KR20160044764A - Composition of foot-and-mouth disease vaccine containing adjuvants potentiating immune response and method manufacturing the same - Google Patents

Abstract

The present invention relates to a foot-and-mouth disease vaccine capable of enhancing the immunity of an animal and effectively defending against attack of a virus by including an adjuvant for enhancing immunity, and a method for producing the foot-and-mouth disease vaccine composition. And can effectively defend against the attack of viruses.

Description

[0001] The present invention relates to a vaccine composition for vaccination against foot-and-mouth disease comprising adjuvant, which enhances immunity,

The present invention relates to a foot-and-mouth disease vaccine composition comprising an adjuvant for enhancing immunity and, more particularly, to an adjuvant for enhancing an immunity of an animal, To a possible foot-and-mouth vaccine composition and a method for its production.

Foot-and-mouth disease (FMD) is a virus-borne disease that infects animals with split hoofs and is characterized by rapid replication and rapid spreading of the disease. Due to its economic importance, This is a very important animal disease that can propagate between countries, and its presence in the trade of livestock products is the most important check factor.

The foot-and-mouth disease pathogen is a single stranded bipolar RNA virus, belonging to the genus Piconaviridae and Aphthovirus, and has seven different serotypes (A, O, C, Asia, SAT1, SAT2 and SAT3) Being classified.

Among the O-type foot-and-mouth disease viruses, there are more than 8 regional types, and foot-and-mouth disease vaccination can prevent foot-and-mouth disease.

The issue of extermination and control of foot-and-mouth disease has recently been reported in large-scale outbreaks in 1997, in Taiwan, in cows and pigs in 2001, and in cows and pigs in Korea and Japan in 2010 and 2011 The massive outbreak caused enormous economic losses, making it notorious as a national disaster disease, and adding to the notion that only the disposal policy is not the best for disease control, adding to the controversy about vaccination against vaccination.

The most recent large-scale foot-and-mouth disease outbreak in Korea has killed about 3.5 million people in 2010 and 2011 at that time. Looking at the economic losses caused by foot-and-mouth disease in Korea in 2010 ~ 2011, 5% of the cattle were slaughtered, 5% of the total number of breeders were slaughtered, 3,300,000 pigs were killed, and 34% The damage rate was significantly higher in pigs.

Since then, Korea has vaccinated all susceptible animals raised to prevent further outbreaks of foot-and-mouth disease, and is currently vaccinated.

Currently, vaccine virus stocks are being recommended as the largest antigens in the World Standards Institute. However, identifying and responding to the prevalent viruses in the outbreak area is very important for the country's prevention, Development of vaccines suitable for the conditions is also required.

For example, foot-and-mouth disease vaccines have traditionally been developed with vaccines based on cattle, and the criteria for evaluating vaccines against pigs are still unclear.

Given the high immunity of cows in Korea, the selection of adjuvants for immune enhancement in pigs with relatively low immunity is very important.

As an adjuvant used in a foot-and-mouth disease vaccine, it is most commonly used as an adjuvant by commercializing it as an emergency foot-and-mouth vaccine (Vaccine, 1999 Apr. 9:17 (15-16), 1858-68) using ISA206. Bhatti has a problem that there is a limit to the immunity enhancement of foot-and-mouth disease vaccine, especially in pigs.

Thus, a foot-and-mouth disease vaccine (Vaccine 31, 2013: 31, 332732) and a foot-and-mouth disease vaccine in a pig using ISA 201 (J. Biosci. Med., 2013 : 1, 2225), and so on.

SUMMARY OF THE INVENTION The present invention has been made to overcome the problems of the prior art as described above, and it is an object of the present invention to provide an adjuvant for enhancing immunity in a domesticated animal such as cattle, pig, To provide a foot-and-mouth disease vaccine composition.

Another object of the present invention is to provide a method for producing the foot and mouth vaccine composition.

To achieve the above object, the present invention provides a foot-and-mouth disease vaccine composition comprising a foot-and-mouth disease virus antigen and an adjuvant, wherein the adjuvant comprises a liposome and a double water type comprising water, dimethyloctadecylammonium in-oil-in-water (WOW) double oil form) emulsion.

