KR100693858B1 - 5 Low pathogenic avian influenza viral vaccine for preventing H5 subtype avian influenza viral infection - Google Patents

Abstract

In order to prevent H5 serotype high pathogenic avian influenza virus infection in an animal or human, the present invention was prepared using the H5N3 serotype avian influenza virus isolated from wild migratory birds having the same pathogenicity as the high pathogenic avian influenza virus and a non-pathogenic deficiency. It is about vaccines.

Avian influenza, H5N3 avian influenza virus, H5 highly pathogenic avian influenza virus, vaccine, vaccine

Description

Low pathogenic avian influenza viral vaccine for preventing H5 subtype avian influenza viral infection}

1 compares the protective protein amino acid sequences of the low pathogenic avian influenza A / Wild bird feces / Korea / CSM-2 / 02 (H5N3) strain and the high pathogenic avian influenza A / CK / Korea / ES / 03 (H5N1) strain.

FIG. 2 is a graph showing growth curves in the oviposition of low pathogenic avian influenza A / Wild bird feces / Korea / CSM-2 / 02 (H5N3) strains.

In order to prevent H5 serotype high pathogenic avian influenza virus infection in an animal or human, the present invention was prepared using the H5N3 serotype avian influenza virus isolated from wild migratory birds having the same pathogenicity as the high pathogenic avian influenza virus and a non-pathogenic deficiency. It is about vaccines.

Avian Influenza is a disease caused by Orthomyxovirus, which usually varies rapidly from cases where there is no rapid transmission and no clinical symptoms to 100% mortality when infected. . The highly pathogenic avian influenza is classified as List A disease by the International Water Bureau (OIE), and the first case of human infection by avian influenza virus in Hong Kong in 1997 as well as domestic and foreign poultry industry is reported. It is a disease that is very important in the public health aspects.

Avian influenza virus (AIV) is a virus belonging to type A. There are 15 types of HA and 9 types of NA depending on the hemagglutinin (HA) and neuraminidase (NA) genes in the outer membrane of the virus. There are various serotypes, such as dividing, and it is not cross-protection between different serotypes. Among the various serotypes of AIV, all highly pathogenic avian influenza (HPAI) has been shown to be due to H5 or H7 serotypes.

The first scientifically identified Highly Pathogenic Avian Influenza (HPAI) outbreak was caused by H5N1 type Avian Influenza Virus (AIV) in Scotland in 1959 and later in the UK, Canada, Australia, Germany, USA, Ireland, A total of 21 HPAIs were generated by the end of 2003 due to H5 or H7 serotypes among AIV subtypes in countries around the world including Mexico, Pakistan, Hong Kong, Italy, Chile, the Netherlands and Belgium.

In Korea, a total of 19 H5N1 type HPAIs were generated from December 2003 to March 21, 2004, affecting not only domestic poultry industry but also related secondary industries due to the shrinking consumption concerns about human infection. The government's quarantine protection for the eradication of HPAI was severely damaged and ended by direct expenses of about 150 billion won. Even more problematic is HPAI caused by H5N1 type in Southeast Asia, such as Thailand, Vietnam, Indonesia, Malaysia, etc. from 2004 to present, and especially in Thailand and Vietnam. Infection cases continue to emerge, raising global concerns.

In the case of HPAI, in most countries, the policy is to eradicate infected animals by killing them (choosing them). Korea also implemented a policy of killing from 2003 to 2004 to end the disease. However, as in the case of Mexico and Italy, which are currently using avian influenza vaccine, Korea cannot insist on eradicating HPAI early, and if it becomes widespread throughout the country, it will insist only on the policy of killing as in the past due to huge compensation and environmental problems. You will not be able to. Therefore, while trying to prevent the recurrence of HPAI, there is a need to develop a low-pathogenic vaccine virus as a vaccine strain that has a protective effect against H5 serotype HPAI in case.

Avian influenza vaccines developed to date include inactivated oil vaccines against H5N2 sera in Mexico (Intervet Mexico, Mexico) and H5 recombinant fowl vaccines based on H5 serotypes based on chicken pox virus. pox-vectored AI vaccine; Merial Select, USA) (Avian Diseases, 2003. 47: 1002-1005), and the so-called Diva vaccine developed and used in Italy (Differentiating Infected from Vaccinated Animals; DIVA, Avian Pathology, 2003. 32 (1): 47-55). The Italian Diva vaccine is a vaccine developed using the low-pathogenic H7N3 virus as a vaccine strain to protect H7N1 HPAI. There are advantages to it. However, avian influenza has various serotypes as described above, and since the serotypes are different, they are not protected. Therefore, avian influenza vaccine has high protective ability against H5 type H5N1 defense against HPAI, which has been popular in Asia since late 2003. There is a need to develop a vaccine using low-pathogenic vaccines with high safety during the manufacturing process.

