KR101360112B1 - A novel airborn transmissible low pathogenic avian Influenza virus (H9N2) K040110/2010 and vaccine for low pathogenic avian Influenza comprising the same - Google Patents

Abstract

The present invention relates to a novel H9N2 type low pathogenic avian influenza virus A / Korean native chicken / Korea / K040110 / 2010 (H9N2) strain capable of air propagation, and an influenza virus immunogenic composition comprising bacterium or antigen thereof as an active ingredient. will be.

Description

A novel airborn transmissible low pathogenic avian Influenza virus (H9N2) K040110 / 2010 and vaccine for low pathogenic avian Influenza containing the same}

The present invention relates to a novel H9N2 type low pathogenic avian influenza virus A / Korean native chicken / Korea / K040110 / 2010 (H9N2) strain capable of air propagation, and an influenza virus immunogenic composition comprising bacterium or antigen thereof as an active ingredient. will be.

Influenza A virus is a member of the genus Osmyxovirus, a member of the Osmyxoviridae family, and contains a negative sense single stranded RNA (Lamb, RA and Krug, R. 1996. In Field Virology. 3 ^rd Edn. Eds BN Field, DM Knipe, PM Howley. Philadelphia, Lippincott-Raven. p.1353-1395), there are 16 different subtypes of hemagglutinin (HA) and 9 different subtypes of neuraminidase (NA), which can be distinguished by antigen and genetically (Choi et al., 2004. Vet. Rec. 154, 274-275; Fouchier et al., 2005. J. Virol. 79, 2814-2822).

Antigenicity is different among these different subtypes, so cross-reaction or cross-immunity is poor, and frequent antigenic variation due to stool and urine occurs between the same subtypes, thus preventing many influenza viruses.

In addition, such mutations cause expansion of host range, propagation and pathogenic changes of existing influenza viruses as well as antigenic variations related to prevention, thereby making it more difficult to control influenza viruses. In particular, the new influenza virus produced by mutations could lead to pandemic influenza, such as the Spanish flu, which is the world's biggest disaster, causing 50 million deaths in two years.

 Due to the importance of the above, a variety of researches are being actively conducted around the world on the principle of variation of these influenza viruses, and various kinds of influenza virus strains having various characteristics are utilized as a means of research.

In particular, influenza virus strains that are generated through mutations and have novel characteristics showing differences in propagation and pathogenicity with existing influenza virus strains are used for the production of immunogenic compositions effective for research strains and influenza diseases having such characteristics. High value as an antigen producing strain.

Antigen-producing strains used for research on influenza virus and production of immunogenic compositions have been isolated and developed in various types for humans and livestock.

In the case of H9 type low pathogenic avian influenza virus strains for poultry, it is marketed in the form of inactivated immunogenic agents and vaccines in combination with immunogenic compositions for single and other infectious diseases.

However, influenza viruses are highly antigenic as described above, and vaccine strains isolated or developed in the past do not provide sufficient protection against recent influenza strains (Palese P. Making better influenza virus vaccines? Emger Infect Dis 2006). 12 (January (1)): 61-5).

Therefore, there is a need for the development of immunogenic agents and vaccines that provide effective defense against new influenza virus strains that are currently popular in Korea. In the case of influenza viruses, immunogenic agents and vaccines can be effectively produced when inactivated by conventional methods using virus strains.

Therefore, the development and isolation of novel influenza strains is a key step in the development of immunogenic agents and vaccines of the above type.

As a related prior patent, Korean Patent Publication No. 1020070060049, 'Method for producing influenza vaccine', relates to a method for producing industrial-scale influenza virus or antigen for prevention, diagnosis, immunotherapy or therapeutic purposes, in particular, influenza vaccine , More particularly, a method for producing cell vaccines derived from Madin-Darby Canine Kidney (MDCK) and cell cultures of human vaccines comprising influenza types A and B,

In another related prior art, Korean Patent Publication No. 1020070092474, 'H3 Equine Influenza A Virus,' refers to HA (hemagglutinin) having SEQ ID NO: 1 or SEQ ID NO: 1 that does not have valine at position 78 or arginine at position 159. An isolated H3 equine influenza virus is described comprising an HA having an amino acid sequence substantially identical to.

The present invention solves the above problems, and the object of the present invention is to provide a strain for producing an antigen used in the production of an immunogenic composition effective against low pathogenic avian influenza disease.

Another object of the present invention is to provide a method for analyzing the characteristics of the existing low pathogenic influenza and the novel low pathogenic avian influenza different in propagation power and pathogenicity.