In addition, the present invention relates to a method for preparing a vaccine preparation, comprising the steps of (a) injecting a foot-and-mouth disease virus antigen and a buffer in the order of a foot-and-mouth disease virus antigen and a buffer solution while stirring an adjuvant in the form of an underwater type emulsion comprising a liposome and dimethyl dioctadecylammonium, ) Stirring the mixture of the adjuvant, foot-and-mouth virus antigen and buffer after the step (a), and (c) allowing to stand after the step (b).

The foot-and-mouth disease vaccine composition according to the present invention has the advantage of enhancing immunity in animals and effectively defending against attack of viruses.

In addition, the foot-and-mouth disease vaccine composition according to the present invention can be used as a foot-and-mouth vaccine composition against various endemic animals such as cattle, pigs, sheep, goats, deer and the like.

FIG. 1 shows the results of immunization of pigs with various vaccines using foot-and-mouth disease vaccine antigens and measurement of protective ability after inoculation with attack. Clinical symptoms and virus outbreaks were investigated by the day after the attack. In the case of oil vaccine, the clinical index was low when ISA201 and Emulsigen-D or gel were included.
FIG. 2 shows the results of immunization with emulsigen-D and aluminum hydroxide (AL) vaccine in lactic acid by using foot-and-mouth disease vaccine antigen, Inoculation group, and B is a non-vaccinated control group.
FIGS. 3A and 3B are graphs showing the levels of post-immune neutralizing antibodies against serotypes in a vaccine prepared by mixing emulsifier-di and aluminum hydroxide containing seven foot-and-mouth disease virus antigens in the lactic acid.

The present invention relates to a foot-and-mouth disease vaccine composition comprising a foot-and-mouth disease virus antigen and an adjuvant, wherein the adjuvant is a water-in-oil-in-water type comprising liposomes and dimethyloctadecylammonium, To provide a foot and mouth vaccine composition that is in the form of an emulsion.

While FMD vaccines generally use oil vaccines, oil vaccines have the advantage of maintaining a long-lasting immune response and relatively rapid immune response, while the oil vaccine is more effective than the commonly used, It has the disadvantage that the inoculation stress can be confirmed and the local reaction can be seen more.

In the case of foot-and-mouth disease vaccines, which are widely used at present, only oil vaccines can be used for both cows and pigs. However, since vaccines using gels can also be used globally, There is also.

Selection of the best adjuvant with high immunity is one of the most important factors in terms of enhancing the immunity of the vaccine since the efficacy test for confirming the immunity of the vaccine is the most important part of the important vaccine.

The foot-and mouth vaccine composition of the present invention may further comprise aluminum hydroxide in the gel form of the adjuvant.

In the foot and mouth vaccine composition of the present invention, the adjuvant may be Emulsigen-D.

In the foot-and-mouth disease vaccine composition of the present invention, the foot-and-mouth disease virus antigen is selected from the group consisting of foot and mouth disease virus O serotype, foot and mouth virus A serotype, foot and mouth disease virus Asia 1 serotype, foot and mouth disease virus C serotype, foot and mouth disease virus SAT 1 serotype, And / or the foot-and-mouth virus SAT3 serotype.

In the foot-and-mouth disease vaccine composition of the present invention, the foot-and-mouth disease virus antigen may be O serotype, which is a typical foot-and-mouth disease virus such as O / Andong / SKR / 2010 (AD-P) virus.

The foot-and mouth vaccine composition of the present invention may further comprise a buffer such as Tris-NaCl, a phosphate buffer solution or the like.

The foot-and mouth vaccine composition of the present invention can be formulated according to the administration method to be used. The injectable vaccine composition is sterile, and may be free of pyrogens and particulate matter.

The foot-and mouth vaccine composition of the present invention may be used with isotonic agents or solutions. Additives for the isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose.

In addition, the foot and mouth vaccine composition of the present invention may include a vasoconstrictor.

