KR101654023B1 - Live vaccine composition, inactivated vaccine composition and oral vaccine comprising the same from currently isolated attenuated porcine epidemic diarrhea virus - Google Patents

Abstract

The present invention relates to an attenuated porcine epidemic diarrhea virus (PEDV) and a vaccine composition against the PEDV comprising the same and, more specifically, to: an attenuated PEDV (Accession No. : KCTC 12886BP); a vaccine composition and a vaccine comprising the attenuated PEDV; a dispenser capable of administering the vaccine to pigs, and a kit comprising the same and a composition for preventing PEDV infection; and a method for inducing protective immune response against porcine epidemic diarrhea (PED). A live attenuated vaccine and the inactivated vaccine of the present invention have more excellent safety and immunogenicity than existing vaccines, and are more effectively used for preventing pigs from being infected by new varieties according to genetic modification of the PEDV, thereby being able to be used for preventing the PED infection and inducing protective immune response against the PED. In addition, the live attenuated vaccine and the inactivated vaccine of the present invention can minimize damages of economic loss caused by weight loss of pigs when the pigs are recovered after being infected by the PED which is concerned by hog raising farms by effectively reducing the death rate of pigs caused by the recently spread PEDV and remarkably reducing diarrhea symptoms, and thus induce stabilization of the hog raising farms and satisfy both producers and consumers, thereby having remarkably high industrial availability.

Description

TECHNICAL FIELD [0001] The present invention relates to a vaccine composition for vaccination against porcine epidemic diarrhea virus and a vaccine composition for oral administration using the vaccine composition and an inactivated vaccine composition comprising the same,

The present invention relates to a highly virulent porcine epidemic diarrhea virus vaccine and a porcine epidemic diarrhea vaccine composition containing the vaccine, which are popular in Korea since the end of 2013, Accession No. KCTC 12886BP), a vaccine oral administration composition comprising the vaccine composition, and a composition for preventing infectious influenza virus infection of a live vaccine and an inactivated vaccine, to an effective defense immune response inducing method against swine diarrheal disease.

Porcine Epidemic Diarrhea (PED) is one of the most important viral diseases that cause enormous damage to the swine industry in Asian countries including Korea. It is not only Asia but also North America and South America, including Peru, Chile has also reported serious damage reports.

This disease is especially caused by the dehydration of diarrhea due to the occurrence of pediatric pre - hay fever in winter, and it has seasonal characteristics mainly in winter and early spring. Recently, however, the disease has also occurred in other seasons than the winter season, and the onset pattern is becoming more diverse. After the first reports of UK and Belgian PED viruses in Europe at the end of the 1970s, Europe did not occur in the 1990s, but China and Japan caused the disease in the 1980s and caused massive damage to piglets. It occurs mainly in the southeastern part of the back ground. In Korea, it first occurred in 1992 and continues to occur. A number of papers have been published that suggest that vaccine-resistant pig virus-associated diarrhea viruses, which start from China at the end of 2010 and are not prevented by vaccination, have killed about 700,000 piglets, To date, it has recorded about 8 million infant deaths in the United States and has affected the economy in the United States. In Korea, low infections (160 cases, 55,109 cases) were reported from 2005 to the first half of 2013, but high infections and damage from the end of 2013, when the influenza pandemic strain was introduced from the United States (Number of outbreaks: 231, number of infections: 46,540).

However, the pig farmers in the first place are avoiding reporting this disease because they belong to three kinds of livestock infectious diseases in Korea and because of the reporting duty and restriction of the livestock movement of the farms, the secondary economic loss of farms occurs. Experts in pig farming claim that up to 40% of the entire pig farm is infected with this disease, and estimates of 20-25% infection can be estimated by comparing experts' opinions with reported accurate statistics, You can judge that you have seen the damage. This variant is introduced at the end of 2013, which is similar to ours in Japan, and caused damage to 1.5 million piglets in Japan and the same period in Taiwan by the beginning of 2015, causing hundreds of thousands of damages. This is causing the economic wave that consumers are burdened with pork consumption.

The porcine epidemic diarrhea virus (PEDV) is an RNA virus belonging to the genus Coronaviridae, which is mutated very rapidly due to the nature of the RNA virus and has a genetic variation between the wild type and the vaccine strain causing disease in domestic farms It is known. The causative agent of this disease is coronaviridae coronavirus, and PEDV has various forms with an envelope size of (60-) 120-160 (-200) nm. Although it is known that the gene consists of about 20,000-30,000 genes composed of spike protein gene and surface gene transcriptional gene, systematic and molecular biological approach to this virus has not yet been developed yet . PEDV is composed of about 5 structural proteins. In other words, the surface glycoprotein (or spike, S) involved in the contact of the virus with host cells and hemagglutination and membrane fusion. Integral membrane protein (M) is a 50-60 kDa protein that crosses the virus surface three times. In addition, the fourth-largest Hemagglutinine-Esterase protein (HE) of about 65 kDa is protruded on the surface and is known to function like receptor binding and hemagglutination. There is also a nucleocapsid protein and another small protein that is not known for its function. However, there is no study on these structural proteins, and so far, it is a reality that depends on speculation based on similarity with other coronaviruses. Among these structural proteins, spike protein plays an important role in virus infection. It is known that the antigenicity of PEDV is determined by spike. The antigen specificity is checked by neutralization test using S or complement fixation test using M. Therefore, identifying the role of spike protein in PEDV infection and its relationship with trypsin is an important research area for the prevention of PED.