Therefore, the present inventors conducted a study to develop a H5N1 type HPAI protective vaccine using a low pathogenic avian influenza virus in preparation for the regeneration or reintroduction of H5N1 type HAI-influenza influenza (HPAI), which is prevalent in Asia. Using a strain of avian influenza virus of H5N3 serotype without isolated pathogenicity, a vaccine strain showing high protection against HPAI of H5N1 type was selected, and the safety and efficacy of the vaccine strain were confirmed in the host animal chicken and the present invention was completed.

Accordingly, an object of the present invention is to provide a vaccine against avian influenza virus infection prepared using a low-pathogenic avian influenza virus (H5N3) with the same protective serotype as the virus in order to prevent H5N1 serotype avian influenza virus infection in an animal or human. It is.

In order to achieve the above object, the present invention exhibits a high protective capacity against the highly pathogenic avian influenza H5N1 serotype virus, and when used as a vaccine, is a vaccine virus that can distinguish between infected and vaccinated individuals, and has no pathogenicity isolated from wild migratory birds. A H5N3 serotype avian influenza virus is provided and provides a vaccine against avian influenza virus infection prepared using the same.

Hereinafter, the present invention will be described in more detail.

In the present invention, A / Wild bird feces having the same protective serotype among viruses isolated from wild migratory birds of domestic migratory birds in order to select AIV strains showing high protective ability against HPAI isolates of domestically generated H5N1 serotype in 2003-2004 The amino acid sequences of the hemagglutinin (HA) gene, a protective gene of / Korea / CSM-2 / 02 (H5N3), and seven other total gene sequences were analyzed and are shown in SEQ ID NOs: 1-9.

Comparison of amino acid sequence homology of the HA gene, a major defensive gene, showed low-pathogenic A / Wild bird feces / Korea / CSM-2 / 02 (H5N3) strains isolated from wild migratory birds in Cheonsu Bay area in 2002 and H5N1 in Korea. A / CK / Korea / ES / 03 (H5N1) strain, a highly pathogenic isolate of serotype, showed a relatively high homology of 92.9% (see Figure 2).

In addition, the nucleotide sequence of the amino acid segment of the HA gene, which determines the pathogenicity of avian influenza virus, is RKKR / GLFGA for the highly pathogenic H5N1 strain, and RETR / GLFGA for the low pathogenic H5N3 strain. Dogs were found to have high and low pathogenic characteristics, respectively (see FIG. 2).

In addition, the present invention was investigated the optimal growth conditions in the egg breeding of low-pathogenic A / Wild bird feces / Korea / CSM-2 / 02 (H5N3) strain in order to prepare an effective high pathogenic avian influenza defense vaccine. 10 ^7.5 EID ₅₀ /0.1ml titer of H5N3 virus was decimally diluted with PBS, and then inoculated into 30 specific pathogen-free eggs (SPF) for each dilution from 10 ^-2 to 10 ^-5 times. After incubating five eggs per allowance for 24, 36, 48, 60, 72 and 84 hours, respectively, the virus growth curve was measured by measuring virus titer, and then the final 10 ^2.5 EID ₅₀ /0.1 ml titer of H5N3 virus was detected. Conditions for harvesting the virus at 72-84 hours by inoculation with SPF eggs were established (see FIG. 3).

The method for producing the deadly vaccine using the low pathogenic H5N3 serotype virus according to the present invention

1) culturing A / wild bird feces / Korea / CSM-2 / 02 (H5N3) strain for 72 hours in the absence of specific pathogens (SPF column) to collect the ureteric fluid;

2) measuring the titer of the virus collected in step 1) by hemagglutination;

3) concentrating the virus 10-fold by centrifuging at 18,000 rpm for 3 hours (2 ¹⁰ HA);

4) inactivating the concentrated virus of step 3) by adding 0.1% formalin for 12 hours at 20 ° C .; And

5) preparing the dead poison oil vaccine by mixing the inactivated virus solution of step 4) and an oil adjuvant (Sepic) ISA70 in a 3: 7 weight ratio;

It includes.

The present invention will be described in more detail with reference to the following Examples. However, these examples are intended to illustrate the invention, it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Reference Example 1> Analysis and comparison of amino acid sequence of HA gene of low pathogenic A / Wild bird feces / Korea / CSM-2 / 02 (H5N3) and high pathogenic A / CK / Korea / ES / 03 (H5N1)

Low-pathogenic H5N3 serotype avian influenza virus A / Wild bird feces / Korea / CSM-2 / 02 (H5N3) strain isolated from wild migratory birds and A / CK / Korea / ES / 03, the first HPAI isolate in Korea at the end of 2003 (H5N1) strain was inoculated into the ureter cavity of a specific pathogen-free egg (SPF column) and incubated at 37 ° C. The ureteric fluid was aseptically collected and stored at -70 ° C.