Still another object of the present invention is to prepare a strain for the low-pathogenic influenza and a novel low-pathogenic avian influenza different from the propagation and pathogenicity of the existing low-pathogenic influenza for the purpose of research and manufacture of goods.

 It is another object of the present invention to provide a method for controlling a disease state caused by a low pathogenic avian influenza virus infection in poultry.

In order to achieve the above object, the present invention provides a H9N2 type low pathogenic avian influenza A virus deposited with accession number KCTC 11938BP.

The virus of the present invention was deposited on May 23, 2011 with accession number KCTC 11938BP to the Korea Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, Korea.

In one embodiment of the present invention, the virus is preferably avian influenza A virus isolated from domestic poultry, but is not limited thereto.

In addition, a detailed description of the isolation method of avian influenza virus of the present invention is disclosed in Example 1.

As a preferred starting material in the isolation method of the virus of the present invention, it can be isolated from diseased algae, in particular from the respiratory tract of poultry or excreta excreted by such algae. It should not be excluded that other organs may be used as starting materials for the isolation of the virus of the invention.

In another aspect, the present invention provides a vaccine for protecting poultry against clinical symptoms caused by low pathogenic avian influenza virus infection, which comprises the influenza virus of the present invention and a pharmaceutically acceptable carrier or diluent.

The low pathogenic avian influenza virus of the present invention may be included in the vaccine in an inactivated form. If the low pathogenic avian influenza virus is inactivated form, the properties of the low pathogenic avian influenza virus that induce the aforementioned diseases are completely lost.

The vaccine of the present invention can be prepared according to conventional methods commonly used, for example, for commercially inactivated avian influenza virus vaccines. Preparation of veterinary vaccine compositions is particularly described in "Handbunch der Schutzimpfungen in der Tiermedizin" (ed. Mays, A. et al., Verlag Paul Parey, Berlin und Hamburg, Germany, 1984) and "Vaccines or Veterinary Applications" (ed. Peters, AR). Et al., Butterworth-Heinemann Ltd, 1993 .

Briefly, the substrate susceptible to infection is inoculated with the H9 type low pathogenic avian influenza virus of the present invention in a live form and propagated until the virus replicates at a predetermined infectious concentration or antigen content (mass). The avian influenza virus bearing the substance can then be harvested and formulated into a pharmaceutical composition with prophylactic activity.

All substrates that can be used for replication of the avian influenza virus as defined above can be used to produce a vaccine according to the invention if necessary after the avian influenza virus has adapted to the substrate.

Suitable substrates include primary (algal) cell cultures (e.g. chicken embryo fibroblast (CEF), or chicken kidney cell (CK)), mammalian cell lines (e.g. For example, Madin. Darby canine kidney, MDCK cell line. Usually, after inoculation of the cells the virus is propagated in medium containing trypsin for 3-10 days, after which the cell culture supernatant is collected and, if necessary, filtered or centrifuged to remove cell debris.

Alternatively, after the avian influenza virus of the present invention is propagated in embryonated eggs, the avian influenza virus material may be collected by a conventional method.

The present invention also provides a vaccine comprising avian influenza virus in an inactivated form against avian influenza infection. The main advantage of inactivated vaccines is that long levels of high levels of protective antibodies persist. This property makes inactivated vaccines particularly suitable for breeder vaccination.

The purpose of inactivating the harvested virus after the propagation step is to prevent the regeneration of the virus. In general, this can be done by chemical or physical means. Chemical inactivation can be performed, for example, by treating the virus with enzymes, formaldehyde, β-propiolactone, ethylene-imine or derivatives thereof. If necessary, the deactivated compound is later neutralized. Formaldehyde inactivated material may be neutralized with, for example, thiosulfate. Physical inactivation may preferably be carried out by irradiating the virus with sufficient energy, for example UV light. If necessary, the pH can be adjusted to a value of about 7 after treatment .

Vaccines containing inactivated avian influenza viruses may include, for example, one or more of the aforementioned pharmaceutically acceptable carriers or diluents suitable for this purpose. Preferably, the inactivated vaccine of the present invention comprises one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include vegetable oils such as aluminum hydroxide, -phosphate, -oxide, mineral oil, vitamin E acetate, and oil-in-water or water-in-oil emulsions based on saponins.

Inactivated vaccines are usually administered parenterally, ie intramuscularly or subcutaneously.