The most obvious way to prevent damage from swine diarrhea infection is through disinfection and disinfection of animals related to livestock and livestock. The second is vaccination through proper vaccination. In Korea, PED The disease is designated as a third class infectious disease and is managed. Live vaccine, inactivated vaccine and oral vaccine are commercialized and distributed to farm households. However, these vaccines are mainly vaccines developed 14-15 years ago in the early 2000s, and the recently emerging PED virus from the US in 2013 is a highly genetically engineered and genetically modified vaccine that is currently used in the country. (In July 2014, the Ministry of Agriculture, Forestry and Fisheries Quarantine Headquarters announced the evaluation of efficacy for five Korean companies and imported PED vaccines). In addition, existing vaccine strains have genetic differences as well as immunogenicity and are known to form antibodies only after several vaccinations (Korean J. Vet. Serv. 32: 3 201-207). In addition, live vaccines currently distributed are not only inferior in immunogenicity but also vaccinated through oral administration. Oral administration method is a very non-scientific method in which a vaccine diluted solution is fed to pigs and fed to pigs. The digestion of gastric juice and digestive enzymes Is a vaccine that does not have scientific evidence of whether the vaccine virus is transmitted to the small intestine and causes an immune response.

Korean Patent No. 10-1281361 Korean Patent Publication No. 10-2008-0082883

Therefore, the present inventors have been studying PED virus strains for the production of vaccine against new influenza viruses, which have excellent immunogenicity and safety and are able to reflect genetic mutations, in a recent fashionable mutant strain, and isolate new strains of pandemic strains of diarrhea virus , And confirming their defense power, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a vaccine for vaccination against porcine epidemic diarrhea virus (PEDV) (accession number: KCTC 12886BP).

It is another object of the present invention to provide a live vaccine composition of swine epidemic diarrhea comprising the virus strain.

Another object of the present invention is to provide a vaccine which comprises the above live vaccine composition.

It is another object of the present invention to provide a method of inducing a protective immune response against swine epidemic diarrhea in a swine comprising administering to a swine an immunologically effective amount of a vaccine characterized in that it comprises the live vaccine composition Thereby providing an induction method of an immune response.

Another object of the present invention is to provide an inactivated vaccine composition of a swine fever diarrhea virus comprising the virus strain.

Another object of the present invention is to provide a vaccine which further comprises the inactivated vaccine composition and an immunity enhancer.

A further object of the present invention is a method of inducing a protective immune response against swine epidemic diarrhea in a swine comprising administering to the swine an immunologically effective amount of a vaccine characterized in that it comprises the inactivated vaccine composition Thereby providing a method for inducing a protective immune response.

Another object of the present invention is

(1) a dispenser capable of administering the vaccine to the pig;

(2) a kit for preventing swine influenza virus infection comprising the vaccine of (3) or (6).

To achieve the above object, the present invention provides a porcine epidemic diarrhea virus (accession number: KCTC 12886BP).

In order to achieve another object of the present invention, there is provided a live vaccine composition for a swine diarrheal disease comprising the virus strain.

In order to accomplish another object of the present invention, there is provided a vaccine comprising the above live vaccine composition.

In another aspect, the present invention provides a method of inducing a protective immune response against swine epidemic diarrhea in a swine comprising administering to a swine an immunologically effective amount of a vaccine comprising the live vaccine composition Lt; RTI ID = 0.0 > immune < / RTI > response.

In order to accomplish another object of the present invention, there is provided an inactivated vaccine composition for a porcine epidemic diarrhea comprising the virus strain.

In order to accomplish still another object of the present invention, the present invention provides a vaccine further comprising the inactivated vaccine composition and the immunity enhancer.

In one embodiment of the present invention, the virus inactivation may be achieved by adding formalin.

In accordance with another aspect of the present invention, there is provided a method of inducing a protective immune response against swine epidemic diarrhea in a swine comprising administering to the swine an immunologically effective amount of a vaccine comprising the inactivated vaccine composition Or a pharmaceutically acceptable salt thereof.

In order to achieve another object of the present invention,

(1) a dispenser capable of administering the vaccine to the pig;

(2) a composition for preventing a porcine epidemic diarrhea virus infection comprising the vaccine.

In one embodiment of the invention, the dispenser is capable of dispensing its contents; If the composition is inoculated intramuscularly, it can prevent a swine epidemic diarrhea infection.

The live vaccine or inactivated vaccine and the oral vaccine using the vaccine of the present invention of the present invention have superior safety and immunogenicity compared to the conventional vaccine and prevent infection of a new strain due to genetic mutation of the pandemic seasonal diarrhea virus .

