KR20090088121A - The method of preparing an inactivated vaccine comprising antigen-antibody complex and the use of thereof as veterinary composition - Google Patents

Abstract

A method for producing inactivated vaccine having antigen and antibody is provided to enhance the immune reaction and prevent animal diseases caused by body infection of virus. A vaccine is mixed with oil and antigen-antibody complex. A method for producing the antigen-antibody complex comprises: a first step of inoculating inactivated virus antigen in an animal to obtain antiserum; and a step of sterilizing, diluting and mixing antigen and antibody. The virus is RNA type virus or DNA type virus. The RNA type virus is newcastle disease virus, influenza virus, infectious broncheitis virus, and avian pneumovirus.

Description

The method of preparing an inactivated vaccine comprising antigen-antibody complex and the use of flowers as veterinary composition}

The present invention relates to a method for the preparation of an inactivated vaccine using an antigen-antibody complex and to a veterinary composition useful for the prevention of animal diseases caused by infection of the virus using the complex prepared therefrom.

[1] Whitfill CE, Haddad EE, Ricks CA et a1., Avian Disease , 39, pp 687, 1995

[Reference 2] Jim E. Eyles et al., Vaccine , 25, pp73017306, 2007

[3] Alexander DJ, Disease of poultry, 10th edn., Pp 541-569, 1997

[4] Song CS et al., Korean J. Vet . Res . , 40 (3), pp563-573, 2000

Selmons et al., Avian . Dis . , 18 (1), pp119-124, 1974

Document 6 Webster RG et al., Microbiol Rev. , 56 (1), pp152-179, 1992

Document 7 Capua I et al., Avian Pathol . , 32, pp 47-55, 2003

Document 8 Capua I and Marangon S, Avian Pathol . , 32, pp335-343, 2003

9 Swayne DE et al., Avian Pathol . , 28, pp 245-255, 1999

10. David cavanagh and Syed A. Naqi, Disease of poultry 11 ^th edition, 2003

[Reference 11] Jane KACook et al., Avian pathology 28, pp 477-485, 1999

[Document 12] J. Ignjatovic and S. Sapats, Rev. sci . tech . Off . Int . Epiz , 19 (2), pp 493-508, 2000.

[13] CS Song et al., Avian pathology , 27, pp 409-416, 1998

[Reference 14] Cox, RJ, et al., Vaccine , 12, pp993-999., 1994

[Document 15] Donald HB, Isaacs A, J. Gen. Microbiol ., 10 (3), pp457-64, 1954

Adjuvant (adjuvant) is a word derived from the Latin word adjuvare, which means to help or augment, a substance used to enhance the immune response of a vaccine or to reduce its antigen content. Collectively. The exact mechanism of these adjuvant agents is not known, but to date, the phagocytosis of phagocytic cells due to an increase in cytokine or the like is caused by direct activation of the phagocytic cells to increase antigen phagocytosis or indirectly to induce an inflammatory response at the site of inoculation. It is known to induce activation. Most inactivated vaccines add an adjuvant, a substance that enhances the immunogenicity of the antigen, since purely isolated antigens do not have strong immunogenicity on their own. Many of the adjuvant agents used are composed of bacterial membrane components and Freund's adjuvant is typical and consists of an emulsion of oil and water containing sterile Mycobacteria. However, these types of adjuvant are not suitable for human use because they cause excessive inflammatory response. In the case of mineral salts, aluminum salt is the most classic and universal adjuvant and is approved for human use, but the development of next-generation adjuvant is steadily developed due to side effects that cause granulomas and hypersensitivity reactions at the inoculation site. have. In addition, ISCOMs (immunostimulating complexes), which consist of saponins, lipids and cholesterol, have been developed and used. In contrast, emulsion-based adjuvant, which is used only in some cases because of severe side effects in humans, is widely used in veterinary medicine, has the advantage of delaying the outflow of antigen and strongly stimulating the production of antibodies, Montanide ISA adjuvant, Specol, etc. This is representative. However, because adjuvant used in veterinary medicine is intended for inoculation of edible livestock, it has to meet various requirements that safety and residue are important, easy to use and not expensive (Whitfill CE, Haddad EE, Ricks CA et. a1., Avian Disease , 39, pp 687, 1995).