The vaccine of the present invention comprises, as an active ingredient, an avian influenza virus in an effective amount, i.e., an amount that immunizes avian influenza virus material that will induce immunity in vaccinated birds or their offspring against attack by toxic viruses (antigen administration). . Immunity is defined herein to induce a significantly higher level of protection in avian populations after vaccination compared to the non-vaccinated group.

Typically, the inactivated vaccine of the present invention can be administered with an antigen equivalent to a dose of 10 ⁷ -10 ⁹ EID ₅₀ per bird.

The low pathogenic influenza virus vaccine of the present invention can be used effectively in chickens, but other poultry such as turkey, guinea fowl and quail can also be vaccinated effectively. Chickens include edible chickens, cloned cattle, and laying eggs.

The age of the animals receiving the live or inactivated vaccine according to the present invention is the same as the age of animals receiving the commercially available inactivated avian influenza virus vaccine. Vaccination of parental livestock, such as breeders of edible chickens, can be performed with the live, attenuated or inactivated vaccine of the present invention, or a combination of both. The advantages of this type of vaccine program include the direct protection of one day old offspring provided by antibodies from the maternal line that are delivered vertically to the offspring.

The present invention also encompasses vaccine combinations comprising one or more vaccine components of other pathogens infected with poultry in addition to the low pathogenic avian influenza virus of the present invention.

Preferably the vaccine component in the vaccine combination is a live, attenuated or inactivated form of the pathogen infected with the poultry.

In particular, the present invention provides a vaccine combination in which all vaccine components are in an inactivated form.

Vaccine combinations include infectious bronchitis virus (IBV), avian reovirus, Infectious bursal disease virus (IBDV), fowl adenovirus (FAdV), and hypogonadal virus (egg). It is preferred to include one or more (inactivated) vaccine strains of drop syndrome 76 'virus, EDS 76' virus and turkey rhinotracheitis virus (TRTV).

By providing a novel H9 type low pathogenic avian influenza virus, a vaccine comprising the same, and a method of preparing the same, it is possible to effectively protect poultry from recent low pathogenic avian influenza virus infections.

1 is a diagram showing the results of the electrophoresis after the chain polymerase reaction to the M protein of influenza virus in order to confirm the presence of influenza virus from the chick chickenan inoculated by tanning from the domestic chicken.
Figure 2 shows the S1 protein of chicken infectious bronchitis virus and M protein of Newcastle virus to confirm the presence of chicken infectious bronchitis virus (IBV) and New Cattle virus (NDV) from chicken chicks inoculated by inoculating the chaebol collected from native chicken Figure shows the results of electrophoresis after the chain polymerase reaction on and F protein.
Figure 3 is a table showing the results of analyzing the proliferation of avian influenza virus K040110 / 2010 isolates through animal experiments.
Figure 4 is a table showing the results of analyzing the propagation power of the K01310 / 2001 isolate strain that has been commercially used as the antigen of avian influenza virus through animal experiments.
Figure 5 is a table showing the results of analyzing the propagation of avian influenza virus K040110 / 2010 isolates through animal experiments.
Figure 6 is a table showing the results of the analysis of the protective ability against the test killing vaccine K040110 / 2010 using avian influenza virus K040110 / 2010 through animal experiments.
FIG. 7 shows the antibody titers of influenza virus in serum collected two weeks and three weeks after inoculating a test deadly vaccine prepared using avian influenza virus K040110 / 2010 in 6-week-old chickens using HI test. to be.
Figure 8 is a table showing the results of the analysis of the protective ability against avian influenza virus K040110 / 2010 and K01310 / 2001 through animal experiments.

Hereinafter, the present invention will be described in more detail by way of non-limiting examples. However, the following examples are illustrated to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1: Isolation and Identification of Viruses from Chickens

In 2010, dead organs and cecum tonsils were collected from native chicken farms suspected of low pathogenic avian influenza infection.

After diluting the sample, dilution with 10% weight / volume (w / v) in PBS, centrifugation, and filtering the supernatant to 0.45 pore size to exclude bacterial contamination 10 days old specific pathogen free, SPF) was inoculated with chicken eggs and incubated in a 37 ℃ thermostat.

After incubation for 3 days, allantoic fluid was collected from chicken eggs and reacted with 10% SPF chicken erythrocytes in a 1: 1 ratio to determine the hemagglutination activity of the virus in the urinary cavity. It was.