Figure 1 shows the PEDV positive samples identified by RT-PCR.
Fig. 2 is a graph showing the cytopathic effect (CPE) by PEDV-CUP-B1406 strain.
FIG. 3 is a diagram showing phylogenetic tree results based on analysis of gene homology with other PEDV strains of whole spike protein of PEDV-CUP-B1406 strain.
FIG. 4 is a diagram showing the result of confirming the virus activity through continuous passage of PEDV-CUP-B1406 virus. FIG.
FIG. 5 is a graph showing the results of confirming the neutralizing antibody value of PEDV-CUP-B1406 strain-inoculated pigs.
FIG. 6 is a graph showing antibody titers in mouse blood against PEDV-CUP-B1406 strain inactivated vaccine by ELISA method. FIG.
FIG. 7 is a graph showing neutralization antibody titer of Vero cells in mouse blood antibody against PEDV-CUP-B1406 strain inactivated vaccine.
FIG. 8 is a graph showing the levels of IFN-γ in mouse blood against PEDV-CUP-B1406 strain inactivated vaccine by ELISA.
FIG. 9 shows the results of comparing weight changes after inoculation with live pigs, inactivated vaccines, and oral vaccines against piglets born after inoculation.
FIG. 10A is a graph showing a result of intestinal biopsy of piglets after inoculation with the piglets. (A: Company (SM98) inactivated vaccine)
FIG. 10B is a graph showing a result of intestinal biopsy of piglets after the challenge with the piglets. (B: PEDV-CUP-B1406 vaccine inoculation of domestic isolate inactivated)
FIG. 10c is a graph showing a result of intestinal biopsy of piglets after inoculation with the piglets. (Negative: negative control, positive: positive control)

The present invention provides a porcine epidemic diarrhea virus (PEDV) vaccinator (Accession No: KCTC 12886BP).

The porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is a single-stranded RNA virus with an envelope that causes severe diarrhea in pigs .

A vaccine shorter or attenuated virus refers to a virus which, when administered to an animal, indicates that the clinical symptoms of the disease are absent or diminished. The vaccine shippers of the present invention can be produced by methods known in the art, for example, Lt; / RTI >

The present invention provides a live vaccine composition of swine epidemic diarrhea comprising the virus strain.

The present invention provides a vaccine characterized in that it comprises the live vaccine composition.

A live vaccine composition refers to a composition comprising a live virus or active ingredient thereof.

Vaccine means a pharmaceutical composition containing at least one immunologically active ingredient that induces an immunological response in an animal. The immunologically active component of the vaccine may contain appropriate elements of a live virus or dead virus (subunit vaccine), whereby these elements destroy the entire virus or its growth culture, and then the desired construct (s) Or by isolation and purification processes induced by appropriate manipulation of a suitable system such as bacteria, insects, mammals or other species, or by appropriate purification of the pharmaceutical composition (Polynucleotide vaccination) by induction of the above synthetic process in an animal in need of vaccination by direct incorporation of the genetic material. The vaccine may comprise one or more of the elements described above.

The vaccine may be in any form known to those skilled in the art, for example, but not limited to, a solid form suitable for the form or suspension of liquids and injections. Such formulations may also be emulsified or encapsulated into liposomes or soluble glass, or may be prepared in the form of aerosols or sprays. They may also be included in transdermal patches. For liquids or injections, it may contain, if necessary, propylene glycol and sodium chloride in an amount sufficient to prevent hemolysis (e.g., about 1%).

The vaccine of the present invention may further comprise a pharmaceutically acceptable carrier or diluent. The term "pharmaceutically acceptable" as used herein refers to a non-toxic composition which is physiologically acceptable and which, when administered to a pig, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, .

Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. The carrier may be an aqueous solution, or a non-aqueous solution, suspension or emulsion. As immunosuppressants for increasing the immunogenicity, there may be used orthopedic or amorphous organic or inorganic polymers and the like. Immune adjuvants are known to play a role in promoting immune responses through chemical and physical linkage to antigens in general. The immunosuppressants used in this study were atypical aluminum gels, oil emulsions, dual oil emulsions and immunosols. Various plant-derived saponins, levamisole, CpG dinucleotide, RNA, DNA, LPS and various types of cytokines have been used to promote the immune response. Such an immunogenic composition can be used as a composition for inducing an optimal immune response by a combination of various adjuvants and an immune response promoting additive. In addition, as a composition to be added to the vaccine, a stabilizer, an inactivating agent, an antibiotic, a preservative, etc. may be used. Depending on the route of administration of the vaccine, the vaccine antigen may be mixed with distilled water, buffer solution or the like.

The vaccine may be administered through the route of administration such as oral, muscular, subcutaneous, etc., but is not limited thereto, and may be administered through a muscle administration route.

The present invention provides a method of inducing a protective immune response against swine epidemic diarrhea in a swine comprising administering to the swine an immunologically effective amount of a vaccine characterized in that it comprises the live vaccine composition Lt; / RTI >

The immune response may be an antibody, B cell, helper T cell, suppressor T cell, cytotoxic T cell, and gamma-delta T cell specifically directed against antigens or antigens contained in the vaccine composition or vaccine comprising the vaccine But are not limited to, one or more of production or activation, efficacy of a therapeutic or protective immunological response in a host to increase tolerance to a new infection or decrease the clinical severity of the disease. Preferably a protective immune response.

