KR20210045901A - A vaccine composition comprising Lactobacillus Plantarum and Fowl Adenovirus - Google Patents

Abstract

The present invention provides a vaccine composition or a feed composition against a virus comprising Lactobacillus plantarum and fowl adenovirus KCTC 12425BP. The vaccine composition has a synergistically high antigen amount reduction effect compared to an inactivated vaccine that does not contain lactic acid bacteria, and can have a high ability to inhibit the expression of clinical symptoms and/or an ability to inhibit virus release.

Description

A vaccine composition comprising Lactobacillus Plantarum and Fowl Adenovirus}

The present invention relates to a vaccine composition against Lactobacillus Plantarum and poultry adenovirus KCTC12425BP.

Adjuvant is a material that has the function of strengthening the immune response to an antigen or regulating the immune response in a specific direction. Adjuvants can be roughly divided into two ways depending on the mechanism of action. The first is a'depot' type of antigen delivery system such as Alum, emulsion, and liposome, and the second is a TLR agonist, and/or cytokine. It is a method of directly stimulating immune cells by adding an immunostimulant that has immune activity such as such. Until recently, studies on various types of adjuvants have been reported, but adjuvants that have been approved for use in human vaccines due to problems such as safety, stability, efficacy, and cost are extremely rare. In the case of animal vaccines, more diverse types of adjuvants are used than human vaccines, but in the poultry industry, mineral oil emulsions dominate the adjuvant market due to the overwhelming advantage of its efficacy and cost.

Complete Freund's Adjuvant (CFA), an oil emulsion containing inactivated and dried Mycobacterium cells developed in 1937, was an effective adjuvant, but had the disadvantage of being very reactive in tissues. After that, incomplete Freund's Adjuvant (IFA), excluding Mycobacterium from CFA, has been developed and is widely applied to vaccines for animals. Oil emulsion is in various forms of Oil-in-Water (O/W) or Water-in-Oil-in-Water (W/O/W) in addition to Water-in-Oil (W/O) form such as IFA. It is used, and the efficacy, safety, or immunity maintenance period is different for each type, so it is very important to select and apply the appropriate type according to the target infectious disease and breed.

W/O emulsion is an adjuvant most widely used in inactivated vaccines for poultry by slowing the release of antigens through the depot effect at the inoculation site and continuously inducing an immune response. W/O emulsion using mineral oil induces a stronger immune response than O/W emulsion, but can cause problems such as local granuloma formation during vaccination, and because antigen is released slowly, it induces rapid immunity at the beginning of vaccination. There is a drawback that it is difficult. In addition, when the antigen dose is small, there is a limitation in that it is difficult to obtain sufficient immunogenicity for disease defense.

Lactic Acid Bacteria (LAB) is a type of gram-positive, anaerobic or microaerobic bacteria and is isolated from fermented foods such as kimchi, soybean paste, or yogurt. For the purpose of increasing the productivity of livestock by enhancing resistance to harmful microorganisms in the intestine of animals, the poultry industry has mainly used lactic acid bacteria in the form of probiotics. In addition, according to a recent study, peptidoglycan (PGN), which constitutes the cell wall of Gram-positive bacteria such as lactic acid bacteria, is mixed with an antigen and when inoculated into an animal in the form of an adjuvant, it acts as an immunostimulating agent to prevent the immunity of the antigen. It is known to greatly increase the originality.

Fowl Adenovirus (FAdV) infection is an infectious disease caused by Group I FAdV, and is characterized by infecting liver cells as a target to form an inclusion body. FAdV, which belongs to Group I, has a total of 12 serotypes, and pathogenicity varies greatly among these serotypes. The major serotypes that cause damage to the domestic poultry industry are known to be caused by type 4, type 8, and type 11. In particular, type 4 is accompanied by hypocystic edema (Hydropericardium syndrome; HPS), resulting in morbidity and mortality compared to other serotypes. It is known to be relatively high.

The main route of infection of FAdV is both vertical transmission and horizontal transmission within the flock due to maternal infection. It is known to have an incubation period of about 5 to 18 days, but it can be judged that the possibility of vertical infection is high if it occurs at a young age before 10 days of age. It is a disease that is highly susceptible to younger ages, and because vertical infection is possible, it causes problems such as an increase in mortality and a decrease in productivity in posterior chicks after infection with the breeder, causing a big blow to the poultry industry. Accordingly, the Zadok oil vaccine has been developed and used in the front line of the farm, but it is important to keep the antibody titer high in the maternal group as the maternal transfer antibody delivered from the maternal has the most important effect in the early defense of the next generation chick. .