Antibodies are globulin-based proteins formed in lymphoid tissue by B lymphocytes or B cells and are one of the most important substances involved in specific immunorecognition. Antibodies have two different functions: first, to specifically recognize and bind to specific sites of an antigen that triggers an immune response, to neutralize the action of the antigen, and second, to remove the antigen by binding to the antigen. It is the role of attracting various substances and immune cells. For example, antibodies can bind to toxins to alter the simple chemical composition of the toxin to neutralize toxicity, or attach to invading microorganisms, eliminating their motility and preventing them from invading somatic cells. In addition, antigens surrounded by antibodies cause chemical chain reactions with complement, a series of proteins found in the plasma, causing direct degradation of invading microorganisms or attracting phagocytic cells. By applying the specificity and high affinity of these antigenic antibody responses and the diversity of antibodies that can distinguish tens of millions of antigens, many kinds of antibody medicines have emerged today, including diagnostics and therapeutics (Jim E. Eyles). et al., Vaccine , 25, pp 73017306, 2007).

Hemaglutination inhibition test (HI test) is the most commonly used method for antibody testing against Newcastle disease, avian influenza or infectious bronchitis virus. The hemaglutination inhibition test (HI test) quantifies the presence of antibodies that inhibit the hemagglutination ability of the virus. It is a method of analysis. This method is widely used for serological analysis of the disease because of its relatively high accuracy and simple test method.

Newcastle disease virus is a single-stranded RNA virus belonging to the genus Avulavirus genus of the family Pararamyxoviridae. Newcastle disease viruses have an envelope, which contains the Haemaglutinin-Neuraminidase (HN) protein, which allows the virus to bind to host cells, and the F (Fusion) protein, which causes the envelope to fuse with the host cell. F protein and HN protein are glycoproteins and are distributed on the surface of the envelope (Alexander DJ, Disease of poultry, 10th edn., Pp541-569, 1997).

Newcastle disease is a lethal, highly contagious livestock epidemic in poultry that causes 100% mortality when infected with unvaccinated chickens, and respiratory and digestive symptoms and laying rates in laying hens without proper vaccine. It is a deadly disease that causes economic damage from degradation. In Korea, outbreak advisories are issued annually by the authorities, but the number of occurrences continues to increase and occurs nationwide, causing serious damage to poultry farmers. The main symptoms of Newcastle disease begin to slumber, show respiratory symptoms such as runny nose and cough, and die from green diarrhea. In addition, if the vaccine is not properly administered, laying hens or breeders with low antibody titers may have lowered or stopped egg production, and even if they are vaccinated. Sometimes (Song CS et al., Korean J. Vet . Res . , 40 (3), pp563-573, 2000).

Newcastle disease vaccines are largely divided into live and killed vaccines. Live vaccines can be classified into mesogenic strain, lentogenic strain and apathogenic strain depending on the residual virulence of the virus used as a vaccine strain (Alexander DJ, Disease of poultry, 10th edn). , pp 541-569, 1997). B1 and LaSota (including Clone) among the poisonous strains are representative of Newcastle disease live vaccine vaccine which has been used most widely in Korea as well as in the world, and it is proliferated in the respiratory tract of chickens at the time of inoculation. Although different, it is generally known to cause a vaccination response that is easily detectable after vaccination. In order to minimize the vaccination response during the live vaccination, it is mainly proliferated in the gastrointestinal mucosa, and it is known to induce a vaccination response that is easily detected after vaccination. have. In order to minimize vaccination response during recent vaccination, various live organisms using V4 strain, Ulster 2c strain, VG / GA strain, and NDV-6 / 10 strain, such as respiratory nonpathogenic (entrepreneurial) vaccine strains, which are mainly grown in the gastrointestinal mucosa, are used. Toxins have been developed. However, these respiratory non-pathogenic vaccines have the advantage that there is little vaccination response at the time of vaccination, while the vaccination efficacy is somewhat lower than that of attenuated strains such as B1 strain, and the viscera belonging to the genotype VII type which is popular in Korea recently. Sexually ill Newcastle disease virus defenses are considered to be ineffective. Newcastle disease live vaccines are frequently identified recently in broiler farms in winter, especially in the case of 2-3 live vaccines, causing newcastle disease and becoming epidemic locally (Song CS et al., Korean J. Vet . Res . , 40 (3), pp563-573, 2000).