In addition, the foot and mouth vaccine composition of the present invention may further comprise a stabilizer including gelatin and albumin. The stabilization may allow the formulation to stabilize at room or ambient temperatures for extended periods of time, such as LGS or polyvalent cations or polyvalent anions for the vaccine preparation.

The foot-and mouth vaccine compositions of the present invention may be administered orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, intravenous, intravenous, intravenous, intravenous, intravenous, intravenous, intravenous, intravenous, intravenous, inhalation, via inhalation, intravenous, intraabdominal, intraperitoneal, subcutaneous, intramuscular, May be administered by other routes, including intranasal, intrathecal, and intraarticular, or a combination thereof.

The foot-and-mouth disease vaccine composition of the present invention can be administered for veterinary use, in a suitably acceptable formulation according to conventional veterinary practice. The veterinarian can easily determine the dosing regimen and route of administration that are most appropriate for a particular animal.

The foot-and-mouth disease vaccine composition of the present invention may be administered by conventional means such as a syringe, needle-free infusion device, "microprojectile bombardment gone guns", or other physical methods such as electroporation ("EP"), hydrodynamic method ≪ / RTI >

In addition, the present invention relates to a method for preparing a vaccine preparation, comprising the steps of (a) injecting a foot-and-mouth disease virus antigen and a buffer in the order of a foot-and-mouth disease virus antigen and a buffer solution while stirring an adjuvant in the form of an underwater type emulsion comprising a liposome and dimethyl dioctadecylammonium, ) Stirring the mixture of the adjuvant, foot-and-mouth virus antigen and buffer after the step (a), and (c) allowing to stand after the step (b).

The method for producing a foot and mouth vaccine composition of the present invention may further include repeating the steps (b) and (c) when the oil layer and the water layer are separated after the step (c).

The method for producing the foot-and mouth vaccine composition of the present invention may further include the aluminum hydroxide in the gel form of the adjuvant.

The preparation method of foot-and-mouth disease vaccine composition of the present invention may be such that the adjuvant is Emulsigen-D.

The method for producing a foot-and-mouth disease vaccine composition of the present invention is characterized in that the foot-and-mouth disease virus antigen is selected from the group consisting of foot-and-mouth disease virus O serotype, foot-and-mouth disease virus A serotype, foot and mouth disease virus Asia 1 serotype, foot and mouth disease virus C serotype, Type and / or foot-and-mouth virus SAT3 serotype.

In order to investigate the production conditions having optimal antigenicity and immunogenicity using O / Andong / SKR / 2010 (AD-P) virus, a foot-and-mouth disease virus that occurred in Korea in 2010, And the test was conducted to compare the efficacy thereof.

Immunologic reactivity and defense were confirmed by concentrating on relatively low immunity pig experiments rather than cows with comparatively good immunity. The vaccine efficacy of cattle was confirmed by using lactic acid, which shows immunologically similar responses to foot and mouth disease vaccines .

Currently, Korea is using double oil emulsion (DOE) type oil vaccine. Some vaccines use oil adjuvants from Marcoll or Montanide-ISA206.

Recent experiments have shown that in pigs and lactic acid to compare their efficacy, including ISA201 (Seppic, France), newly developed Emulsigen-D and Carbigen (MVP technologies, USA) Immune and defense experiments were conducted.

In addition, it was observed that the vaccine effect was increased by incorporating aluminum hydroxide (Al (OH) ₃ ) which helps protein adsorption and stability of the vaccine.

Hereinafter, the content of the present invention will be described in detail through experimental examples. However, the following examples are intended to further illustrate the present invention, and the scope of the present invention is not limited thereto.

Example 1 Production of foot-and-mouth disease test vaccine antigen

The virus was cultured in a young hamster kidney cell line (BHK-21), and the virus was harvested 24 hours after infection and treated with virus-inactivating agent BEI (Binary ethylenimine) After that, the cells were concentrated with PEG (polyethylene glycol 6000), treated with a sucrose gradient, and the fraction was harvested by ultracentrifugation to purify viruses belonging to foot-and-mouth disease virus antigen. And a vaccine for safety testing.

