, and the culture was 3,000 in a 4 ° C centrifuge. Centrifugation at xg for 30 minutes.

Viral genomes were extracted using RNA extraction kits in 200 μl of each supernatant. 5 'and 3' RACE was performed to identify 5 'and 3' terminal sequences of the extracted viral genome, respectively. In addition, RT-PCR or nested PCR was performed using the primer pairs of Table 1 to identify internal sequences of respective genomes.

After each amplification product was extracted from the gel, each nucleotide sequence was analyzed. Homology of the entire genome sequence of the 11 genomes of the vaccine column and each attenuated strain of each passage was performed using the DNAMAN version 6.0 program, and systematic analysis was performed on the DNAsis MAX DNA basic module.

After sequencing the entire genomes of the 11 genomes, a systematic analysis was performed. As a result, no systematic differences were observed between the primary vaccine column and each passage. That is, it was confirmed that there was no rearrangement by a large genome mutation or mixed infection according to serial passage (see FIG. 5).

Rotavirus RNA was extracted from each vaccine column and passage line, and the entire nucleotide sequence of each of the 11 genomes was sequenced. The nucleic acid sequence was replaced with an amino acid sequence. It was confirmed that there was a tendency to increase. Substitution of this amino acid sequence was presumed to be related to the attenuation of the vaccine strain (see Figures 6-8).

Example  3. Selection of Vaccine Protectors

The protective agent necessary for producing the swine rotavirus trivalent attenuated mixed live vaccine according to the present invention was prepared as shown in Table 3 below based on the protective agent composition proven to be stable and effective in the existing TR2 vaccine.

Ingredient content Lactose 74.62 g KH ₂ PO ₄ 0.517 g K ₂ HPO ₄ 0.854 g Sodium L-Glutamate 10 g gelatin 5 g Distilled water 1,000 ml

Example  4. Each Vaccine  And preparation of trivalent vaccines

Each baeksinju 3 and the composition of the vaccine is based on the TR2 composition components that are sold in conventional, each baeksinju to 10 ^6. to contain ⁰ TCID ₅₀ or more protective agent and composition was as shown in Table 4.

Experimental Example  7. Each Vaccine  And safety testing of trivalent vaccines

To examine the safety of each lot of each vaccine strain and trivalent vaccine, (i) intraperitoneally inoculate 0.5 ml of 8 mice weighing 15-20 g for each lot for 7 days, and (ii) for each lot. 7 ml of 2 ml intramuscular of 2 guinea pigs and 300 mg of guinea pigs and 2 guinea pigs intraperitoneally for 7 days, (iii) 5 ml and 4 to the muscles of 2 pigs of 4 to 6 weeks of age for each lot. Two 6-week-old pigs were orally inoculated with 5 ml and the clinical symptoms were observed for 14 days.

Experimental results, as shown in Tables 5 to 8 (Table 5: 174-1V-80, Table 6: PRG942V-80, Table 7: K71V-80, Table 8: Trivalent mixed live vaccine) of the abdominal cavity, guinea pigs of the mouse Abnormal symptoms of inoculation sites were observed by inoculation into muscles, abdominal cavity, and pig muscles, but survival was confirmed without any clinical symptoms.

Bell Lot number Inoculation method The number of animals Vaccine amount Clinical symptoms mouse T316Rota-174-1-Lot1 Intraperitoneal 8 0.5 ml none T316Rota-174-1-Lot2 Intraperitoneal 8 0.5 ml none T316Rota-174-1-Lot3 Intraperitoneal 8 0.5 ml none guinea pig T316Rota-174-1-Lot1 Intramuscular 2 2 ml none T316Rota-174-1-Lot2 Intramuscular 2 2 ml none T316Rota-174-1-Lot3 Intramuscular 2 2 ml none T316Rota-174-1-Lot1 Intraperitoneal 2 2 ml none T316Rota-174-1-Lot2 Intraperitoneal 2 2 ml none T316Rota-174-1-Lot3 Intraperitoneal 2 2 ml none pig T316Rota-174-1-Lot1 Intramuscular 2 10 ml none T316Rota-174-1-Lot2 Intramuscular 2 10 ml none T316Rota-174-1-Lot3 Intramuscular 2 10 ml none

