KR20210045899A - A vaccine composition comprising Lactobacillus Plantarum and H5N9 avian influenza virus - Google Patents

Abstract

The present invention provides a vaccine composition or a feed composition against avian influenza virus, including Lactobacillus Plantarum and H5N9 highly pathogenic avian influenza virus KCTC13027BP.

Description

A vaccine composition comprising Lactobacillus Plantarum and H5N9 avian influenza virus}

The present invention relates to a vaccine composition against avian influenza virus comprising Lactobacillus Plantarum and H5N9 type highly pathogenic avian influenza virus KCTC13027BP strain.

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.

Highly Pathogenic Avian Influenza (HPAI) has caused enormous damage to the domestic poultry industry over 7 times since its first outbreak in 2003. In particular, the highly pathogenic avian influenza that occurred between 2016 and 2017 continued for 253 days, killing 37.8 million poultry, the largest ever, and the damage was so great that it was estimated that the amount of direct and indirect socioeconomic damage was estimated to be over 400 billion won. Until now, Korea has been responding to the inflow and spread of HPAI in Korea with strong killing and quarantine measures, but the urgent time when large losses are expected in the country despite the reinforced quarantine policy, as can be seen from the outbreaks from 2016 to 2017. Can come.

In this situation, one of the countermeasures to respond to AI across the country is the emergency vaccine policy. There has been a lot of discussion on the selection and application of emergency vaccines until now, but due to the characteristics of emergency vaccines that are already used at the time HPAI was introduced, rapid increase in antibody titer and rapid formation of defense capability accordingly are the most important considerations. Is one of them. In the case of HPAI, live vaccines are excluded from priority consideration for emergency vaccines due to the possibility of recombination with outdoor strains. Therefore, the main consideration is the Zadok Oil Vaccine, and this Zadok Oil Vaccine currently occupies more than 95% of the global AI vaccine market, but as described above, there is a disadvantage that it is difficult to form a rapid defense function in the early stage of the vaccine compared to the live venom vaccine.

The research team is required to develop an H5N9 highly pathogenic avian influenza vaccine that overcomes the limitations of the existing Zadok oil vaccine, which is difficult to induce rapid immunity in the early stage of the vaccine.

One aspect provides a vaccine composition against avian influenza virus comprising Lactobacillus Plantarum and H5N9 type highly pathogenic avian influenza virus KCTC13027BP strain.

One aspect provides a feed composition having a protective effect against avian influenza virus, including Lactobacillus Plantarum and H5N9 type highly pathogenic avian influenza virus KCTC13027BP strain.

One aspect provides a vaccine composition against avian influenza virus comprising Lactobacillus Plantarum and H5N9 type highly pathogenic avian influenza virus KCTC13027BP strain.

In the vaccine composition, Lactobacillus plantarum may be accession number KCTC12946BP. 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 H5N9 type highly pathogenic avian influenza virus KCTC13027BP strain 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 H5N9 type highly pathogenic avian influenza virus may be deposited under the accession number KCTC 13027BP.

The vaccine composition contains an effective amount of H5N9 type highly pathogenic avian influenza virus as an active ingredient, i.e., an H5N9 type highly pathogenic avian influenza virus substance that will induce immunity in vaccinated individuals or their offspring against attack by a toxic virus, for example, antigen administration. Is included in an immunizing amount. 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 H5N9 type highly pathogenic avian influenza virus, _{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 Antigen equivalent to the dose of 10^9 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 May contain an antigen equivalent to the dose of _{EID 50.}

The vaccine composition may include a combination of the H5N9 type highly pathogenic avian influenza virus 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 of preparing a vaccine composition against avian influenza virus comprising the step of providing Lactobacillus Plantarum to H5N9 type highly pathogenic avian influenza virus KCTC13027BP.

The manufacturing method may include the step of adding a pharmaceutically acceptable excipient to the H5N9 type highly pathogenic avian influenza virus KCTC13027BP.

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

One aspect provides a feed composition having a protective effect against avian influenza virus, including Lactobacillus Plantarum and H5N9 type highly pathogenic avian influenza virus KCTC13027BP.

The vaccine composition against avian influenza virus including Lactobacillus Plantarum and H5N9 type highly pathogenic avian influenza virus KCTC13027BP according to the present invention is by adding Lactobacillus plantarum, which is a lactic acid bacterium, to be inactivated. It may have improved efficacy compared to vaccines.

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.

1 shows the titer of HI antibody according to the time course of 1 week after the vaccine administration of the H5N9 type dead poison antigen.
2 shows mortality information over time after vaccine administration of H5N9 type dead poison antigen.

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: Preparation of a vaccine containing H5N9 dead poison antigen and L.p dead cells

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. 10-1879936 “New Avian Influenza H5N9 Recombinant Virus and Vaccine Composition Containing Same” Patented virus KCTC13027BP (H5N9) was used.

