KR101908905B1 - Recombinant influenza virus to form cross-protection against multiple subtypes h9 and h5 of influenza viruses and vaccine comprising the same - Google Patents

Abstract

The present invention relates to a novel influenza virus effective for cross-reacting with influenza viruses of different subtypes H9 and H5, a vaccine containing the same as an active ingredient, and a method for preventing or treating an influenza virus infection disease ≪ / RTI > The recombinant influenza virus of the present invention includes a chimeric hemagglutinin gene prepared by fusing the hemagglutinin HA1 region and the HA2 region derived from two different subtypes, And exhibits a cross-reactive effect against a virus having a clade, it can be very usefully used as a virus vaccine having a wide range of neutralizing antibody effects.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a recombinant influenza virus and a vaccine containing the recombinant influenza virus. 2. The recombinant influenza virus vaccine according to claim 1, wherein the influenza virus is a human recombinant influenza virus. 2. The recombinant influenza virus vaccine according to claim 1, }

The present invention relates to a novel influenza virus, which is effective for cross-immunization against influenza viruses of multiple subtypes of H9 and H5, a vaccine containing the same as an active ingredient, a vaccine for the H9 and H5 influenza virus infection , A composition for the diagnosis of influenza virus containing the virus or an antigen thereof, or a method for detecting the virus.

Influenza viruses are RNA envelope viruses with a particle size of about 125 nm in diameter. The virus consists essentially of a core of ribonucleic acid (RNA) bound to an internal nucleocapsid or nucleoprotein surrounded by a viral envelope with a lipid bilayer structure and an exogenous glycoprotein. The inner layer of the viral envelope consists mainly of matrix proteins and the outer layer consists mainly of host-derived lipid material.

The influenza virus genome is contained on eight single strands of RNA encoding 11 proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2 and PB2). The segmentation nature of the genome allows the exchange of whole genes between different viral strains during cell co-culture. Eight RNA segments are HA that encode hemagglutinin; NA encoding neuraminidase; An NP encoding a nuclear protein; M encoding two matrix proteins (M1 and M2) by using different decoding structures from the same RNA segment; NS encoding two unique nonstructural proteins (NS1 and NEP) by using different decoding structures from the same RNA segment; PAs encoding RNA polymerases; PB1 encoding RNA polymerase and PB1-F2 protein (inducing apoptosis) by using different detoxification structures from the same RNA segment; And PB2 encoding an RNA polymerase.

These influenza viruses are highly polymorphic particles consisting of two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and hemagglutinin is a highly polymorphic particle composed of two And the fusion of virus-cell membranes during cell infiltration of the virus, and these surface proteins, in particular hemagglutinin, are known to determine the antigen specificity of the influenza subtype.

Influenza viruses are classified as type A, type B, and type C based on differences in antigen. Influenza A viruses are described by nomenclature including subtype or type, geographic origin, strain number and isolation date, for example, A / Beijing / 353/89. There are at least 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9). All subtypes are found in algae, but H1-H3 and N1-N2 are found in humans, pigs and horses (Reference: Murphy and Webster, Orthomyxoviruses, in Virology, ed. Recently, it has been reported that H18 and NA N11 have also been found in recent HA subtypes (Suxiang Tong et al., (1999)). , "New World Bats Harbor Diverse Influenza A Viruses" PLoS Pathogens, (October 2013)].

As influenza is a common infectious disease, influenza viruses are highly variable and have the potential to spread from one species to another, thus addressing the global spread of highly pathogenic influenza is becoming a big challenge. With regard to the field of veterinary medicine, influenza viruses infect almost all mammals and thus belong to a wide range of host diseases. Pigs, and chickens are infected with consumable diseases or other viruses and germs to cause a great economic damage to farmers. Therefore, they are regularly vaccinated with viruses and sadox vaccines.

The present inventors have made efforts to develop a vaccine capable of more effectively preventing and treating influenza A having various subtypes. As a result, hemagglutinin HA1 region derived from two different subtypes (H9 and H5) In the case of recombinant influenza virus containing the chimeric hemagglutinin gene prepared by fusing the HA2 region with the H9N2 serotype and the H5N8 serotype in the case of the influenza A virus, .

Korean Patent Laid-Open No. 10-2010-0102593 Korean Patent Laid-Open No. 10-2008-0113217

It is therefore an object of the present invention to provide a recombinant influenza virus comprising the chimeric hemagglutinin gene of two subtypes H9 and H5.

It is another object of the present invention to provide a novel recombinant virus vaccine which forms a cross-over immune response against the multiple subtypes H9 and H5.

It is still another object of the present invention to provide a method for preventing or treating an influenza virus infection disease using the composition.

It is still another object of the present invention to provide a composition for the diagnosis of influenza virus containing the virus or antigen.