Bell Lot number Inoculation method The number of animals Vaccine amount Clinical symptoms mouse T316Rota-PRG942-Lot1 Intraperitoneal 8 0.5 ml none T316Rota-PRG942-Lot2 Intraperitoneal 8 0.5 ml none T316Rota-PRG942-Lot3 Intraperitoneal 8 0.5 ml none guinea pig T316Rota-PRG942-Lot1 Intramuscular 2 2 ml none T316Rota-PRG942-Lot2 Intramuscular 2 2 ml none T316Rota-PRG942-Lot3 Intramuscular 2 2 ml none T316Rota-PRG942-Lot1 Intraperitoneal 2 2 ml none T316Rota-PRG942-Lot2 Intraperitoneal 2 2 ml none T316Rota-PRG942-Lot3 Intraperitoneal 2 2 ml none pig T316Rota-PRG942-Lot1 Intramuscular 2 10 ml none T316Rota-PRG942-Lot2 Intramuscular 2 10 ml none T316Rota-PRG942-Lot3 Intramuscular 2 10 ml none

Bell Lot number Inoculation method The number of animals Vaccine amount Clinical symptoms mouse T316Rota-K71-Lot1 Intraperitoneal 8 0.5 ml none T316Rota-K71-Lot2 Intraperitoneal 8 0.5 ml none T316Rota-K71-Lot3 Intraperitoneal 8 0.5 ml none guinea pig T316Rota-K71-Lot1 Intramuscular 2 2 ml none T316Rota-K71-Lot2 Intramuscular 2 2 ml none T316Rota-K71-Lot3 Intramuscular 2 2 ml none T316Rota-K71-Lot1 Intraperitoneal 2 2 ml none T316Rota-K71-Lot2 Intraperitoneal 2 2 ml none T316Rota-K71-Lot3 Intraperitoneal 2 2 ml none pig T316Rota-K71-Lot1 Intramuscular 2 10 ml none T316Rota-K71-Lot2 Intramuscular 2 10 ml none T316Rota-K71-Lot3 Intramuscular 2 10 ml none

Bell Lot number Inoculation method The number of animals Vaccine amount Clinical symptoms mouse T3163Rota-Lot1 Intraperitoneal 8 0.5 ml none T3163Rota-Lot2 Intraperitoneal 8 0.5 ml none T3163Rota-Lot3 Intraperitoneal 8 0.5 ml none guinea pig T3163Rota-Lot1 Intramuscular 2 2 ml none T3163Rota-Lot2 Intramuscular 2 2 ml none T3163Rota-Lot3 Intramuscular 2 2 ml none T3163Rota-Lot1 Intraperitoneal 2 2 ml none T3163Rota-Lot2 Intraperitoneal 2 2 ml none T3163Rota-Lot3 Intraperitoneal 2 2 ml none pig T3163Rota-Lot1 Intramuscular 2 10 ml none T3163Rota-Lot2 Intramuscular 2 10 ml none T3163Rota-Lot3 Intramuscular 2 10 ml none

Experimental Example  8. Each Vaccine  And in pigs given a trivalent vaccine Serum neutralizing antibody  inspection

To test the serum neutralizing antibody titers of each lot of each vaccine strain and trivalent vaccine, 2 ml of each lot was injected into the muscle of 8 piglets, 4-6 weeks of age, and 2 weeks after the first vaccination The method was repeated. Two piglets were used as the vaccinated control group. Blood was collected 28 days after the first inoculation and serum neutralizing antibodies were used for the test.

The test serum was then inactivated for 30 minutes at 56 ° C., and then 0.05 ml of serum was diluted in binary in cell maintenance culture in 96-well microplates. Each vaccine strain was mixed with 10X trypsin at a ratio of 1: 9, and sensitized at 37 ° C. for 30 minutes, diluted with 2 × 10 ² TCID ₅₀ /0.1 ml of virus content, and sensitized with binary diluted antibody solution at 37 ° C. for 60 minutes. I was. 0.1 ml of each well of a 96-well plate in which MA104 cells were monolayer cultured, and then 0.1 ml of 2X trypsin was added and cultured at 37 ° C. for 5 days. Observation was performed daily under a microscope to determine the positive and negative effects of the cell degeneration effect. The serum titer was expressed as the reciprocal of the highest serum dilution factor that inhibited the cell degeneration effect.