Newcastle disease poisoning vaccines are largely divided into gel vaccines and oil vaccines. Recently, multivalent mixed poisoning oil vaccines, which can prevent three or more diseases such as Newcastle disease, chicken infectious bronchitis, and spawning hypoplasia, have been developed and used worldwide.

Influenza virus is an RNA virus belonging to the family Orthomyxoviridae, and its serotypes are classified into three types, type A, type B and type C. Types B and C have been identified only in humans, and type A has been identified in humans, horses, pigs, other mammals, and various types of poultry and wild birds (Selmons et al., Avian) . Dis . , 18 (1), pp 119-124, 1974; Webster RG et al., Microbiol Rev. , 56 (1), pp 152-179, 1992).

The serotypes of influenza A viruses are classified according to the two types of proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA), and 144 types (HA proteins) 16 species and 9 NA proteins). HA acts as a virus to attach to somatic cells, and NA allows the virus to penetrate into cells (Alexander DJ, Vet . Microbiol . , 74 (1-2), pp3-13, 2000).

The normal natural host of influenza A virus is known as wild waterfowl such as ducks and seagulls.As a result of epidemiological investigations of influenza infection in wild birds around the world, there are 16 existing HA and 9 NA influenza species. The virus was confirmed to be infected in wild birds (Selmons et al., Avian Dis. , 18 (1), pp119-124, 1974). Avian influenza virus, classified as type A, is a common common virus, a non-pathogenic avian influenza that causes mild respiratory symptoms when infected with chickens, and low-pathogenic birds that cause mortality and spawning degradation of 1-30%. Low pathogenic avian influenza (LPAI) and high pathogenic avian influenza (HPAI) with more than 95% high mortality and also called "bird flu" (Alexander DJ, Vet . Microbiol. , 74 (1-2), pp3-13, 2000). The highly pathogenic avian influenza is classified as Class A by the International Water Office (OIE) and domestically infected with animal species I.

The live virus vaccine among the avian influenza vaccines is almost impossible to develop due to the characteristics of the virus that is easily mutated. The vaccines developed to date can be largely classified into a deadly vaccine and a recombinant vaccine. In Italy and Hong Kong in 1999, the highly pathogenic avian influenza outbreaks have prolonged and spread throughout the country.In addition, avian influenza vaccines have been used as a means of combating high pathogenic avian influenza in combination with selective killing policies. Has been positively evaluated as effective in controlling highly pathogenic avian influenza (Capua I et al., Avian Pathol . , 32, pp 47-55, 2003; Capua I and Marangon S, Avian Pathol . , 32, pp 335-343, 2003; Swayne DE et al., Avian Pathol . , 28, pp 245-255, 1999). Vaccines positively evaluated in Italy (serum type A / H7N1) and Hong Kong (serum type A / H5N1) are all serotoxins and are heterologous serotypes with the same HA type but different NA type (serum type A / H7N3, This is a case where serotoxin vaccine was prepared with serotype A / H5N2) and an antibody test was used to distinguish it from an outdoor infection. However, this deadly vaccine cannot be distinguished from vaccine and outdoor infection by the Agar Gel Precipitation (AGP) test, which is the standard diagnostic method for avian influenza A. Fluorescent antibodies that distinguish NA type are large-scale antibodies. It is pointed out that it is not suitable for monitoring test. In addition, the now-developed dead venom vaccine can reduce the amount of virus released into the feces during high-pathogenic avian influenza infection, but it does not completely prevent the spread of the disease (Capua I et al., Avian Pathol . 32, pp 47-55, 2003; Capua I and Marangon S, Avian Pathol . , 32, pp 335-343, 2003; Swayne DE et al., Avian Pathol . , 28, pp 245-255, 1999).

Infectious bronchitis (IB) is an acute epidemic of chickens with a very high propagation caused by IB virus (IBV). IB does not have a high mortality rate when infected, but has a high morbidity rate and causes a wide variety of productivity declines, such as coughing, runny nose, lowering of birth rate, lowering of egg laying rate accompanied by external and internal dizziness, as well as chronic mortality due to respiratory sequelae accompanied by Escherichia coli. Causing huge economic damage to the poultry industry worldwide (David cavanagh and Syed A. Naqi, Disease of poultry 11 ^th edition, 2003). IBV is an RNA virus that has a characteristic that genes constituting virus particles are easily changed, similar to influenza virus. Therefore, a wide variety of IBV serotypes have been reported and reported worldwide, and it is estimated that there will be dozens of species including the mutated mutant virus in each serotype. In addition, the cross-reactions and cross-immunity between these serotypes are not good, and the clinical symptoms of outdoor infections do not take a clear course of disease, such as highly pathogenic avian influenza or Newcastle disease. This is accompanied.