In other words, foot-and-mouth disease virus was cultured in serum-free DMEM (Gibco) in BHK-21 cells. After incubation with virus at 37C for 1 hour, cells were cultured for 24 hours.

The virus was then inactivated with 0.003 N of BEI and the antigen was concentrated by addition of PEG 6000 (Sigma Aldrich).

A sucrose density gradient of 15-45% was treated and centrifuged to isolate the foot-and-mouth disease virus antigen. The fractions containing the antigen were harvested and used as the antigen of foot-and-mouth disease vaccine.

Example 2 Production of foot-and-mouth disease vaccine composition

The oil adjuvants were Emulsigen-D (MVP Technologies, USA) and Montanide ^TM ISA201 VG (ISA201; Seppic, France ), Montanide TM ISA206 VG; was used (ISA206 Seppic, France), aluminum hydroxide gel the gel component in addition some oil vaccine; foot-and-mouth disease by inserting (AL, RehydragelHPA General Chemical, USA ) A test vaccine was prepared.

Foot-and-mouth disease antigens were prepared as vaccines containing 10 ug of antigen per animal, diluted with Tris-NaCl buffer (pH 7.6) and added to each adjuvant to adjust to the appropriate concentration of antigen.

A vaccine containing the antigen and various adjuvants Carbigen, Emulsigen-D, ISA201, ISA206, Emulsigen-D + aluminum hydroxide (gel) and ISA201 + aluminum hydroxide (gel) was prepared.

The vaccine was prepared in the form of water-in-oil-in-water (WOW).

That is, a constant temperature water bath was set at a temperature of 30 ° C to maintain the constant temperature, and sterilized stirrer was used to prevent contamination of other heterogeneous substances such as bacteria. At this time, it may be washed several times with 70% ethanol and sterile distilled water if necessary.

Then, the antigen was added to the suspension while stirring the adjuvant at 500 rpm. The adjuvant, the antigen, and the Tris-NaCl buffer were added in the order of the dose shown in Table 1 below.

After the addition of the adjuvant, the antigen and the Tris-NaCl buffer solution, the mixture was stirred at 2,500 rpm for 10 minutes, and then allowed to stand at 4 ° C to confirm that the oil and water layer of the vaccine were not separated. Respectively.

At this time, if the oil and the water layer are separated, it can be repeated again. The foot-and-mouth spread vaccine composition prepared above was stored at 4 [deg.] C in the dark and used as a vaccine composition for immunogenicity and attack tests.

In Table 1 above, Carbigen is not an oil but a nanoparticle component.

≪ Example 3 > Immunogenicity test and attack test in pigs

22 pigs immunized with foot-and-mouth disease were vaccinated with 2 ml (10 μg) of foot-and-mouth disease vaccine composition (Carbigen, Emulsigen-D, ISA201, ISA206, Emulsigen-D + aluminum hydroxide) prepared in Example 2, Two pigs were used as negative controls.

All pigs were inoculated intradermally with O / Andong / SKR / 2010 (read-out open-air) virus with 10 ^5.0 TCID ₅₀ /0.1 ml foot-pad.

The clinical indices after the inoculation were determined as follows. (i) Body temperature rise 40 ° C (1 point),> 40.5 ° C (2 points),> 41 ° C (3 points); (ii) anorexia (1 point) or unheated food and leftovers (2 points); (iii) lull (1 point) or clerical difficulty (2 points); (iv) fever and hoof pain (1 point) or unable to stand with infected legs (2 points); (v) hoof blisters, symptomatic hoof counts (total 4 points); And (vi) a total of 3 points totaling the incision and tongue lesion (1 point), gum or lip lesion (1 point), or nose (1 point).

Neutralizing antibodies were collected from 0, 7, 14, 21, and 28 days after vaccination, respectively. As a result, in the test group vaccinated with Carbigen, low neutralizing antibodies were detected up to 28 days after vaccination.