Experimental results showed that the antibody titers in serum induced by each vaccine tested using the attenuated single live vaccine of three vaccine strains were 2 ^{7.75 ± 0.46} fold for the 174-1V-80 vaccine and 2 ^{7.6 ± 0.744 for the} PRG942V-80 vaccine. Pear and K71V-80 vaccine strains were identified as 2 ^{8 .25 ± 0.46} fold.

Trivalent attenuated mixed live vaccine test vaccines containing the three vaccine strains were tested for blood neutralizing antibody titers generated after inoculation into pigs using the respective vaccine strains. As a result, 2 ^{7.87 ± 0.64} was compared to 174-1V-80 vaccine strains. Pear, 2 ^{7.75 ± 0.707} times compared to PRG942V-80 vaccine, and 2 ^{8 ± 0.53} times compared to K71V-80 vaccine.

The blood neutralizing antibody titer generated after inoculation of the three attenuated vaccines with attenuated live vaccines in pigs was tested using the existing vaccine vaccine, swine rotavirus A1 strain. Although the neutralizing antibody titer was low, the K71V-80 vaccine showed high neutralizing antibody titer against A1 strain. In addition, the blood neutralizing antibody titer generated after inoculation of the mixed trivalent attenuated mixed live vaccine test vaccine into pigs was examined using a pig rotavirus A1 strain, which is an existing vaccine, and was higher than that of the single vaccine.

Based on the above results, the neutralizing antibody values induced by each single vaccine test and mixed vaccines thereof were determined to be 2, which is the test standard of the Ministry of Agriculture, Food and Rural Affairs and Quarantine. ^{More than seven} times, each vaccine was confirmed to be suitable as an attenuated vaccine.

In summary, when the interference reaction between each single vaccine and mixed vaccine was confirmed, the antibody titer of other antigens increased to a certain level when inoculated with a single vaccine, but the antibody titer was lower than that of the same antigen. On the other hand, the three swine rotavirus combination vaccines showed similar or higher results than the single inoculation, indicating that the three mixed vaccines were superior to the single vaccines (Table 9).

Experimental Example  9. To verify the efficacy of swine rotavirus vaccine Attack virus Half effective amount

Virus titers of three vaccine strains, K71 (G5P [7]), 174-1 (G8P [7]), and PRG942 (G9P [23]) strains were examined and then diluted 10.

After diluting 3 ml of the three vaccine column concentrations or 3 ml of PBS into oral piglets not given colostrum, 2 days old piglets were examined for clinical symptoms such as diarrhea and mortality.

Experimental results showed that the diarrhea-induced half-effective amount (50% Effective Dose; ED ₅₀ ) was found to be 5.6-1 ¹⁰ FFU / ml for the primary isolate, 3.1 x 10 ² FFU / ml for the PRG942 ^primary isolate, and 3.1x10 ¹ FFU / for the K71 primary isolate. confirmed in ml.

Experimental Example  10. Vaccine Caution In piglets  Safety verification

In order to test the vaccine candidate careful safety in piglets, 80 for viral-based station Cain 10 corresponding to 100 times the amount of each attenuated the baeksinju the pigs are not fed with colostrum, when using a commercially available vaccine ^{5. 0} TCID Two weeks after oral inoculation with ₅₀ , an amount corresponding to 100 times the ED ₅₀ of each primary isolate was challenged.

For 14 days after vaccination, the clinical symptoms such as diarrhea and mortality were examined.

As a result of the experiment, as shown in Table 10, no clinical symptoms such as diarrhea were observed during the 2-week period after vaccination and during the 2-week period of vaccination (total 4 weeks). Diarrhea is completely prevented.