It is estimated that there are more than a dozen serotypes of IBV that are in worldwide prevalence, and cross-protection between serotypes is largely unacknowledged. has been reported to also cause the protective effect (Jane KACook et al, Avian pathology , 28, pp477-485, 1999;...... J. Ignjatovic and S. Sapats, Rev sci tech Off Int Epi z, 19 (2), pp493-508, 2000), IBV isolates with such a wide range of cross-protective capabilities are used mainly in the development of the next generation of IB vaccines. However, every time a new serotype is found, it is not easy to develop and apply a vaccine based on the same serotype, and it is not easy to select isolates that exhibit extensive cross-protection. Therefore, in recent years, when a new type of IBV is isolated, it is a protective type by examining genotype or testing whether an existing IB vaccine can protect a new type of IBV rather than attempting to classify serotypes. and the preferred method of determining the classification IBV scheme (J. Ignjatovic and S. Sapats, Rev . sci. tech. Off. Int. Epiz, 19 (2), pp493-508, 2000) the.

In the case of IB vaccine was the first vaccine developed saengdok in the 1950s was the development of Zadok vaccine for the immunization of hens added subsequently (J. Ignjatovic and S. Sapats, Rev . Sci. Tech. Off. Int. Epiz, 19 (2) , pp 493-508, 2000). The only sera type used worldwide is the mechachuses type, and in some regions the vaccine of type 4/91 is used together (Jane KACook et al., Avian) . pathology 28, pp 477-485, 1999; J. Ignjatovic and S. Sapats, Rev. sci . tech . Off . Int . Epiz , 19 (2), pp 493-508, 2000). In Korea, Mechachuses vaccines (H120, Ma5) were effective for the prevention of respiratory malformation IB until the early 1990s.However, despite the widespread vaccination, the damage caused by IB persists. In 1997, the mutant IB Zadok oil vaccine was developed and completed industrialization, and is currently marketed under the trade name BBNE (CS Song et al., Avian) . patholog y 27, pp 409-416, 1998). However, it is urgent to supplement the efficacy since the deadly vaccine cannot completely prevent the infection of IBV.

Therefore, the present inventors use 'antibody' as a new adjuvant for adjuvant, and more specifically, to boost the immune response by the antigen of the boosting vaccine through the technique of preparing the vaccine using the antigen-antibody complex. At the same time, the present invention has been completed by reducing the amount of antigen contained in the vaccine, thereby reducing the production cost of the vaccine.

In order to achieve the above object, the present invention provides a method for producing an inactivated vaccine, characterized in that using the antigen-antibody complex.

In another aspect, the present invention provides a veterinary composition for the prevention of animal diseases caused by viruses containing an inactivating vaccine using the antigen-antibody complex obtained by the production method as an active ingredient.

Viruses as defined herein include RNA-type viruses or DNA-type viruses.

RNA-type viruses as defined herein include newcastle disease virus, influenza virus, infectious broncheitis virus, avian pneumovirus, porcine genital and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus, coxskie virus or reticuloendotheliosis virus.

DNA-type viruses, as defined herein, include ausjeszky's disease virus and adeno virus.

Hereinafter, the present invention will be described in detail.

The antigen-antibody complex of the present invention is a virus antigen inactivated by a conventional method, preferably an RNA virus, more preferably a Newcastle virus, a small pathogen-free small animal, preferably a small animal such as a chicken or a rabbit. A first step of obtaining an antisera containing the antibody through a further inoculation after about 1 to 3 weeks, preferably about 2 weeks after the first inoculation; The antigen-antibody complex of the present invention may be obtained by filtering and diluting the same, and mixing the antigen with the antibody corresponding to about 0.5 to 2 times the amount of the antigen, preferably the same amount.

Furthermore, the vaccine of the present invention can be obtained by mixing the antigen-antibody complex prepared by the above method with an oil, preferably an ISA75 oil.

The present invention provides a veterinary composition for preventing animal diseases caused by viruses containing the vaccine obtained by the above manufacturing process as an active ingredient.