Primer HA-F of SEQ ID NO: 10 (5'-TATTCGTCTCAGGGAGCAAAAGCAGGGG-3 ') and primer HA-R of SEQ ID NO: 11 to specifically analyze the H5N3 and H5N1 HA gene sequences Gene fragments corresponding to about 1.7 kb were amplified using '-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3'), and RNA was extracted with Qiagen's RNAeasy mini kit, followed by Qiagen's one-step RT-PCR kit (one-). Reaction step for cDNA synthesis at 50 ° C 30 minutes and 95 ° C 15 minutes using step RT-PCR kit), 30 cycles for conditions of 94 ° C 45 seconds denaturation, 53 ° C 15 seconds annealing, 72 ° C 1 minute 30 seconds extension After the reaction, the reaction was finally amplified at 72 ° C. for 5 minutes, and sequencing was performed using the amplified gene fragments. Gene sequencing and other detailed methods of primers used in RT-PCR were performed according to the method of Lee et al. (2005) (Journal of Virology, 79, 3692-3702, 2005).

The amino acid sequence of the hemagglutinin (HA) gene, which is a protective protein of the low pathogenic avian influenza H5N3 serotype virus, and other seven whole gene sequences are shown in SEQ ID NOs: 1 to 9.The high pathogenic avian influenza H5N1, which was prevalent in Korea in 2003-2004 The results of comparing the HA gene and amino acid sequence of the serotype virus are shown in FIG. 2. From the results of FIG. 2, the sequences of the two genes showed relatively high homology of 92.9%.

In addition, a comparison of the entire gene sequence of the H5N1 type of highly pathogenic avian influenza (Journal of Virology, 79, 3692-3702, 2005) with the entire gene sequence of low pathogenic avian influenza isolated from migratory birds selected as vaccine strains The results are shown in Table 1 below.

Gene Homology between Highly Pathogenic H5N1 Virus and Low-pathogenic H5N3 Virus PB2 PB1 PA HA NP NA M NS Gene homology (%) 85.6 88.5 91.7 91.1 94.4 54.6 93.1 94.4

   * A / chicken / Korea / ES / 03 (H5N1)

   ** A / wild bird feces / Korea / CSM-2 / 02 (H5N3)

Example 1 Preparation of Dead Poison Vaccine Using Low Pathogenic H5N3 Serotype Virus

To prepare the deadly vaccine using the low pathogenic H5N3 serotype virus, A / wild bird feces / Korea / CSM-2 / 02 (H5N3) strains were cultured for 72 hours in the absence of specific pathogens (SPF column) to collect the urinary fluid. The titer was measured by hemagglutination (2 ⁷ HA), centrifuged at 18,000 rpm for 3 hours, 0.1% formalin was added, and then inactivated at 20 ° C. for 12 hours, followed by inactivation of the virus solution and oil adjuvant. (Oxa adjuvant; Seppic Co., Ltd.) ISA70 was mixed in a weight ratio of 3: 7 to prepare a dead poison oil vaccine.

<Comparative Example 1> Preparation of deadly poison vaccine using high pathogenic H5N1 strain

Except for using the highly pathogenic H5N1 strain A / CK / Korea / ES / 03 (H5N1) was performed in the same manner as in Example 1 to prepare a deadly vaccine using a highly pathogenic H5N1 strain for comparative analysis with low pathogenic H5N3 strain.

Experimental Example 1 Immunogenicity of Highly Pathogenic AIV H5N1 and Low Pathogenic H5N3 to Highly Pathogenic H5N1

In order to confirm the immunogenicity of the highly pathogenic H5N1 strain A / CK / Korea / ES / 03 (H5N1) of the dead venom vaccine prepared in Example 1, a total of 18 5-week-old SPF chickens, 0.5 ml per animal, were used as muscles. Three weeks after the first inoculation with the dead venom vaccine prepared in Example 1 and Comparative Example 1, 10 animals were collected per group, and the antibody was examined and the highly pathogenic A / CK / Korea / ES / 03 (H5N1) strain. 10 ^6.6 ELD ₅₀ /0.1ml challenged after the challenge was investigated. Three weeks after the first inoculation, the inoculation vaccines prepared in Example 1 and Comparative Example 1 were inoculated twice with 8 mice per group, and the protective effect was examined in the same manner as in the first time. Defensive immunity was evaluated as having a protective effect if the virus was not re-isolated from the oral cavity and total excretory cavity on the 5th or 7th day of challenge. Ten and eight SPF chickens not vaccinated at all were used as controls for comparison with the primary and secondary inoculation groups, respectively. The control group died completely within 2 to 3 days, and attempted virus re-separation from the dead bodies. In addition, the presence of mortality was observed for 2 weeks after challenge and evaluated as having a protective effect when no mortality occurred. The results are shown in Table 2 below.