Furthermore, an object of the present invention is to provide an influenza virus (accession number: KCTC), which is characterized by detecting an influenza virus (accession number: KCTC 13008BP) in an infected or infected cell through an antigen-antibody reaction using the virus or its antigen 13008BP).

In order to achieve the object of the present invention as described above, the present invention provides recombinant influenza virus (accession number: KCTC 13008BP) comprising a chimeric hemagglutinin gene of two subtypes H9 and H5.

In one embodiment of the present invention, the chimeric hemagglutinin gene is HA1 derived from A / CK / Korea / 04163/04 (H9N2); The HA2 gene region derived from A / MD / Korea / W452 / 2014 (H5N8) may be fused.

In one embodiment of the present invention, the A / CK / Korea / 04163/04 virus is an avian influenza virus classified mainly since 2004, and has been widely used as an anti- And the like. The A / MDk / Korea / W452 / 2014 virus is a highly pathogenic avian influenza virus isolated in 2014.

In one embodiment of the present invention, the HA2 gene region derived from the A / MDk / Korea / W452 / 2014 (H5N8) includes a portion including an ectodomain, a transmembrane region and a cytoplasmic region, (A / MDk / Korea / W452 / 2014) lacking a multibasic cleavage site, one of the features of the highly pathogenic avian influenza virus that occurred in 2014, And may be a single chimeric H9 / H5 HA structure. (The cleavage site used the A / CK / Korea / 04163/04 virus gene sequence because the HA1 part used the A / CK / Korea / 04163/04 virus part)

In one embodiment of the present invention, the chimeric hemagglutinin gene may be a polynucleotide of SEQ ID NO: 3.

The present invention also provides an influenza virus vaccine comprising the virus as an active ingredient.

In one embodiment of the present invention, the vaccine may be a live attenuated vaccine, a sadox vaccine or a subunit vaccine (immunogenic fragment).

In one embodiment of the present invention, the virus may cross-react with all influenza A viruses with all H5 serotypes including H9N2 serotype, H5N1 and H5N8.

The present invention also provides a method for preventing or treating an influenza virus infection disease by administering the vaccine to an individual other than a human.

The present invention also provides a composition for the diagnosis of influenza virus comprising the virus or antigen thereof.

Furthermore, the present invention relates to an influenza virus (accession number: KCTC 13008BP), which is characterized by detecting influenza virus (Accession No .: KCTC 13008BP) in infected or infected cells through an antigen-antibody reaction using the virus or antigen thereof ). ≪ / RTI >

The recombinant influenza virus of the present invention (Accession No .: KCTC 13008BP) comprises hemagglutinin HA1 derived from two subtypes H9 and H5, a chimeric hemagglutinin gene prepared by fusing the fusion peptide region and the HA2 region (Cross-protection) effect on viruses with various subtypes and clades by the inclusion of the same group of avian influenza viruses Differential diagnosis by screening of antibodies against neutralizing antibodies and verification of neutralizing antibody ability can be made.

1 (A) is a schematic representation of the hemagglutinin gene structure of common influenza virus, (B) is a schematic representation of the chimeric hemagglutinin gene (cH9 / H5 HA) structure of the present invention will be.
FIG. 2 is a schematic diagram showing a process of producing the recombinant influenza virus of the present invention by a reverse genetics system. FIG.
FIG. 3 is a graphical representation of vaccine schedules for mice and chickens as experimental animals to confirm the efficacy of the recombinant influenza virus of the present invention.
FIG. 4 is a graph showing the results of immunization of recombinant influenza viruses of the present invention with a recombinant influenza virus vaccine or a control vaccine after mouse first or second inoculation, followed by homologous virus (H9N2), heterologous virus (H5H8) (H5N2) was infected at a dose of 100 MLD ₅₀ in mice for 14 days: (A) weight change and survival rate of mice to the allogeneic virus (H9N2); (B) Weight change and survival rate of mice against heterologous virus (H5H8); (C) Weight change and survival rate of mice against heterologous virus (H5N2).
FIG. 5 shows the results of the ELISA method for determining the IgY antibody titer of chicken and mouse against homozygous (H9N2) and heterozygous (H5H8) virus after separating serum between the first or second inoculation of the recombinant influenza virus vaccine or the control vaccine Results.
Figure 6 shows the results of (A) lung, (B) nasal swab and (C) cloacal swab viral titer after vaccination with H9N2 and cHA H9 / H5N2 vaccine with chicken (H9N2) and heterozygous (H5H8) .
Figure 7 shows the viral titers in organ for the heterozygous virus (H5H8) after vaccination of chicken with H9N2 and cHA H9 / H5N2 vaccine.
Figure 8 shows the viral titer against the heterozygous (H5N1) virus in the organs after the first or second inoculation of the recombinant influenza virus vaccine or the control vaccine.