Veterinary compositions containing the vaccine of the present invention as an active ingredient may further comprise suitable excipients and diluents according to conventional methods.

Excipients and diluents that may be included in the veterinary composition containing the vaccine of the present invention as an active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, Liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben and mineral oil.

The veterinary composition containing the vaccine of the present invention as an active ingredient may further include a filler, an anticoagulant, a lubricant, a humectant, a spice, an emulsifier, a preservative, and the like. The veterinary composition according to the present invention may be administered to an animal. It may then be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient, the formulations being powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols , Soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile external preparations, and the like.

Veterinary compositions containing the vaccine of the present invention as an active ingredient may vary depending on the age, sex, and weight of the animal, but may be administered once to several times in an amount of 0.1 to 100 mg / kg. In addition, the dosage of the vaccine may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

Inactivated vaccines using the antigen-antibody complexes can be administered to animals alone and in combination with other feed additives in an edible carrier.

Inactivated vaccines using the antigen-antibody complexes can also be easily administered in separate oral formulations as top dressings, either by injection or transdermally or in combination with other ingredients. Typically, a single daily dose or divided daily doses can be used, as is well known in the art.

The invention is applicable to a number of animal diets, including mammals, poultry and fish. More specifically, the diet is commercially important mammals such as pigs, cattle, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), furry animals (eg mink and fox). , And zoo animals (eg monkeys and tailless monkeys), as well as livestock (eg cats and dogs). Commercially important poultry typically include chickens, turkeys, ducks, geese, pheasants and quails. Commercially raised fish, such as trout, may also be included.

The present invention relates to a method of preparing an inactivated vaccine using an antigen-antibody complex. More specifically, the present invention utilizes an antibody as a novel adjuvant to produce an antigen of an inactivated vaccine and enhances the immune response induced by boosting the immune sensitized animal. It can be used in veterinary compositions useful for the prevention of animal diseases caused by infection of the virus by inducing high levels of antibody titers in the in vivo experiments used.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Example 1 Preparation of a Vaccine-Containing Vaccine Against Chicken Newcastle Virus

1-1. Construction of homologous and heterologous antibodies

Experiments were carried out as follows to produce antibodies against chicken Newcastle disease virus.

After inactivating Newcastle virus (LaSota) for 4 hours at 37 ° C with BEI (binary ethylenimine), mixed with Freud incomplete adjuvant (Sigma) in the same amount, SPF (specific pathogen free) Chickens and rabbits (New Zealand White, Samtaco) were vaccinated. Antisera was prepared by sacrifice at 2 weeks and then highly immunized and at 2 weeks after the sacrifice of animals.

1-2. Preparation of Antigen-Antibody Complex Solution

Antisera obtained from chickens and rabbits in Example 1-1 was filtered and sterilized by a micro filter (0.45 um), diluted in binary with PBS, and then Newcastle virus antigen (La Sota) of 512 (2 ⁹ ) HA units. Note) and each was mixed in the same amount (4 ml). Controls were prepared using PBS instead of antisera.

1-3. Preparation of Oil Vaccine

The experiment was carried out as follows to prepare an oil vaccine against chicken Newcastle virus.

7.5 ml of the antigen-antibody complex solution of Example 1-2 was dropped and mixed with 2.5 ml of ISA75 oil (Seppic, France) dropwise and mixed with a magnetic bar to prepare an oil vaccine.

Example 2. Vaccine Preparation Against Newcastle Virus

2-1. Construction of homologous antibodies

After inactivating Newcastle virus (La Sota), treated with BEI (binary ethylenimine) for 4 hours at 37 ° C, in the same amount with Freud incomplete adjuvant (Sigma), specific pathogen free (SPF) ) Chickens were inoculated with muscle. Antisera was prepared by sacrificing chickens two weeks later and then highly immunized and then two weeks later chickens were sacrificed.

2-2. Preparation of Antigen-Antibody Complex Solution

The antisera obtained from the chicken in Example 2-1 was filtered and sterilized by a micro filter (0.45 um) and diluted in binary by PBS to prepare antiserum of 2 ⁷ and 2 ² HI titers and 512 (2 ⁹ ). The same amount (4 ml) was mixed with Newcastle virus antigen (La sota strain) of HA unit, respectively. Controls were prepared using PBS instead of antisera.

2-3. Preparation of Oil Vaccine

Experiments were carried out as follows to produce an oil vaccine for chickens sensitized with the Newcastle virus live read vaccine.