Experimental Example  11. Vaccine Caution In piglets  Efficacy Verification

To test the efficacy of vaccine strains in piglets, each attenuated vaccine strains, 174-1V-80, PRG942V-80, and K71V-80, were 100 times the ED ₅₀ of each primary pathogenic isolate in _50- day-old piglets that were not fed colostrum. (174-1V-80 for 5.6x10 ³ FFU, PRG942V-80 for 3.1x10 ⁴ FFU, K71V-80 for 3.1x10 ³ FFU) and 100 times of ED ₅₀ of each primary isolate after 2 weeks after oral inoculation. 174-1 strain 5.6x10 ³ FFU, PRG942 strain 3.1x10 ⁴ FFU, K71 strain 3.1x10 ³ FFU) were orally challenged. The animals were bred for 14 days after challenge.

For 28 days after vaccination and vaccination, the clinical symptoms such as diarrhea and mortality were examined daily. In addition, feces were taken daily for a total of 29 days before vaccination and after vaccination and vaccination, followed by cryopreservation. Serum was separated from blood collected weekly before vaccination and after vaccination and vaccination, followed by 30 ° C at 56 ° C. Immobilized for a minute and then frozen.

Around 28 days after the experiment was completed, each experimental piglet was euthanized, and the necropsy was examined for the presence of gross lesions. The organs of the digestive organs were collected and fixed in 10% neutral formalin. Sections of 3 μm thickness were prepared using the immobilized tissues, and hematoxylin and eosin stains were used to examine lesions under an optical microscope.

As a result, the blood serum collected weekly maintained ^2-4 times the antibody titer compared to the negative control until 1 week after vaccination of 174-1V-80, PRG942V-80 and K71V-80, but after 2 weeks Animals showed more than 2 ⁷ fold neutralized serum fold. That is, after neutralizing antibody titer was induced by each vaccine strain, the titer was boosted by challenge of virus challenge (Fig. 9).

On the observation of lesions, no abnormality was observed in the 174-1V-80 vaccine strain by visual inspection of gastrointestinal abnormalities. In addition, no abnormalities were observed in histological examination of pathological histological lesions of the digestive organs obtained by autopsy of the pigs in the non-vaccinated group without any vaccine or pathogenic virus.

7 days after the inoculation of 174-1 primary pathogenic piglets, the microscopic examination of pathological histological lesions of the digestive organs obtained by autopsy revealed that the villi of the duodenum, jejunum and ileum were severely atrophy. And fused, and the crypt was severely multiplied. On the other hand, two weeks after administration of the 174-1V-80 attenuated test vaccine containing the vaccine protection in piglets, the primary pathogenic strain (174-1) was autopsied 14 days after challenge (28 days after vaccination). The presence of pathological histological lesions of the digestive organs obtained through light microscopy showed a significant decrease in atrophy and fusion of the villi of the duodenum, jejunum and ileum, and the growth of the groin (Table 11).

After the inoculation of PRG942V-80 attenuated test vaccine with vaccine protection in piglets, the primary pathogenic strain (PRG942) was challenged and visually examined for gastrointestinal abnormalities.

7 days after inoculation of PRG942 primary pathogenic strain in piglets, histological examination of pathological histological lesions of the digestive organs obtained by autopsy revealed that the villi of the duodenum, jejunum and ileum were severely contracted and fused. It was. On the other hand, two weeks after administration of the PRG942V-80 attenuated test vaccine containing the vaccine protective agent in piglets, the primary pathogenic strain (PRG942) was inoculated about 14 days (28 days after vaccination). Histopathologic examination of the presence of histopathologic lesions revealed that the atrophy and fusion of the villi of the duodenum, jejunum and ileum, and the proliferation of the scrotum were significantly alleviated (Table 12).

After inoculation with the K71V-80 attenuated test vaccine containing vaccine protection in piglets, the primary pathogenic strain (K71) was challenged and visually examined for gastrointestinal abnormalities.

The microscopic examination of pathological histological lesions of the digestive organs obtained by autopsy around 7 days after inoculation of the K71 primary pathogenic strain in piglets showed that the villi of the duodenum, jejunum and ileum were severely contracted and fused. It was. Pathology of digestive organs obtained by autopsy at 14 days (28 days after vaccination) of the first pathogenic strain (K71) after inoculation with the K71V-80 attenuated single vaccine strain containing vaccine protection in piglets Examination of histological lesions by light microscopy showed a significant reduction in atrophy and fusion of the villi of the duodenum, jejunum, and ileum, and the growth of the groin (Table 13).