1.3 Vaccine manufacturing

To evaluate the efficacy of the Lactobacillus-added Zadok Oil Vaccine, H5N9 was inoculated into the allantoic cavity of a 10-day-old chicken fetus egg, and 72 hours later, allantoic fluid was collected and the presence or absence of virus was confirmed by Hemagglutinin assay. The collected virus was used as an antigen after inactivating for 24 hours at room temperature with 0.2% (v/v) formalin.

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, H5N9 dead poison antigen and L.p dead cells were mixed according to the ratio of each test to prepare a mixed antigen for each group.

The prepared mixed antigen was mixed with ISA70VG (SEPPIC, France) 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, a W/O emulsion vaccine was prepared, and then 2 minutes (1 ml) of the vaccine were inoculated into the muscles using 5 6-week-old chickens that have never been exposed to avian influenza virus. 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 Lactobacillus-added H5N9 W/O vaccine 0/10 0/10 Lactobacillus-free H5N9 W/O vaccine 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: Vaccine initial challenge vaccination test

In order to confirm the initial defense ability of the vaccine, the virus concentration _{was divided into two stages: 10^8.2 and 10^7.2 EID 50} /chicken, and a test group was planned in which the addition of lactic acid bacteria dead cells was different for each antigen (see Table 2 below). ).

group Adjuvant used Virus concentration
(EID ₅₀ ) Lactobacillus concentration
(CFU) Receipt of disclosure One ISA70VG 10 ^8.2 10 ^8.4 8 2 - 8 3 10 ^7.2 10 ^8.4 8 4 - 8 5 - - - 8

As a test axis, 40 8-week-old SPF chickens (Namduck SPF, Korea) were used, and sterilized PBS was used as a control. Subsequently, depending on the test group, 0.5 ml of test vaccine or control vaccine was inoculated into the breast muscle of 8-week-old SPF chickens, respectively, and blood was collected from the jugular vein 1 week after vaccination, and serum was separated therefrom. The isolated serum was subjected to hemagglutinin inhibition (HAI) using the same antigen as the vaccine.

Afterwards, 10^6.0 EID ₅₀ /100ul/bird of challenge vaccination virus was injected into the nasal cavity of the sinus axis. Virus excretion test was performed 2, 3, 5, 7 days after the injection of the challenge virus, the samples discharged from the oral cavity and the common excretory cavity were collected using a cotton swab, and RNA was extracted from the collected samples to detect the virus. The extracted RNA was evaluated for the presence and amount of viral genes using Real-time RT-PCR.

As a result, the lactic acid bacteria-added group showed higher antibody titer than the non-added group in the results of blood collection at week 1 after vaccination, and low mortality and low virus excretion were observed in the challenge vaccination test.

Livestock Chicken (Log10(HI titer)): Mean±SD vaccine 10^8.2 + L.p 10^8.2 10^7.2 + L.p 10^7.2 Mock 1 week later 6.625±0.517 2.75±1.58 0.875±0.834 0.0±0.0 0.0±0.0

group Number of virus discharged objects/total number
(Average virus discharge Log10(EID50/ml) 10^8.2 + L.p 10^8.2 10^7.2 + L.p 10^7.2 Mock Sampling location Oral cavity Total excretion Oral cavity Total excretion Oral cavity Total excretion Oral cavity Total excretion Oral cavity Total excretion 2 days after vaccination 0/8
(0) 0/8
(0) 1/8
(0.3) 0/8
(0) 0/8
(0) 0/8
(0) 8/8
(3.6) 3/8
(1.9) 8/8
(4.2) 7/8
(4.3) 3 days after vaccination 0/8
(0) 0/8
(0) 2/8
(0.7) 0/8
(0) 0/8
(0) 0/8
(0) 5/8
(1.5) 1/8
(0.3) - - 5 days after vaccination 0/8
(0) 0/8
(0) 0/8
(0) 0/8
(0) 0/8
(0) 4/8
(1.7) 2/5
(1.2) 2/5
(1.2) - - 7 days after vaccination 0/8
(0) 0/8
(0) 0/8
(0) 0/8
(0) 0/8
(0) 0/8
(0) 0/3
(0) 0/3
(0) - -

As a result of the above, it was confirmed that the sadok oil vaccine added with lactic acid bacteria dead cells could suppress the expression of clinical symptoms and virus excretion during the initial H5N9 infection in chickens compared to the sadok oil vaccine without the same concentration of lactic acid bacteria.

[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 avian influenza virus comprising Lactobacillus Plantarum and H5N9 type highly pathogenic avian influenza virus KCTC13027BP. The method of claim 1,
The vaccine is a vaccine composition for poultry protection. The method of claim 1,
The H5N9 type highly pathogenic avian influenza virus KCTC13027BP is a vaccine composition that is in an inactivated form. The method of claim 1,
The vaccine composition is a vaccine composition that further comprises an adjuvant.

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