An oil vaccine was prepared by dropping and mixing 2.5 ml of ISA75 oil (Seppic, France) drop by drop while appropriately stirring 7.5 ml of the antigen-antibody complex solution of Example 2-2 using a magnetic bar.

Experimental Example  Chicken Newcastle On the virus  Hemagglutination test

1-1. Anticoagulant Hemagglutination Test

In order to measure hemagglutination titers, experiments were performed using Cox et al. (Cox, R. J et al., Vaccine 12, pp993-999, 1994) as follows.

Hemagglutination inhibition (HI) titers of antisera obtained from chickens and rabbits were measured in Example 1-1, respectively. The hemagglutination inhibition test was performed by diluting the serum in PBS in 96-well microplates in binary format, then mixing the same amount (25 uL) of Newcastle virus antigen in 4HA units and sensitizing for 30 minutes at room temperature, followed by 25 uL of 1% chicken erythrocytes. After the addition, the mixture was mixed well, and after standing at room temperature for 40 minutes, the reading was performed. Hemagglutination inhibition titer was the dilution factor of the final well in which aggregation occurred.

As a result, both antiserum produced in chicken and rabbit showed HI titer of 2 ⁹ .

1-2. Viral hemagglutination testing by antigen-antibody complex formation

A hemagglutination test was performed to confirm the viral hemagglutination change by the antigen-antibody complex obtained in Example 1-2 (Donald HB, Isaacs A, J. Gen. Microbiol ., 10 (3) pp457- 64, 1954).

0.25 ml of 1% chicken erythrocytes were added to 0.25 ml of the binary-diluted antibody-virus complex solution in the microplate wells and the hemagglutination titer was measured after 40 minutes of reaction at room temperature. Hemagglutination titer was the dilution factor of the final well in which aggregation occurred.

As a result, as shown in Table 1, the hemagglutination capacity of the Newcastle virus antigen (2 ⁹ HA units) was completely inhibited by rabbit antiserum of 2 ⁷ to 2 ⁴ HI titers. In addition, in the case of chicken antiserum, antiserum of 2 ³ HI titer diluted twice more than rabbit antiserum was confirmed that the hemagglutination ability of the virus was completely inhibited.

Experimental Example  2. Inoculation and Immunogenicity Assessment of Vaccines Containing Antigen-Antibody Complexes

Evaluation of the immunogenicity of the vaccine using the antigen-antibody complex The experiment was performed as follows.

The vaccine containing the antigen-antibody complex obtained in Example 1-3 was intramuscularly inoculated into the breast muscle at 7 ml of SPF chicken by 7 weeks of age. HI titers were measured by blood collection two, three and six weeks after vaccination. Hemagglutination inhibition test was performed by diluting the serum in binary format with PBS in 96-well microplates, then mixing the same amount (25 uL) of Newcastle virus antigen in 4 HA units and sensitizing for 30 minutes at room temperature, followed by 25 uL of 1% chicken. Erythrocytes were added and mixed well, then allowed to stand at room temperature for 40 minutes before reading. Hemagglutination inhibition titer was the dilution factor of the final well in which aggregation occurred.

As shown in Table 2, the HI titer was lower in chickens vaccinated with the antibody compared to the control group without the antibody, but the HI titer was increased as the high concentration of the antibody was mixed regardless of the antibody type. When the antibody was mixed with about 2 ⁷ HI titers, it was found that HI titers were formed at almost the same level as the control group. In particular, in the case of rabbit antiserum, despite the heterologous antibody, high levels of antibody titers were formed from 3 weeks after inoculation, thereby confirming the effectiveness of the heterologous antibody.

Experimental Example  3. Newcastle Virus With live vaccine Sensitized  Hemagglutination Test in Chickens

3-1. Anticoagulant Hemagglutination Test

In order to measure hemagglutination titers, experiments were performed using Cox et al. (Cox, R. J et al., Vaccine , 12 pp993-999, 1994) as follows.

Hemagglutination inhibition (HI) titers of antisera obtained from the chickens of Example 2-1 were measured. Hemagglutination inhibition test was performed by diluting the serum in binary format with PBS in 96-well microplates, then mixing the same amount (25 uL) of Newcastle virus antigen in 4HA units and sensitizing for 30 minutes at room temperature, followed by 25 uL of 1% chicken erythrocytes. After the addition, the mixture was mixed well, and after standing at room temperature for 40 minutes, the reading was performed. Hemagglutination inhibition titer was the dilution factor of the final well in which aggregation occurred.