A polymerase chain reaction (PCR) was performed on the matrix (M) protein gene, which is a specific protein of influenza virus, to the intraluminal virus with hemagglutinin (forward primer sequence AGCAAAAGCAGGTAG, reverse direction). A primer sequence AGTAGAAACAAGGTAGTTTTT) electrophoresis was confirmed that the presence of avian influenza virus in the sample obtained by checking the genome of the gene size. (Fig. 1)

Example 2: Other Pathogen Negative Testing in Novel Avian Influenza Virus Field Isolates

To determine the presence of other pathogens capable of proliferation in the intestinal urea, the S1 protein gene (Infectious bronchitis virus IBV) of chicken infectious bronchitis virus (forward frieta, nucleotide sequence TAGTGACCCTTTTGTGTGCACTAT, reverse frieto and sequence GTTTGTATGTACTCATCTGTAAC) , Electrophoresis was performed after reverse transcription polymerase chain reaction (RT-PCR) on F (Fusion) protein gene (New-Prison), Newcastle disease (NDV) protein (forward-prior nucleotide sequence GCTGATCATGAGGTTACCTC, reverse-prior sequence AGTCGGAGGATGTTGGCAGC). The genome of the gene size could not be identified. Therefore, it was confirmed that there are no other pathogens except avian influenza virus in the trachea and cecum tonsil of native chicken. (Fig. 2)

Example  3: Pathogenicity analysis through animal experiment

To investigate the in vivo virulence of avian influenza virus isolated from native chickens, 10-fold dilutions of virus with PBS were inoculated intravenously with 10 ml of 10 ⁶ EID ₅₀ per 10-week-old SPF chickens according to OIE regulations. . The clinical symptoms and mortality were observed for 10 days after challenge, and 3 chickens died between 4 and 9 days after inoculation. The mortality was 30%, and when the IVPI index was calculated according to OIE regulations, it was 1.15. It was confirmed that it belongs to the low pathogenic avian influenza virus.

Example  4: proliferative analysis through animal experiment

To confirm the pathogenicity and proliferation of the avian influenza virus isolated from native chickens, the experimental group consisted of three-week-old chicks and nine-week-old chicks that were not vaccinated, and were inoculated with the A / Chicken vaccine. / Korea / 01310/01 Commercialized vaccine prepared using the vaccine strain as an antigen, Great Microorganism, Korea) The experimental group was composed of 9-week-old chickens and a total of three experimental groups. The experimental system belonging to all experimental groups was inoculated at a dose of 0.1 ml of 10 ^6.0 EID ₅₀ per dose. Three, five and seven days after the inoculation, the oral cavity and total excretory swabs were swabbed and the swabs used for collection were stored in PBS containing 1% of gentamicin antibiotics. After mixing the PBS and swabs well, the amount of virus released from the oral cavity and total excretory cavity was determined by measuring the titer of the virus contained in PBS. (Figure 4)

As a result, it was confirmed that the experimental group vaccinated with the commercialized vaccine showed a virus release rate of 80%, and the existing commercialized vaccine did not effectively protect the virus isolated from domestic chickens.

Example 5 Analysis of Air Propagation of Existing Avian Influenza Virus Vaccine Using Animal Experiments

A / Chicken / Korea / 01310/01, which has been commercialized and used as the antigen of avian influenza killed vaccine, is not directly contacted with the virus inoculation group and the virus inoculation group, but the air is raised in the kennel. The experimental group was composed of the propagation group. The virus inoculated group was inoculated at a dose of 0.1 ml of 10 ^6.0 EID50 per dose. After 1, 2, 3, 4, 5, and 6 days of inoculation, swabs of the oral cavity and total excretory cavity were swabbed, and the swabs used for collection were stored in PBS containing 1% of gentamicin antibiotics. After mixing the PBS and swabs well, the virus was detected in the oral cavity and the total excretory cavity by detecting the gene of the virus contained in the PBS. (Fig. 5)

As a result, influenza virus strains that were previously isolated and used as vaccine strains were not able to spread by air because no virus was released in the experimental group belonging to the air propagation group.

Example 6 Analysis of Air Propagation of Novel Avian Influenza Virus Strains through Animal Experiments

In order to check the propagation power of avian influenza virus isolated from native chickens, the virus inoculation group, direct contact group living together with the virus inoculation group, and direct contact with the virus inoculation group are impossible. The experimental group was constructed. Viral inoculation group was inoculated at a dose of 0.1 ml of allowance 10 ^6.0 EID ₅₀ . 3, 5, 7 and 9 days after inoculation swabs of the oral cavity and total excretory cavity were swabbed and the swabs used for harvesting were stored in PBS containing 1% gentamicin antibiotics. After mixing the PBS and swabs well was confirmed the presence of virus discharged from the oral cavity and total excretory cavity by detecting the gene of the virus contained in the PBS (Fig. 5).