Such protection is evidenced by a reduction or absence of clinical signs typically exhibited by the infected host, a faster recovery time or a lower duration or a lower virus titer in the tissues or fluids or feces of the infected host.

The effective amount is an amount of a vaccine capable of inducing an immune response to reduce the frequency or severity of a pandemic influenza virus infection in a pig, and can be appropriately selected by those skilled in the art. For example, an effective amount of the vaccine is 1 x 10 ⁴ to 1 x ₁₀ ⁷ TCID ₅₀ / ml per day, preferably 1 x 10 ⁵ to 1 x 10 ⁶ per day TCID < / RTI > ₅₀ / ml.

The immune response inducing method may be, but is not limited to, inoculating the vaccine with oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous routes. Preferably, it may be intramuscular inoculation of the vaccine during primary and secondary inoculation.

The present invention provides an inactivated vaccine composition for swine epidemic diarrhea comprising the virus strain.

The present invention provides a vaccine, further comprising the inactivated vaccine composition and an immunity enhancer.

An inactivated vaccine composition means a composition comprising a dead virus or an active ingredient thereof, and the vaccine comprising the composition can be prepared by methods known in the art. For example, if the virus is highly proliferating, the virus can be obtained and then treated with formalin, beta proprio lactone (BPL), binary ethyleneimine (BEI) or gamma radiation, or by other methods known to those skilled in the art Deactivated. The inactivated virus is then mixed with a pharmaceutically acceptable carrier (such as a saline solution) and optional adjuvants. Preferably, it may be inactivated at a final concentration of 0.1% formalin.

Immune enhancers or adjuvants include any absorption-promoting agent that refers to a compound or mixture that promotes the enhancement of the immune response and the rate of absorption after inoculation. But are not limited to, auxiliary molecules such as, for example, aluminum hydroxide, mineral oil, or other oils or vaccines, or those that are generated by the body after each induction by such additional components, such as, but not limited to, interferon, interleukin Or growth factor.

A method of inducing a protective immune response against swine epidemic diarrhea in a swine comprising administering to the swine an immunologically effective amount of a vaccine characterized in that it comprises the inactivated vaccine composition, Provides a derivation method of the reaction.

The effective amount is an amount of a vaccine capable of inducing an immune response to reduce the frequency or severity of a pandemic influenza virus infection in a pig, and can be appropriately selected by those skilled in the art. For example, an effective amount may be 1 x 10 ⁴ to 1 x ₁₀ ⁷ TCID ₅₀ / ml per day, preferably 1 x 10 ⁵ to 1 x 10 ⁶ per day for a virus strain containing the inactivated vaccine composition TCID < / RTI > ₅₀ / ml.

The present invention

(1) a dispenser capable of administering the vaccine to the pig;

(2) a composition for preventing a porcine epidemic diarrhea virus infection comprising the vaccine.

The dispenser may be, but is not limited to, a syringe or needleless device such as a Piget or Biojector, and preferably a syringe.

The vaccine composition of the present invention and vaccines comprising the same can be used in combination with a kit for preventing swine epidemic diarrhea virus infection, i.e., packaged in predetermined amounts together with instructions for use. The composition for preventing swine epidemic virus infection can be in a form known to those skilled in the art, for example, in the form of a solid suitable for a liquid or an injectable form or suspension, but is not limited thereto. It may also contain an excipient which, depending on its form, will provide a solution having an appropriate concentration upon dissolution, if desired.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

Outdoor week  Isolation and identification of viruses

<1-1> Identification of virus

In April 2014, we collected severe fecal diarrhea in the piglet pigs in Boryong, Chungnam Province, and collected fecal samples and small intestine samples from dead pigs. The samples were homogenized in PBS (phosphate buffer saline, pH 7.4) ) And centrifuged at 3,000 rpm for 10 minutes to recover the supernatant. The recovered supernatant was aseptically filtered using a syringe filter (pore size 0.2 μm, Sartorius), and a sample was prepared. An RNA sample was collected from the sample using a viral RNA extraction kit (Qiagen) (SEQ ID NO: 1, GGT ACC ATG) and PED M_AS (SEQ ID NO: 2, GGA TCC TTA GAC TAA ATG AAG CAC) primers that specifically react with PEDV, PCR was performed. As a result, a DNA of about 850 bp, which is a PEDV specific band, was detected as shown in Fig.