As a result, the antiserum produced in chicken showed HI titer of 2 ⁹ .

3-2. Virus Type Aggregation Test by Antigen-Antibody Complex Formation

The antigen obtained in the above Example 2-2, - to determine the virus titer hemagglutination change by the antibody complex was performed hemagglutination test (Donald HB, Isaacs A, J. Gen Microbiol 10 (3) pp457-64 , 1954).

Hemagglutination tests were performed to confirm the changes in viral hemagglutination titers due to antigen-antibody complex formation. 0.25 ml of 1% chicken erythrocytes were added to 0.25 ml of the binary-diluted antibody-virus complex solution in the microplate wells and the hemagglutination titer was measured after 40 minutes of reaction at room temperature. Hemagglutination titer was the dilution factor of the final well in which aggregation occurred.

As shown in Table 2, the hemagglutination capacity of the Newcastle virus antigen (2 ⁹ HA units) was completely inhibited by the antiserum of 2 ⁷ HI titers and decreased to 2 ⁶ HA units due to the antiserum of 2 ² HI titers. Could confirm.

Experimental Example  4. Newcastle Virus Live poison  Immunization with the vaccine Sensitization

4-1. immune Sensitization

Newcastle virus virulent vaccine of B1 strain (communication microorganism) was immunized by inoculation with 0.5 ml of 4 weeks old SPF chicken (positive strain).

4-2. Dose Vaccination and Immunogenicity Assessment of Vaccines Including Antigen-Antibody Complexes

Three weeks after live poisoning vaccine sensitization, the oil vaccine using the antigen-antibody complex obtained in Example 2-3 was intramuscularly inoculated into the pectoral muscle by 0.5 ml in SPF chickens. HI titers were measured by blood collection at 3, 4, 5 and 7 weeks after vaccination. The hemagglutination inhibition test was performed by diluting the serum in PBS in 96-well microplates in binary format, then mixing the same amount (25 uL) of Newcastle virus antigen in 4HA units and sensitizing for 30 minutes at room temperature, followed by 25 uL of 1% chicken erythrocytes. After the addition, the mixture was mixed well, and after standing at room temperature for 40 minutes, the reading was performed. Hemagglutination inhibition titer was the dilution factor of the final well in which aggregation occurred.

As a result, as shown in Table 3 and FIG. 1, the antibody titers of all test groups mixed with high and low concentrations of the antibody were similar or higher than those of the control group inoculated with the vaccine without the antibody. In particular, the antibody titer is higher when a low concentration of antibody is mixed than when a high concentration of antibody is mixed, as opposed to the result of a single dose of antibody mixed oil vaccine. That is, in the group vaccinated with the oil vaccine mixed with the low concentration of antibody, the antibody titer of 7 weeks after inoculation was formed about 4 times higher than that of the control group, thereby confirming the role of the immune adjuvant.

1 is a diagram showing the change in antibody titers after vaccination using the antigen-antibody complex in chickens sensitized with live read vaccine.

Claims (7)

A method for producing an inactivated vaccine, characterized by using an antigen-antibody complex. The method of claim 1, wherein the vaccine mixes the antigen-antibody complex with an oil. 3. The antibody-containing antiserum of claim 2, wherein the antigen-antibody complex is further inoculated about 1 to 3 weeks after the first inoculation of the inactivated virus antigen in a small pathogen-free small animal by a conventional method. The first step to do; Filter sterilization and dilution thereof, the method comprising the step of mixing the antigen and the antibody. Veterinary composition for the prevention of animal diseases caused by viruses containing an inactivating vaccine comprising the antigen-antibody complex obtained in claim 1 as an active ingredient. The veterinary composition according to claim 4, wherein the virus is an RNA virus or a DNA virus. The method of claim 5, wherein the RNA-type virus is Newcastle disease virus, influenza virus, infectious broncheitis virus, avian pneumovirus, pig genital and respiratory syndrome virus A veterinary composition that is porcine reproductive and respiratory syndrome virus, coxskie virus or reticuloendotheliosis virus. 6. The veterinary composition according to claim 5, wherein the DNA type virus is auzeszky's disease virus and adeno virus.

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