As a result of the experiment, all direct contact groups were directly spread within 3 days and all were infected. The airborne group began spreading within 3 days and all the individuals were infected at 9th day. It was confirmed that it is possible.

Example  7: New  Of avian influenza virus strains Seed virus stock Production

The seedling virus stock was prepared twice from the chicken eggs using the urea solution collected from chicken embryos inoculated with intestinal or caecum-foam emulsions. In order to check the viral load of the seed virus stock, the seed content of the seed was inoculated into the egg, and the virus content of the seed virus stock was found to be 10 ^9.0 EID ₅₀ /.

Example 8: Deadly Poisoned Vaccine Using Novel Influenza Virus Field Isolates making

K040110 / 2010 After adding 0.2% formalin to the culture broth, treated at 37 ° C for 15 hours and then inactivated at 4 ° C for 1 week, or treating intestinal uremic fluid with 0.01M BEI (Binary ethylenimine) at 3 ° C. Treatment with 0.2% B-PL in time or intestinal lumen fluid was inactivated at 25 ° C. for 3 hours to prepare an antigen for deadly vaccine.

Example  9: New separation stock  Inactivated vaccine Safety test

K040110 / 2010 In order to confirm the safety of the deadly inoculated vaccines produced by inactivation of isolates, 15 (3dose) were inoculated into 15 3 3 week old SPF chicken thigh muscles. The clinical symptoms, weight gain and mortality were examined for 3 weeks after inoculation, and the number of gross lesions at the 3, 4, and 5 weeks was examined to determine the formation of gross lesions at the site of inoculation. The clinical results, weight gain, mortality and the formation of gross lesions at the site of inoculation showed no difference compared to the non-vaccinated control group, demonstrating the safety of the manufactured vaccine.

Example  10: New separation stock  Efficacy test of inactivated vaccine

K040110 / 2010 Animal experiments were conducted on 6-week-old SPF chickens to confirm the efficacy of test deadly vaccines inactivated by isolates. The vaccine group was divided into the control group and the vaccine group was inoculated with the prepared test vaccine for 1 minute. Two and three weeks after vaccination, H9 influenza specific antibody titers were confirmed by hemagglutination inhibition test. When the antibody titer was indicated by the log value for 2, the antibody titer after 2 weeks of the vaccine was 6 or more, and the antibody titer after 3 weeks was 7 or more, indicating that sufficient antibody inversion was observed. (Fig. 7)

Three weeks after the vaccine, influenza viruses K040110 / 2010 and K01310 / 2001 were intranasally inoculated at a dose of 0.1 ml of 10 ^6.0 EID ₅₀ per horse to confirm the protective ability of the test vaccine. Five days after challenge, the experimental system was autopsied. The tracheal and cecum tonsils were collected and emulsified and diluted in PBS containing 1% of gentamicin antibiotics. Diluted emulsion solution was inoculated into the embryonated eggs to check for the presence of virus. (Fig. 8)

In the unvaccinated experimental group, the virus was found in all organs and cecal tonsils when challenged with influenza virus K040110 / 2010, but in the experimental group that was vaccinated, the virus remained in all samples even after challenge with K040110 / 2010. It was confirmed that 100% of defense ability was not detected.

In addition, in the experimental group not vaccinated, the virus was detected in 80% of organs and cecal tonsils when challenged with influenza virus K01310 / 2001, which was used as a conventional vaccine strain, but in the experimental group vaccinated, K01310 / 2001. No virus was detected in all samples even after challenge. Therefore, it was confirmed that the vaccine prepared using the influenza virus isolate K040110 / 2010 has a 100% protective effect against strains previously used for vaccine production in addition to the same virus.

Korea Biotechnology Research Institute KCTC11938BP 20110530

Claims (11)

delete delete H9N2 type low pathogenic avian influenza A virus K040110 / 2010, deposited under accession number KCTC 11938BP, and a pharmaceutically acceptable carrier or diluent,
The avian influenza A virus is inactivated form, characterized in that the vaccine for poultry protection against diseases caused by avian influenza A virus infection. delete delete 4. The vaccine of claim 3 wherein said vaccine further comprises an adjuvant. The vaccine of claim 3, wherein the vaccine further comprises one or more vaccine components of other pathogens infectious to poultry. delete delete delete delete

Images