In addition, the highest concentration of neutralizing antibody was detected in the ISA201 group, but it was found that the antibody production rate was relatively late after 3 weeks of vaccination.

In the Emulsigen-D group, neutralizing antibodies were produced most rapidly. In addition, when the combination of Emulsigen-D and aluminum hydroxide was inoculated, a high concentration of neutralizing antibody was generated from the 2nd week of vaccination, which is similar to that of ISA206 currently used for vaccination (see Table 2 below) ).

The virus was attacked at the 4th week of vaccination, and after that, the clinical symptoms and the virus detection amount in serum sample and nasal smear sample were observed for 10 days. In the Carbigen inoculation group, severe foot and mouth disease clinical symptoms were observed and a large amount of virus was detected in the serum and nasal smear samples, and the vaccination effect was low (see FIG. 1A).

FIG. 1 shows clinical indices and viremia and viral release concentrations in pigs after foot-and-mouth immunization. In FIG. 1, A represents Carbigen, B represents Emulsigen-D, C represents Emulsigen-D + aluminum hydroxide, D represents ISA201, ISA206, F means a non-inoculated control group.

In addition, when vaccinated with Emulsigen-D, some viruses were excreted in the juice and no symptoms other than attack site lesions were seen (see Table 2 below).

The use of Emulsigen-D in combination with aluminum hydroxide is more effective in preventing emulsigen-D than in the case of using alone, suggesting that aluminum hydroxide improves the defense (see Table 2 below).

On the other hand, the pigs inoculated with ISA201 had no symptoms other than the attack site lesion, and a small amount of virus was detected in the nasal smear samples (see Table 2 below).

In addition, the adjuvant ISA206, which has been used in the past, was not defended in a single pig, exhibited severe clinical symptoms, and some viremia was also confirmed (see Table 2 below).

^a PrioCHECK FMDV NSP (Prionics AG, Schlieren-Zurich, Switzerland) ELISA, 10 days after infection

^b DPV (Days postvaccination, days after vaccination)

^c DPC (Days postchallenge, days since attack)

^d (i) Body temperature rise 40 ° C (1 point),> 40.5 (2 points),> 41 points (3 points); (ii) anorexia (1 point) or unheated food and leftovers (2 points); (iii) lull (1 point) or clerical difficulty (2 points); (iv) fever and hoof pain (1 point) or unable to stand with infected legs (2 points); (v) hoof blisters, symptomatic hoof counts (total 4 points); And (vi) a total of 3 points totaling the incision and tongue lesion (1 point), gum or lip lesion (1 point), or nose (1 point).

^e lesion only in the inoculation site

Table 3 below shows the results of summarizing the index of the defense ability after inoculation after immunization by various adjuvants in porcine using the foot-and-mouth vaccine antigen prepared in Example 2.

^a Real-time RT-PCR: mean copy numbers of viral RNA / 0.1 ml, 10 days post-challenge

^b Not detected

^c Emulsigen-D + aluminum hydroxide

Table 4 below summarizes the virus release levels and defense effects of foot-and-mouth vaccine immunization in pigs.

^a Copy numbers of viral RNA / 0.1 ml, 8 days after inoculation

^b Not detected

≪ Example 4 > Immunogenicity test and attack test in lactic acid

Based on the results of the pig experiment in Example 3 above, the emulsigen-D and aluminum hydroxide were inoculated in parallel to observe the protective ability and antibody-forming ability in the lactic acid.

Vaccination was carried out in the same manner as in Example 3 except that the inoculation amount of the foot-and-mouth disease vaccine composition was changed to 1 ml (5)) in the pig experiment of Example 3, and blood was collected after 0, 7, 14, 21 and 28 days .

Since the foot-and-mouth disease generally does not form the symptoms of foot-and-mouth disease in general, after vaccination with a pig-like amount in the tongue, only viremia and viral release are observed,

Methods for detection of foot-and-mouth disease viruses were performed using quantitative real-time PCR method, a well-known foot and mouth disease detection method, and the Neutralization Test was also conducted using the method recommended by the OIE (Terrestrial Manual, www.oie.int).