Based on the above development background, in order to develop a vaccine that exhibits superior antibody titer than the existing Zadok oil vaccine, the researchers added lactic acid bacteria dead cells to the common type 4 poultry adenovirus (FAdV-4) dead venom oil vaccine, Efficacy was evaluated.

The research team is required to develop a poultry adenovirus dead poison oil vaccine that overcomes the limitations of the existing dead poison oil vaccine with low antibody titer.

One aspect provides a vaccine composition against a virus comprising Lactobacillus Plantarum and KCTC12425BP, a poultry adenovirus.

One aspect provides a feed composition having a protective effect against viruses, including Lactobacillus Plantarum and KCTC12425BP, which is a poultry adenovirus.

One aspect provides a vaccine composition against a virus comprising Lactobacillus Plantarum and poultry adenovirus KCTC12425BP.

In the vaccine composition, Lactobacillus plantarum may be accession number KCTC12425BP. The vaccine composition may comprise an inactivated Lactobacillus plantarum. The Lactobacillus plantarum may be an adjuvant, for example, an adjuvant or an adjuvant in the vaccine composition.

In the vaccine composition, the poultry adenovirus KCTC12425BP may be in an inactivated form. Inactivation can be carried out by chemical and/or physical means. Chemical inactivation can be carried out, for example, by treating the virus with an enzyme, formaldehyde, propiolactone, ethylene-imine or a derivative thereof. Physical inactivation can be carried out by irradiating the virus with radioactivity with sufficient energy, for example UV light.

The poultry adenovirus may be deposited under the accession number KCTC 12425BP.

The vaccine composition comprises as active ingredient poultry adenovirus in an effective amount, i.e., in an amount that immunizes a poultry adenovirus material that will induce immunity in vaccinated individuals or their offspring against attack by virulent viruses, e.g. antigen administration. do. This immunity is defined as inducing a higher level of protection in a population of subjects after vaccination compared to the unvaccinated group. For example, it is defined as meaning an individual having a significantly higher antigenic reduction effect after vaccination compared to the non-vaccinated group. It is also defined as the ability to suppress the onset of clinical symptoms and/or viral excretion after vaccination compared to the unvaccinated group.

The vaccine composition contains poultry adenovirus, an antigen equivalent to a dose of 10^4 to 10^10 EID ₅₀ per individual, an antigen equivalent to a dose of 10^4 to 10^9 EID ₅₀ , 10^5 to 10^9 Antigen equivalent to the dose of EID ₅₀ , 10^5 to 10^8 _{Antigen equivalent to the dose of EID 50} , 10^5 to 10^7 _{Antigen equivalent to the dose of EID 50} , or 10^6 to 10^7 EID It may contain an antigen equivalent to a dose of _50.

The vaccine composition may include a combination of the poultry adenovirus and the Lactobacillus plantarum. In the above combination, the ratio of the virus concentration and Lactobacillus plantarum concentration is, for example, an _{antigen equivalent to a dose of about 10^4 to 10^10 EID 50} : about 10^6 to 10^10 to CFU (Colony forming unit), about 10^4 to 10^9 _{antigen equivalent to a dose of EID 50} : about 10^6 to 10^9.5 to CFU, about 10^5 to 10^9 antigen equivalent to a dose of _{EID 50: about 10} ^7 to 10^9.5 to CFU, about 10^5 to 10^8 _{antigen equivalent to the dose of EID 50} : about 10^7 to 10^9 to CFU, equivalent to the dose of _{10^5 to 10^7 EID 50} Of antigen: about 10^7 to 10^8.5 to CFU, or about 10^6 to 10^7 _{antigen equivalent to the dose of EID 50} : 10^7.5 to 10^8.5 to CFU.

The vaccine composition may include a pharmaceutically acceptable carrier or diluent. The vaccine composition may contain a compound having an auxiliary activity. The compound having the auxiliary activity may be an adjuvant. The adjuvant may be an oil-in-water type or a water-in-oil type emulsion. The adjuvant may be one containing as main components a vegetable oil such as aluminum hydroxide, -phosphate, -oxide, mineral oil, vitamin E acetate, and saponin.