<1-2> Outdoor main separation

RT-PCR of Example 1-1 was used to perform PED outdoor main separation using a sample showing a positive reaction. Vero cells (ATCC, ATCC: Vero-CCL81) were used to isolate the pediatric bovine spongiform encephalopathy. The sample filtrate showing RT-PCR positive was inoculated into a Vero cell in which a monolayer was formed in a cell culture flask (Nunc., 25 cm 2). Cells were washed 2-3 times with serum-free cell culture medium (alpha-MEM, Sigma-aldrich) prior to inoculation and 1 ~ 2 ml of positive filtrate was inoculated into the cells. The cells were adsorbed onto the cells with gentle shaking at 37 ° C for 1 hour for virus infection. After virus adsorption, cells were cultured in a 37 ° C CO 2 incubator supplemented with virus medium (α-MEM containing 0.02% yeast extract, 0.3% TPB and 5 μg / ml of Trypsine) and observed for cytopathic effects Respectively. During the observation period, the culture medium in which the cytopathic effect was more than 80% was collected and used for next subculture while being frozen at -80 ° C. Some culture samples were taken during subculture and RNA was collected and confirmed by the RT-PCR method while confirming the presence of PEDV.

As a result, as shown in Table 1, there were various results on a sample-by-sample basis. In particular, PEDV-CUP-B1406 sample showed a weak positive response in passage 4 and virus was continuously grown from passage 6, Formed a cytopathic effect in Vero cells and finally succeeded in separation and named PEDV-CUP-B1406 strain (see Fig. 2).

<1-3> Genetic analysis of isolated virus

Genetic analysis was performed to distinguish whether the PEDV isolated in Example 1-2 was actually outdoor or vaccinia. Genetic analysis analyzed the spike protein gene, which is known to be the only surface protein of PEDV. For this purpose, primers for PEDV spike protein gene, PED S1_S (SEQ ID NO: 3, GGT ACC ATG AGT TCT TAA ATT TAC) and PED S1_AS (SEQ ID NO: 4, ACC ACC AAA AGC CGC AGA GAC AGT) Were performed to perform gene amplification and then analyzed by gene sequencing analysis of these genes.

In order to analyze how the PEDV isolated in Example 1-2 was actually homologous to other PEDVs after gene sequencing, it was compared with the SM98 and DR13 strains isolated from Korea As a result, the PEDV-CUP-B1406 strain showed 93.1% and 93.7% homology only, respectively, and the homology was significantly different from that of KNU-0905 isolated in 2009 (95.8%). In particular, the PEDV-CUP-B1406 strain showed 97.6-99.6% homology with the recent populations in the US, Canada, Japan, Taiwan, and China, confirming it as a new mutant in Korea that did not occur before the end of 2013 .

Based on these gene homologous results, phylogenetic tree analysis of the phylogenetic tree showed that the different vaccine strains belonged to group 1a, whereas the PEDV-CUP-B1406 strain belonged to group 2b (FIG. 3) ).

&Lt; Example 2 >

PEDV -CUP-B1406 Continuous virus Pass  Virus through culture Potency  Confirm

&Lt; 2-1 >

The PEDV-CUP-B1406 strain isolated in Example 1 was subjected to PEDV subculture in the same manner as in Example 1. Three days after the virus inoculation, if the typical cell-denaturing effect of PEDV as shown in FIG. 2 is observed, the cells and culture medium are recovered and opened three times. The virus is exposed to the culture supernatant, Subculture was carried out, and 1/10 dilution of the stock solution was inoculated.

<2-2> Measurement of PEDV titers

The harvested virus cultures were counted on a scale of 5 generations. Reactive and Muench methods were used for the titration method. Samples taken for the titration were cryopreserved at -80 ° C, and ten samples were subjected to titration at once. Vero cells pre-incubated in 96-well plates (Nunc.) Were washed with sterile PBS (pH 7.4) and incubated with virus-containing medium (α-MEM containing 0.02% yeast extract, 0.3% TPB and 5 μg / ), 0.1 ml of the PEDV sample to be measured for titer is diluted in a decane and 0.1 ml is inoculated into each well and adsorbed at 37 ° C for 1 hour. After the adsorption was completed, the inoculated sample solution was removed, and 0.1 ml of a new virus culture medium was added to each well, and the cell transformation effect was observed every day at 37 ° C in a CO2 incubator. The inverse number of the maximum virus dilution factor was indicated by TCID ₅₀ (50% Tissue Culture Infective Dose) (see FIG. 4).

&Lt; Example 3 >

Cultured PEDV Pathogenicity test of the CUP-B1406 strain

<3-1> Preparation of Virus Care for Pathogenicity Test

Virus concentrations were adjusted to 10 TCID50 / ml for 15, 40, 65, and 95 virus strains that were subcultured to conduct pathogenicity tests according to passage number. Initial culture virus lacking viral titers was diluted by ultracentrifugation (30,000 rpm, 5 hr, 4 ℃) to dilute the concentrated virus with PBS.

<3-2> Pathogenicity test

Pigs were born from sows who were not infected with a pandemic strain of diarrhea and were not vaccinated. PEDV-CUP-B1406 strain viruses prepared from these piglets were orally inoculated with various subculture viruses to confirm clinical signs and viruses. The PEDV infection status of the sows was confirmed by the PEDV antibody in the blood of the sows and RT-PCR of the fecal samples to be negative for PEDV. Five pigs per group and five non-inoculated control groups were divided into three groups according to the number of each subculture. 1.0 ml (10 TCID50 / ml) of PEDV virus prepared according to the number of subcultures was mixed with 5.0 ml of saline and immediately administered orally to each group. Oral administration of 5.0 ml of saline was carried out in the non-inoculated control group. After oral administration, the virus symptoms were observed by RT-PCR on the clinical symptoms and feces of piglets on a daily basis. In addition, diarrhea was scored and the intensity of diarrhea was measured. Scores were measured as 0, 1, 2, 3, 0 for normal feces, 1 for very mild diarrhea, 2 for moderate diarrhea, and 3 for very severe diarrhea.