As a result of the neutralization test, a high concentration of neutralizing antibody was detected from the 7th day after inoculation in the three lactic acid vaccinated birds, and it was continuously increased until 28 days (see Table 5 below).

The virus was attacked 28 days after the vaccination and the viremia and virus release patterns were observed until the 8th day.

In the non-vaccinated animal group, high viremia was observed up to 5 days after the inoculation, and the virus was also excreted in the nasal cavity and oral cavity. In the vaccinated group, almost no virus was detected in the serum, nasal cavity and oral cavity after the inoculation, indicating that the defensive ability against foot-and-mouth disease was formed (see Table 5 below).

^a Days post vaccination.

^b Days post challenge (attacked on 28 dpv after the attack).

^c attack by real-time RT-PCR

^d PrioCHECK FMDV NSP (Prionics AG, Schlieren-Zurich, Switzerland) ELISA, 8 days after challenge

Fig. 2 shows virus release and viremia after vaccination in lactic acid. In Fig. 2, A is an inoculation group and B is a non-vaccine inoculation group.

FIGS. 3A and 3B show the results of comparing levels of post immunization neutralizing antibodies (various serotype neutralization tests) to seven serotypes in the lactic acid (n = 2).

In Figs. 3A and 3B, Om-PanAsia2 is an O type, Om-A22 is an A type, AsMOG is an Asia type, Om-C-Ob type C is Om-SAT1- -SAU, and the SAT3 type is Om-SAT3-SA.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be understood that various modifications and changes may be made thereto without departing from the scope of the present invention.

According to the present invention, enhancement of immunogenicity is confirmed by different adjuvant of foot-and-mouth disease vaccine composition, and by providing and supplying foot-and-mouth disease vaccine composition produced through the present invention, it can contribute to more effective treatment and / or prevention of foot-and- .

Claims (11)

A foot-and-mouth disease vaccine composition comprising foot-and-mouth disease virus antigen and adjuvant,
Wherein the adjuvant is in the form of a water-in-oil-in-water emulsion comprising liposomes and dimethyloctadecylammonium. The foot-and-mouth disease vaccine composition of claim 1, wherein the adjuvant further comprises gel-type aluminum hydroxide. The foot-and-mouth disease vaccine composition according to claim 1, wherein the adjuvant is Emulsigen-D. The method according to claim 1, wherein the foot-and-mouth disease virus antigen is selected from the group consisting of foot-and-mouth disease virus O serotype, foot-and-mouth disease virus A serotype, foot and mouth virus Asia 1 serotype, foot and mouth disease virus C serotype, foot and mouth disease virus SAT1 serotype, SAT3 serotypes. ≪ RTI ID = 0.0 > 11. < / RTI > The foot-and-mouth disease vaccine composition according to claim 4, wherein the foot-and-mouth disease virus antigen is foot-and-mouth disease virus O serotype. The foot-and-mouth disease vaccine composition of claim 1, further comprising a buffer. (a). While the adjuvant in the form of a water-in-oil-in-water emulsion comprising liposomes and dimethyl dioctadecylammonium is stirred, the foot-and-mouth disease virus antigen and the buffer are injected in the order of foot- step;
(b) stirring the mixture of adjuvant, foot-and-mouth virus antigen and buffer after step (a); And
(c). Wherein the step (b) is followed by a step of standing after the step (b). The method according to claim 7, further comprising repeating the steps (b) and (c) when the oil layer and the water layer are separated after the step (c) Way. 8. The method of claim 7, wherein the adjuvant further comprises gel-type aluminum hydroxide. 8. The method of claim 7, wherein the adjuvant is Emulsigen-D. 8. The method of claim 7, wherein the foot-and-mouth disease virus antigen is selected from the group consisting of foot and mouth virus O serotype, foot and mouth virus A serotype, foot and mouth virus Asia 1 serotype, foot and mouth disease virus C serotype, foot and mouth disease virus SAT1 serotype, SAT3 serotypes. ≪ RTI ID = 0.0 > 21. < / RTI >

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