From the above results, 174-1V-80 attenuated live vaccine, PRG942V-80 attenuated live vaccine, and K71V-80 attenuated live vaccine were effective in preventing small intestine lesions by challenge of each primary pathogenic columnar column. It could be confirmed.

Experimental Example  12. By vaccine Attack virus Feces  Virus discharge through Inhibitory ability  inspection

Viral RNA was extracted from feces collected for a total of 29 days before vaccination and after vaccination and challenge, and then RT-PCR was performed using primer pairs specific to the Rotavirus VP6 gene to release virus through feces. Was examined.

Experimental Example  13. Vaccines and Vaccinations In piglets Serum neutralizing antibody  inspection

Serum neutralizing antibodies were tested using immobilized serum taken weekly before and after vaccine and challenge.

The collected test serum was immobilized at 56 ° C. for 30 minutes, and then 0.05 ml of serum was diluted in binary in cell maintenance culture medium in a 96-well microplate. Each vaccine was mixed with 10X trypsin at a ratio of 1: 9 and sensitized at 37 ° C. for 30 minutes, diluted with virus content of ² × 10 ² TCID ₅₀ /0.1ml, and sensitized with binary diluted antibody solution at 37 ° C. for 60 minutes. I was. 0.1 ml of each well of a 96-well plate in which MA104 cells were monolayer cultured, and then 0.1 ml of 2X trypsin was added and cultured at 37 ° C. for 5 days. Observation was performed daily under a microscope to determine the positive and negative effects of the cell degeneration effect. The serum titer was expressed as the reciprocal of the highest serum dilution factor that inhibited the cell degeneration effect.

As a result, IgM, IgG, and IgA antibodies specific for 174-1 weeks in blood showed a tendency to increase after decrease from 1 week after vaccination to 3 weeks after observation. On the other hand, IgG antibody titer increased from 14 days after vaccination to 21 days and then decreased. However, blood IgA antibody titer increased slightly at 3 weeks and decreased at 4 weeks, but was relatively lower than IgG and IgM antibody titers (see FIG. 10 (a)).

The antibody titer of PRG942V-80 vaccine strain in blood was tested by ELISA, and the serum IgM, IgG, and IgA antibodies were observed from 1 week after vaccination, and then increased rapidly after 2 weeks. . On the other hand, IgG antibody titer increased continuously after 2 weeks after vaccination. However, the blood IgA antibody titer was observed from 1 week after vaccination was confirmed to increase slightly to 4 weeks (see Fig. 10 (b)).

The antibody titer of the K71V-80 vaccine strain in the blood was tested by ELISA. The serum IgM, IgG, and IgA antibodies were rapidly elevated at 1 week after vaccination, and high until 3 weeks. Decreased. IgG antibody titers, on the other hand, increased gradually from 1 week of vaccination but were generally lower than IgM. The blood IgA antibody titer was slightly increased at 3 and 4 weeks after vaccination, but was found to be relatively low compared to the IgM antibody titer (see FIG. 10 (c)).

High levels of IgM and IgG antibodies were formed in the blood by vaccination and challenge, and the above results confirmed that the transmission of rotavirus through the blood could be blocked.

Experimental Example  14. Vaccines and Vaccinations In piglets Feces  My antibody is tested

Among feces collected daily before and after vaccination and vaccination, feces of 1 and 2 weeks after vaccination, 1 week of vaccination (3 weeks of vaccination) and 2 weeks of vaccination (4 weeks of vaccination) were selected. Then, the contents of IgM, IgG and IgA in feces specific to each rotavirus were measured by ELISA as follows.

That is, 10 ⁶ viruses were coated on each well of a 96-well plate for ELISA, and then left overnight at 4 ° C., followed by PBS containing 1% bovine serum albumin (BSA) (pH 7.4). Put was blocked for 1 hour at 37 ℃.

Each feces were diluted 10-fold with PBS, centrifuged through a 4 ° C. centrifuge and the supernatants collected. The fecal supernatant was further diluted with PBS containing 1% BSA, and then dispensed into each well of the 96-well plate for ELISA described above, and then sensitized at 25 ° C for 2 hours.