The results showed that diarrhea symptoms were observed from the 1st day after inoculation in 3 piglets (1-2, 1-6, 1-8) that had been orally administered PEDV-CUP-B1406 strain cultured in 15 passages, Was confirmed by RT-PCR (see Table 3). In the internal organ examination by autopsy, four of five (80%) of the diarrhea died, and symptoms such as hydronephrosis, intestinal gas and small intestinal hemorrhage were typical symptoms due to PEDV infection. And calcification was observed in the case of dehydration.

Even in the case of inoculation with PEDV cultured in the 40s, no diarrhea was observed from day 1 after inoculation, but the presence of virus was confirmed by RT-PCR of the feces collected from 2-2 and 2-8 mammal piglets. From the second day of the attack, severe diarrhea was observed, and 3 out of 5 mice died at 6-7th day. Autopsies on the lungs showed typical symptoms of PEDV infection due to diarrhea-induced severe dehydration.

In the PEDV-inoculated group of the 65th passage, virus release through the feces was observed from the second day after inoculation, but the degree of diarrhea was much weaker than that of the group 1 and 2 pigs.

In the case of PEDV cultured in the 95th passage, no clinical signs of diarrhea were observed after inoculation, and a small amount of virus was excreted in the feces from 2 to 4 days after inoculation.

Thus, the PEDV-CUP-B1406 strain cultured in the passage of the 95th strain was used in the following experiment.

<Example 4>

PEDV Immunogenicity of the CUP-B1406 strain

In order to confirm the immunogenicity of PEDV-CUP-B1406 strains in the 95th passage in Example 3, the antibody-forming ability was tested using PEDV-negative pigs. A total of 15 pigs immunized with pig swine diarrhea antibody 8 to 10 kg body weight were divided into 3 groups of 5 pigs per group and tested with group 1 live vaccine, group 2 inactivated vaccine and group 3 control. At the interval of 2 weeks, 1 ml of 10 TCID 50 / ml concentration of live virus was mixed with IMS1313 adjuvant of 10 TCID 50 / ml concentration of inactivated virus, And the antibody value and neutralizing antibody value were confirmed by ELISA. ELISA antibody was prepared by using whole virus as a coating antigen, and neutralization antibody was evaluated by neutralizing test using the general SN test method. The number of cells was adjusted so that the number of Vero cells was 3 x 10 per ml in the growth medium. Serum of collected blood was separated, denatured at 57 ° C for 30 minutes, and then binarized with MEM medium containing 10 ug / ml of trypsin. An equal volume of 200 TCID50 / 0.1 ml PED virus was mixed with each diluted serum and neutralized at 37 DEG C for 90 minutes. 0.1 ml of the neutralized solution and 0.1 ml of the cell suspension were added to the wells of the Miroplate, followed by incubation for 5 days in a 37 ° C CO ₂ incubator. The maximum dilution factor of the serum that inhibits the cell degenerating effect on the cultured cells was used as a neutralizing antibody (see FIG. 5)

&Lt; Example 5 >

PEDV Preparation and immunological characterization of test vaccine using -CUP-B1406 strain

<5-1> Preparation of test vaccine

To prepare the inactivated vaccine, Vero cell line was infected with PEDV-CUP-B1406 strain, and the supernatant of the infected cells was centrifuged at 3,000 rpm to remove cell debris. The virus titer was fixed at the same level, For 2 hours to harvest the virus pellet. The virus pellet was resuspended in PBS and formalin was added to a final concentration of 0.1% and inactivated at 4 ° C for at least 24 hours. In order to confirm virus inactivation, MDCK monolayer was infected and it was confirmed that no plaque was formed. After the inactivation confirmation, the viral solution was mixed with the adjuvant at a ratio of 7: 3, and a test vaccine was prepared by adding Qual A (Sigma Aldrich) to 10 μg / ml. The final concentration of PEDV antigen in the test vaccine is at least 10 TCID50 / ml.

In order to make a live vaccine, the PEDV-CUP-B1406 strain for 95 generations was centrifuged at 3,000 rpm to remove the cell debris. The viral titres were then made the same, and then the live vaccine strain (10 ⁵ TCID ⁵⁰ / ml / dose). The final concentration was also adjusted to 10 ⁶ TCID50 / ml / dose by inoculating the live vaccine strain for oral use.

Positive controls for the comparative tests were K PED inactivated vaccine using SM98 strain, PED live vaccine, and N oral vaccine.