group Vaccine Count Antibody titers in blood at day 21 after vaccine (HI titer)  Result after challenge Virus reisolation Our company District River Total excretion steel Comparative Example 1 Primary 8.6 ± 1.8 0/10 6/10 0/10 Example 1 Primary 2.7 ± 1.9 6/10 6/10 5/10 Control - 0.0 6/10 10/10 10/10 Comparative Example 1 Secondary 11.7 ± 0.6 0/8 0/8 0/8 Example 1 Secondary 7.8 ± 1.2 0/8 0/8 0/8 Control - 0.0 8/8 8/8 8/8

* A / chicken / Korea / ES / 03 (H5N1), 2 ⁷ HA titer

** A / wild bird feces / Korea / CSM-2 / 02 (H5N3), 2 ⁶ HA titer

As a result of Table 2, in order to defend against highly pathogenic H5N1 strains, the vaccine prepared with the same highly pathogenic virus had an immune effect, but it was confirmed that the immune effect could be obtained even by using the same low pathogenic H5N3 strain with the same protective serotype.

Example 2 Preparation of Dead Poison Vaccine Using Low Pathogenic H5N3 Serotype Virus

In actual vaccine production, safety problems such as the outflow of highly pathogenic viruses due to virus contamination or improper inactivation during the manufacturing process are an important aspect of the avian influenza vaccine. However, as shown in Test Example 1, when a low-pathogenic strain is used as a vaccine strain, a full defensive effect can be obtained after two vaccinations. The vaccine was prepared after concentration of low pathogenic strains to high titers.

A / wild bird feces / Korea / CSM-2 / 02 (H5N3) strains were incubated for 72 hours in the absence of specific pathogens (SPF column), and the urinary fluid was collected and the titer was measured by hemagglutination (2 ⁷ HA). ) Centrifuged at 18,000 rpm for 3 hours to concentrate virus 10 times (2 ¹⁰ HA), and then inactivated by adding 0.1% formalin at 12C for 12 hours, then inactivated virus solution and oil adjuvant (oil Adjuvant; Seppic Co., Ltd.) ISA70 was mixed in a weight ratio of 3: 7 to prepare Zadok oil vaccine.

Test Example 2 Immunogenicity of Highly Pathogenic, Low-pathogenic AIV H5N3, to High-pathogenic H5N1

In order to determine the immunogenicity of the highly virulent H5N1 strain of the deadly poison vaccine of Example 2 prepared by 10-fold concentration (2 ¹⁰ HA) of low pathogenic strain A / wild bird feces / Korea / CSM-2 / 02 (H5N3) The protective effect after one-time immunization was evaluated by the same method as in Example 1, and the results are shown in Table 3 below.

group Antibody titers in blood at day 21 after vaccine (HI titer)  Result after challenge Virus reisolation Our company District River Total excretion steel Example 2 8.1 ± 1.3 0/10 0/10 0/10 Non-Vaccine Control 0.0 10/10 10/10 10/10

* A / wild bird feces / Korea / CSM-2 / 02 (H5N3), 2 ¹⁰ HA titer

As described above, the vaccine using the low-pathogenic H5N3 serotype vaccine strain showing a protective effect on the H5 serotype avian influenza virus according to the present invention is prepared for the re-influx or reoccurrence of H5 type prevalent in Asia since 2003. Safe and effective vaccines will greatly contribute to disease control.

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Claims (3)

1) culturing A / wild bird feces / Korea / CSM-2 / 02 (H5N3) strain for 72 hours in the absence of specific pathogens (SPF column) to collect the ureteric fluid; 2) measuring the titer of the virus collected in step 1) by hemagglutination; 3) centrifuging the virus at 18,000 rpm for 3 hours and concentrating 10 times with phosphate buffer (PBS); 4) inactivating the concentrated virus of step 3) by adding 0.1% formalin for 12 hours at 20 ° C .; 5) preparing the dead poison oil vaccine by mixing the inactivated virus solution of step 4) and an oil adjuvant (Sepic) ISA70 in a 3: 7 weight ratio; A method for producing an apototoxic vaccine for avian influenza virus infection using a low pathogenic H5N3 serotype virus comprising a. Highly pathogenic avian influenza H5N1 serotype virus infection prevention vaccine prepared by the method according to claim 1. According to claim 2, wherein the dead vaccine vaccine for preventing avian influenza virus infection, characterized in that it contains at least one protein encoded by the gene sequence of SEQ ID NO: 2 to 9.

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