The vaccine composition may be one further comprising a vaccine component of other pathogens infectious to poultry.

The vaccine may be a vaccine for poultry protection. The poultry can be, for example, chicken, duck, goose, turkey, guinea fowl or quail.

The vaccine composition may be simultaneously inoculated to one or more individuals, specifically poultry. The simultaneous inoculation may be spray or eye drop inoculation. The attenuated vaccine composition can be administered simultaneously to one or more individuals. The vaccine composition for simultaneous inoculation may be administered by a spray inoculation method, an eye drop inoculation method, or a negative water administration method. The vaccine composition for simultaneous vaccination may significantly increase the number of individuals administered the vaccine by single administration compared to the vaccine composition for individual vaccination. The vaccine can be administered simultaneously to about 10 or more individuals. The vaccine can be administered simultaneously to about 100 or more individuals. The vaccine can be administered concurrently to about 1,000 or more individuals. The vaccine can be administered concurrently to more than about 10,000 individuals. The vaccine can be administered concurrently to more than about 50,000 individuals. The vaccine can be administered concurrently to more than about 100,000 individuals. The above-described inoculation method may be an important factor in evaluating the effectiveness of the vaccine composition. The vaccine composition may be administered parenterally or orally. The parenteral administration may be nasal administration, intramuscular administration, and/or subcutaneous administration.

One aspect provides a method for preparing a vaccine composition against a virus comprising the step of providing Lactobacillus Plantarum to poultry adenovirus KCTC12425BP.

The manufacturing method may include adding a pharmaceutically acceptable excipient to the poultry adenovirus KCTC12425BP.

One aspect provides a method of preventing or alleviating diseases caused by viral infection by administering the above-described vaccine composition to poultry.

One aspect provides a feed composition having a protective effect against viruses including Lactobacillus Plantarum and poultry adenovirus KCTC12425BP strain.

Vaccine composition against a virus comprising Lactobacillus Plantarum and poultry adenovirus KCTC12425BP according to the present invention is improved efficacy compared to conventional inactivated vaccines by adding Lactobacillus plantarum, a lactic acid bacteria dead cell. Can have.

The vaccine composition synergistically has a higher antigen amount reduction effect compared to an inactivated vaccine that does not contain lactic acid bacteria, and may have a higher ability to inhibit the expression of clinical symptoms and/or have a higher ability to inhibit virus excretion.

Figure 1 shows the ELISA antibody titers according to the time course of 2 weeks and 3 weeks after the vaccine administration of the poultry adenovirus.

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

Example 1: Materials and Methods

1.1 Lactobacillus isolation

Registration No. 10-1749222 Lactobacillus Plantarum (Lactobacillus Plantarum) KCTC12946BP, which is a patented lactic acid bacterium, was used for the'composition for preventing and treating influenza virus containing lactic acid bacteria as an active ingredient'.

1.2 Virus

Registration No. 10-1564316 “New Poultry Adenovirus and Its Vaccine” Patented virus, FAdV-4 KCTC 12425BP, was used.

1.3 Vaccine manufacturing

To evaluate the efficacy of the Lactobacillus-added Zadok Oil Vaccine, FAdV-4 was cultured in chicken embryonic liver cells (CEL) until a cytopathic effect (CPE) appeared, and then the virus culture solution was 0.2 After inactivation for 24 hours at room temperature with% (v/v) formalin, it was used as an antigen.

Lactobacillus Plantarum (Lactobacillus Plantarum) KCTC12946BP (hereinafter Lp) was incubated in MRS liquid medium (Difco, USA) at 37°C for 24 hours. The cultured lactic acid cells were centrifuged to precipitate the cells, washed twice with sterile PBS (Phosphate Buffered Saline), and then the cell suspension was heated at 100° C. for 30 minutes to inactivate, and then used as an adjuvant.

Thereafter, FAdV dead poison antigen and L.p dead cells were mixed according to the ratio to prepare mixed antigens for each group. The prepared mixed antigen was mixed with KF-40 (SEOJIN chemical, Korea) at a ratio of 30:70 (w/w) to prepare a W/O emulsion, and stored at 4°C for 24 hours, and then used as a vaccine.