K Co., Ltd. used the Goryeo Biotech Pandemic Power Pandemic PED vaccine and the oral vaccine of N Corp. belongs to the genotype group 1a of the oral PED vaccine of Green Cross Co., Ltd., . However, as of the beginning of 2014, Group 2b evaluated the efficacy of the vaccine at the national level due to the domestic pandemic and the vaccine distrust of pig farmers. As a result, the Ministry of Food, Agriculture, Forestry and Livestock Was found to be inferior in efficacy.

<5-2> Mouse Inoculation

PEDV-CUP-B1406 test inactivated vaccine and K-inactivated vaccine were inoculated to the left footpad at a dose of 0.1 ml in female BALB / c mice (ORIENT BIO Co., Ltd.). Groups were divided and booster was given at 3 weeks after the first immunization. Blood samples were collected every week.

<5-3> Identification of antibody titers in mouse blood

The most popular PED antigen (200 ng / well) (genotype type 2b) was coated on 96 wells and incubated overnight at 4 ° C. The next day, the coating solution was suctioned, and each well was washed three times with 200 μl of PBS-T, treated with 100 μl of PBS containing 2% BSA, and incubated at room temperature for 1 hour. Each well was washed three times with 200 μl of PBST, 100 μl of PBS was added to the control group, 100 μl of the serum of the test vaccine group was diluted 200 times with PBS, 100 μl of the undiluted sample was treated with 100 μl of serum, Lt; / RTI &gt; for 1 hour. Each well was washed three times with 200 μl of PBS-T, and the goat anti-mouse IgG: HRP was diluted 1: 1000 in blocking buffer, treated with 100 ul per well, and incubated at 37 ° C for 1 hour. Each well was washed three times with 200 μl of PBS-T, treated with 100 μl of TMB solution, and incubated at room temperature for 10 min. After stopping 100 ul of stop solution, OD value was measured at 450 nm.

The level of total IgG to PED was higher than that of the inoculated vaccine with PEDV-CUP-B1406 strain inoculated with the K inactivated vaccine. Total IgG antibody levels began to show a significant difference from 4 weeks after vaccination and the antibody titers were more than 2 times greater in the vaccine using the PEDV-CUP-B1406 strain compared to the K inactivated vaccine (see Figure 6) .

<5-4> Confirmation of serum neutralizing antibody

The neutralizing antibodies were measured and compared using the sera obtained from the mouse inoculation experiment. The number of cells was adjusted so that the number of Vero cells was 3 x 10 per ml in the growth medium. Serum of collected blood was separated, denatured at 57 ° C for 30 minutes, and then binarized with MEM medium containing 10 ug / ml of trypsin. An equal volume of 200 TCID50 / 0.1 ml of the virus for the production of the test vaccine and the same viruses were mixed with each diluted serum and neutralized at 37 DEG C for 90 minutes. 0.1 ml of the neutralized solution and 0.1 ml of the cell suspension were added to the wells of the Miroplate and cultured at 37 ° C for 5 days. The highest dilution factor of serum that inhibits CPE in cultured cells was defined as neutralizing antibody.

As a result, the neutralizing antibody by PEDV-CUP-B1406 virus was formed at 7th week (Log2), the neutralizing antibody antibody by K inactivation vaccine was 4 (Log2) (See FIG. 7).

<5-5> Measurement of IFN - γ

Both groups were stimulated with formalin inactivated PED and injected with conjugated Ab of IFN-y the following day. After 4 hours, blood samples were collected and the serum was assayed for IFN-γ by ELISA in accordance with the Mouse IFN-γ In vivo Capture Assay Set Manual.

As a result, it was confirmed that the group inoculated with the PEDV-CUP-B1406 vaccine had a higher IFN-γ level than the group inoculated with the K inactivated vaccine (see FIG. 8).

&Lt; Example 6 >

PEDV Of the test vaccine using the -CUP-B1406 strain Sow  Inoculation and Pigeon  Examination of defense strength by attacking inoculation

Table 3 shows the outbreak of PEDV virus in sows born to sows after vaccination with the PEDV-CUP-B1406 strain and the currently marketed K company (SM98) live vaccine and inactivated vaccine and N strain of oral vaccine in pregnant sows After oral challenge with the same composition, the survival rate of the piglets was checked to compare the protective power of the vaccine.

(Group 2), inactivated vaccine (group 4), and N-group oral vaccine (group 5) were inoculated with two live spermatozoa of the PEDV-CUP-B1406 (Group 1), inactivated vaccine (group 3), and oral vaccine (group 5) were inoculated to two pregnant sows twice in the same manner and the serum neutralizing antibody and colostrum antibody values of the sows due to the vaccination were measured Respectively. In addition, five healthy piglets were randomly selected from each sow and were orally administered with the aggressive virus (10 TCID50 / ml) to a 15-year old strain of PEDV-CUP-B1406 strain, Survival and diarrhea were observed. The intestinal tissues of pigs with clinical symptoms were examined by immunochemical staining.

As a result, the serum antibodies of sows inoculated with the PEDV-CUP-B1406 vaccine were higher than those of the existing K-live vaccine and inactivated vaccine and the N-group oral vaccine Respectively. Also, it was observed that the neutralization antibody of PEDV-CUP-B1406 vaccine was highly formed even in the neutralization test using cells (see Table 4). These results indicate that existing commercial vaccines are less immunologically matched with the more prevalent PED viruses (see Table 4).