Thereafter, peroxidase-donated antibodies specific for IgM, IgG and IgA of pigs were dispensed into each well, and the respective antibody titers were measured by ELISA.

As a result, the antibody against fecal 174-1V-80 vaccine strain increased steadily after IgA was observed from 2 weeks after vaccination. However, it was confirmed that the antibody titers of IgM and IgG were rarely observed in feces (see (a) of FIG. 11).

The antibody titer of the PRG942V-80 vaccine strain was tested by ELISA. As a result, IgA was continuously observed from 2 weeks after vaccination, and a high increase was observed especially after vaccination. However, the antibody titer of IgG was slightly observed around day 21 in feces, and it was confirmed that IgM was not observed (see FIG. 11B).

 The antibody titer of the K71V-80 vaccine strain was examined by ELISA. As a result, IgA was observed from 1 week after vaccination and rapidly increased to 3 weeks after 4 weeks. However, antibody titers of IgM and IgG were hardly observed in feces (see FIG. 11 (c)).

The results showed that secreted IgA, which plays a central role in neutralizing rotavirus in the intestine, was observed from day 7 by oral vaccination of each swine rotavirus vaccine strain, thereby effectively defending the attacker. .

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown by the following claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. Should be.

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13429

Deposit Date: 20171215

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13430

Deposit Date: 20171215

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13431

Deposit Date: 20171215

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> MIXED LIVE ATTENUATED VACCINE COMPOSITION FOR PROTECTING PORCINCE          ROTAVIRUS INFECTION <130> 09009 <160> 52 <170> KoPatentIn 3.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP1F forward <400> 1 ggctattaaa gctrtacaat ggggaag 27 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP1F reverse <400> 2 tcccactggg amacgtctgt atat 24 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP1F forward <400> 3 gaattctact cacagtcaaa t 21 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP1F reverse <400> 4 ggtcacatct aagcgctcta atctts 26 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP1-5 reverse <400> 5 cgagttcatt cgccgtcaaa tctgcttc 28 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP1-3 forward <400> 6 tttcactagg agtcccacca gttgatgc 28 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> VP2 forward <400> 7 ggctattraa ggytcaatgg cgtacag 27 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> VP2 reverse <400> 8 tttctataat gcattckttg catt 24 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VP2 forward <400> 9 ataaaytcac aagcagcaaa tga 23 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP2_Rbc reverse <400> 10 gtcatatctc cacartgggg ttgg 24 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP2-5 reverse <400> 11 cataatctcc atctggcagc gtgtctct 28 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP2-3 forward <400> 12 ctggatgcta cagttttcgc ccagatag 28 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VP3 forward <400> 13 ggctttttaa agcaatatta gta 23 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VP3 reverse <400> 14 atggtgtgtc caatggatcc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VP3 forward <400> 15 ggatccattg gacacaccat 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VP3 reverse <400> 16 ggtcacgacc tgaccatggt g 21 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP3-5 reverse <400> 17 attatagtga cctcgcgtgt cctttcca 28 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> VP3-3 forward <400> 18 gatcagccaa agagtttgct gcgttg 26 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP4F forward <400> 19 ggctataaaa tggcttcgct ca 22 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP4F reverse <400> 20 atttcggacc atttataacc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP4F forward <400> 21 ggttataaat ggtccgaaat 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP4F reverse <400> 22 ggycwcaacc tctagacact 20 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP4-5 reverse <400> 23 ccctctacag ttggcgcaag tagtaccc 28 <210> 24 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP4-3 reverse <400> 24 caataggatc atcagcatcc gcttggac 28 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP6F <400> 25 ggctttwaaa cgaagtcttc 20 <210> 26 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GEN-VP6R <400> 26 ggtcacatcc tctcact 17 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP6-5 reverse <400> 27 gagctattcc gtttcgttgc gactctct 28 <210> 28 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP6-3 forward <400> 28 gtgttcccac caggtatgaa ttggacag 28 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VP7 forward <400> 29 gcctttaaaa gcgagaattt 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VP7 reverse <400> 30 ggtcacatca tacaactcta 20 <210> 31 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP7-5 reverse <400> 31 gagctttaat gagcggtgca agtacgac 28 <210> 32 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> VP7-3 forward <400> 32 tgtcgctgta attcaggtag gaggtcca 28 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LAP-NSP1-F <400> 33 gggctttttt ttgaaaagtc 20 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VF5R <400> 34 ggtcacattt tatgctgcct a 21 <210> 35 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> NSP1-5 reverse <400> 35 catgatacat ggtacagcct cgacag 26 <210> 36 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NSP1-3 forward <400> 36 gccactgagg tacacaactg caaatgg 27 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VF3F <400> 37 ggcttttaaa gcgtctcagt c 21 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VF3R <400> 38 ggtcacataa gcgctttcta ttc 23 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NSP2-5 <400> 39 cttcagcagt ggcagtggtt tcaatttc 28 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NSP2-3 <400> 40 cacgcagaca gagtattcgc taca 24 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VF2F <400> 41 atgctcaaga tggagtctac t 21 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VF2R <400> 42 ggtcacataa cgcccctata g 21 <210> 43 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NSP3-5 reverse <400> 43 agtgcctgat caatagtcgc agctttgc 28 <210> 44 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NSP3-3 forward <400> 44 gaggtccatg gaattgtcag atgatgtc 28 <210> 45 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 10Beg16 <400> 45 tgttccgaga gagcgcgtg 19 <210> 46 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 10End722c <400> 46 gaccattcct tccattaac 19 <210> 47 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NSP4-5 reverse <400> 47 cgaacacttc gacgttctca acgctatt 28 <210> 48 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NSP4-3 forward <400> 48 ctatgtgaga ggttgagttg ccgtcgtc 28 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VF1F <400> 49 ggcttttaaa gcgctacagt g 21 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VF1R <400> 50 ggtcacaaaa cgggagtggg 20 <210> 51 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NSP5-5 reverse <400> 51 gatcgcaccc aacgttactt gaaggtc 27 <210> 52 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NSP5-3 forward <400> 52 gtgcgatcaa gtggatttct ccctgact 28