Test Example 1: Vaccine safety verification test

To confirm the safety of the Lactobacillus Dead Cell Vaccine, after preparing the W/O emulsion vaccine, 5 6-week-old chickens that had never been exposed to poultry adenovirus were inoculated with 2 minutes (1 ml) of the vaccine into the muscles. Observed for 14 days.

As a result, it was confirmed that there was no mortality or clinical symptoms during the observation period, and that they survived in a healthy way.

group Clinical symptom ^A Our company FAdV W/O vaccine added with lactic acid bacteria 0/10 0/10 FAdV W/O vaccine without lactobacillus 0/10 0/10 Negative control 0/10 0/10

^{A Number} of clinical symptoms/total number of trials for 21 days after vaccine

Test Example 2: Antibody titer enhancement effect confirmation test

In order to confirm the enhancing effect of the antibody of the vaccine added to lactic acid bacteria according to the concentration of lactic acid bacteria, the virus concentration was set to 10^7.0 EID50/chicken, and the concentration of lactic acid bacteria dead cells was 10^7.3 CFU/chicken, and 10^8.3 CFU/chicken. A test group added at a concentration was planned. (Refer to Table 2)

group Adjuvant used Virus concentration
(EID ₅₀ ) Lactobacillus concentration
(CFU) Receipt of disclosure One KF40 10 ^7.0 - 10 2 10 ^7.3 10 3 10 ^8.5 10 4 - - - 10

As a test axis, 32 8-week-old SPF chickens (Namduck SPF, Korea) were used, and sterilized PBS was used as a control. Then, according to the test group, 0.5 ml of test vaccine or control vaccine was respectively inoculated into the breast muscle of 8-week-old SPF chickens, and blood was collected from the jugular vein 2 and 3 weeks after vaccination, and serum was separated therefrom. The separated serum was subjected to an ELISA test according to the manufacturer's instructions using a commercially available FAdV ELISA Kit (Biocheck, Netherlands) to measure the antibody titer.

Was.

As a result, it was confirmed that in the case of the high concentration of lactic acid bacteria added group, the antibody titer was significantly higher than that of the non-added group in the blood sampling results at the 2nd and 3rd weeks after the vaccine. In the case of the low concentration of lactic acid bacteria, the antibody titer increased compared to the non-added group, but it was not significant. Based on the above results, it was confirmed that the effect of improving the antibody value of the dead poison oil vaccine by the addition of lactic acid bacteria was related to the concentration of lactic acid bacteria, and in the case of the group added with a high concentration of lactic acid bacteria (10^8.3CFU/chicken), compared to the non-added group. It was confirmed that a significant antibody has a synergistic effect.

Livestock Chicken (ELISA titer): Mean±SD vaccine FAdV-4 10^7.0 FAdV-4 10^7.0
+ Lp 10^7.3 FAdV-4 10^7.0
+ Lp 10^8.3 Mock After two weeks 8665±5608 12063±2284 15440±1971 547±133 3 weeks later 6.625±0.916 7.444±1.333 5.25±1.670 463±142

[National R&D project that supported this invention]

This result is funded by the Ministry of Food, Agriculture, Forestry and Food,

Researched with support from livestock disease response technology development project (116104-3)

Assignment identification number: 116104-3

Ministry name: Ministry of Agriculture, Food and Rural Affairs

Research and management professional institution: Food, Agriculture, Forestry and Fisheries Technology Planning and Evaluation Institute

Research Project Name: Livestock Disease Response Technology Development Project

Research Project Title: Functional feed additive for poultry lactobacillus for control of avian influenza and

Development of oil immunity enhancer/lactic acid bacteria adjuvant

Contribution Rate: 1/1

Organizer: CARB Co., Ltd.

Research period: 2016.09.05 ~ 2019.09.04

Claims (4)

A vaccine composition against a virus comprising Lactobacillus Plantarum and poultry adenovirus KCTC 12425BP. The method of claim 1,
The vaccine is a vaccine composition for poultry protection. The method of claim 1,
The poultry adenovirus KCTC 12425BP strain will be in an inactivated form of a vaccine composition. The method of claim 1,
The vaccine composition is a vaccine composition that further comprises an adjuvant.

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