In group 1, group 2 and control group 7, which were inoculated with the live vaccine in sows, five 5-day-old piglets were selected for postnatal day 3 and the offspring protective effect against diarrhea deficiency (Table 5) Diarrhea - induced diarrhea was observed in 10% of 2 - group piglets, and 70% of the control group died due to 3 outbreaks of diarrhea due to diarrhea. Diarrhea also showed mild diarrhea in all subjects. In the positive control group of group 7, the mortality rate was only 20%, and severe vomiting and hydronephrotic diarrhea occurred, resulting in dehydration and death of 6-7 days. However, the survival rate of 100% of the live vaccine produced by PEDV-CUP-B1406 in Group 1 was not observed until day 7, and the diarrhea was also seen between 2 days and 4 days after diarrhea. (See Table 5). In group 3 and group 4 vaccination of piglets by inoculation with piglets, two piglets of group 4 died, and the mortality rate was 80%. The remaining 8 pigs showed moderate diarrhea and decreased weight However, the inactivated vaccine made mainly of PEDV-CUP-B1406 in group 3 did not occur until day 7, and diarrhea also showed very weak diarrhea between 2 days and 5 days, (See Table 6). On the other hand, in group 5 and group 6 pig vaccination against piglets, group 6 piglets died of 3 dead, 70% of the parental defense rate, and 7 of the pigs showed moderate diarrhea, whereas group 5 PEDV-CUP-B1406 The majority of oral vaccines were found to have a mortality rate (survival rate) of 90% due to one outbreak, and the other nine showed a very weak diarrhea between 2 days and 5 days, (See Table 7). PEDV-CUP-B1406 In the case of inoculation of live PED virus against live pigs born by inoculation of live vaccine, inactivated vaccine and oral vaccine made in the main, As in the negative control group, normal body weight recovery was rapidly recovered to normal body weight from day 6 in vaccines made with PEDV-CUP-B1406 (see FIG. 9).

The results of H & E staining for piglets per group (see FIG. 10), live pigs (group 1), inactivated vaccine (group 3), and oral vaccine (group 5) made predominantly PEDV-CUP- Almost all normal findings in small intestine villi and intestinal epithelial cells were compared with positive and negative controls. On the other hand, the length of the mammary epithelial epithelial villi in group 7, which was tested as a positive control, was shortened due to almost total elimination, and the epithelial cells were flattened compared to normal, and the cytoplasm was found to contain fear. Immunohistochemical staining showed that epithelial cells were abruptly removed due to positive PED antigen in the positive control group. In addition, PED antigen was found in small intestinal epithelium in the vaccine group mainly made of PEDV-CUP-B1406, but it was confirmed that disappearance such as epithelial cell disappeared hardly occurs. Because of this phenomenon, And the P antigen could be observed in the epithelial cells in the vaccination group of the K vaccination group. It was confirmed that the withdrawal of the small intestinal epithelium was more severe than that of the vaccine group pigments made of PEDV-CUP-B1406.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Korea Biotechnology Research Institute KCTC12886BP 20150828

<110> Woojin B & G <120> Live vaccine composition, inactivated vaccine composition and          oral vaccine containing the same from currently currently isolated          가사 <130> pN1509-259 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PED M_S <400> 1 ggtaccatgt ctaacggttc tatt 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PED M_AS <400> 2 ggatccttag actaaatgaa gcac 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PED S1_S <400> 3 ggtaccatga ggtcttaaat ttac 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PED S1_AS <400> 4 accaccaaaa gccgcagaga cagt 24

Claims (10)

Pandemic influenza virus (PEDV) vaccine shippers (accession number: KCTC 12886BP). A live vaccine composition of a porcine epidemic diarrhea comprising the porcine epidemic diarrhea virus (PEDV) vaccinator of claim 1 (accession number: KCTC 12886BP). A vaccine characterized in that it comprises the live vaccine composition of the porcine epidemic diarrhea of claim 2. A method of inducing a protective immune response against a swine epidemic diarrhea in a swine comprising administering to the swine an immunologically effective amount of the vaccine of claim 3. An inactivated vaccine composition of a porcine epidemic diarrhea comprising the porcine epidemic diarrhea virus (PEDV) vaccine carrier of claim 1 (Accession No: KCTC 12886BP). A vaccine for the inactivation of porcine polio diarrhea further comprising an immunity enhancer in the inactivated vaccine composition of claim 5. The method according to claim 6,
Wherein the inactivation is achieved by adding formalin to the virus strain. A method of inducing a protective immune response against a porcine epidemic diarrhea in a swine comprising administering to the swine an immunologically effective amount of the vaccine of claim 6. (1) a dispenser capable of administering the vaccine to the pig; And
(2) A kit for preventing swine infectious diarrhea infection comprising the vaccine composition according to claim 2 or 5. 10. The method of claim 9,
The dispenser being able to dispense its contents; Wherein the composition is inoculated intramuscularly.

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