Claims (10)

A vaccine composition for preventing swine rotavirus infection, comprising attenuated strain of swine rotavirus strain 174-1V, attenuated strain of K71V, and attenuated strain of PRG942V. The attenuated strain of swine rotavirus strain 174-1V is 174-1V-80 (Accession Number: KCTC 13429BP), and the attenuated strain of swine rotavirus strain K71V is K71V-80 (Accession Number: KCTC). 13430BP), and the attenuated strain of the swine rotavirus strain PRG942V is PRG942V-80 (Accession Number: KCTC 13431BP) is a vaccine composition for preventing swine rotavirus infection. A live vaccine for preventing swine rotavirus infection, comprising the vaccine composition for preventing swine rotavirus infection of claim 1. The live vaccine of claim 3, further comprising a vaccine protector, an immune enhancer, or a combination thereof. The live vaccine of claim 4, wherein the vaccine protecting agent is a lactose phosphate glutamate gelatin mixture. An inactivated vaccine for preventing swine rotavirus infection, comprising the vaccine composition for preventing swine rotavirus infection of claim 1. 7. The inactivated vaccine for preventing swine rotavirus infection according to claim 6, wherein each virus line of the vaccine composition is formalin-treated. A method of inducing a protective immune response against swine rotavirus infection in a pig comprising administering to the pig an immune effective amount of the live vaccine of claim 3 or the inactivated vaccine of claim 6. Administering the live vaccine of claim 3 or the inactivated vaccine of claim 6 to sows in late pregnancy; And
A method of inducing a protective immune response against swine rotavirus infection in pigs, comprising feeding colostrum of the sows to piglets delivered from the sows. Attenuated strain of swine rotavirus strain 174-1V 174-1V-80 (accession number: KCTC 13429BP), attenuated strain of swine rotavirus strain K71V K71V-80 (accession number: KCTC 13430BP) and attenuated strain of pig rotavirus strain PRG942V Mixed strain consisting of PRG942V-80 (Accession Number: KCTC 13431BP).

Images