In one embodiment, the present invention relates to a recombinant influenza virus comprising a chimeric hemagglutinin gene (Accession No. KCTC 13008BP).

"Hemagglutinin" is one of the surface glycoproteins of influenza virus that mediates the attachment of the virus to the host cell and the fusion of the virus-cell membrane during the infiltration of the virus into the cell, and determines the antigen specificity of the influenza subtype It is surface antigen protein of influenza virus.

In the present invention, the chimeric hemagglutinin gene comprises an HA1 spherical head domain region derived from A / CK / Korea / 04163/04 (H9N2); HA2 gene region derived from A / MDk / Korea / W452 / 2014 (H5N8) is fused. The HA2 part consists of an ecto domain and a transmembrane cytopathic domain, and the ecto domain consists of fusion peptide, a helix loop, and coiled part.

The chimeric hemagglutinin gene of the present invention comprises at least 95%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4% , At least 99.6%, at least 99.8%, or at least 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 1.

In addition, the recombinant influenza virus of the present invention is characterized in that 6 or 7 other genes other than the chimeric hemagglutinin gene are A / PR / 8/1934 (H1N1), which is currently used as a backbone virus of human body vaccine, Derived from the virus A / CK / Korea / 04163/2004 (H9N2) can be used.

In one embodiment of the present invention, the present inventors provided a reverse genetics system by providing the above-mentioned chimeric hemagglutinin gene and seven other genes from A / CK / Korea / 04163/04 (H9N2) To produce the recombinant influenza virus of the present invention, which was named 'cHA H9 / H5N2'. On April 15, 2016, the virus was deposited with KCTC, Korea Research Institute of Bioscience and Biotechnology, and received the deposit number KCTC 13008BP.

In another aspect, the present invention relates to a vaccine that induces an immune response against influenza viruses that can infect a susceptible host animal and cause disease. Preferably, the vaccine of the present invention may comprise the cHA H9 / H5N2 virus (Accession No. KCTC 13008BP) as an active ingredient.

The host animal in which the vaccine of the present invention can cause an immune response may include a human, a dog, a cat, a pig, a horse, a chicken, a duck, a turkey, a pearl and the like, preferably a human and a chicken.

The vaccine of the present invention may be an attenuated live vaccine or a sadox vaccine, a subunit vaccine, a synthetic vaccine or a genetic engineering vaccine, but a live vaccine that induces an effective immune response desirable.

As used herein, the term " virulence vaccine "means a vaccine comprising live viral active ingredients. The term "attenuation" refers to artificially weakening the toxicity of living pathogens, mutating the genes involved in essential metabolism of pathogens, inducing immune system stimulating the immune system without causing disease in the body do. The attenuation of the virus can be achieved by ultraviolet (UV) irradiation, drug treatment, or in vitro high-order serial passaging. Inactivation can also be accomplished by making clear genetic changes, for example by insertion of specific deletions of viral sequences known to provide toxicity or insertion and variation of sequences into viral genomes.

The term " Sadox vaccine "is also referred to as an inactivated vaccine or an inactivated vaccine, and is a vaccine containing a dead virus. Examples thereof include whole-virus vaccines and split vaccines, which are readily manufacturable by known methods. For example, the whole virus vaccine can be prepared by treating the whole virus with formalin, and the divided vaccine can be prepared by treating the virus with ether to crush and obtain only the envelope.

The term "subunit vaccine" is a vaccine prepared by extracting only an antigen component that can cause an immune function among components of a virus, thereby minimizing adverse effects by inducing the immune formation against the antigen site necessary for viral defense. For example, the chimeric hemagglutinin (HA) protein of the recombinant influenza virus of the present invention can be extracted and used.

A "genetic engineering vaccine" may be a modification or removal of a specific gene that causes virulence of the virus.

The virus polypeptides (chimeric hemagglutinin) or fragments thereof used as the vaccine of the present invention can also be produced using methods known in the art. For example, viral polypeptides may be synthesized using solid phase synthesis methods and may be generated using recombinant DNA techniques.

The recombinant virus used as a vaccine of the present invention can be produced, for example, by a reverse genetic system as in FIG. Plasmids and reagents using a vPHW vector in a vero cell line of about 70% can be used to induce transfection so that the influenza virus can be recombined. The HA gene may be a chimeric H9 / H5 HA gene as shown in FIG. 1 (B), except for the backbone A / PR / 8/1934 (H1N1) or A / CK / 04 < / RTI > virus can be used. The recombinant virus can be obtained after inoculation of the virus prepared by such a process into fertilized eggs of about 9 to 10 days or after 36 to 48 hours from intracellular infection.

The vaccine of the present invention may also include naked nucleic acid compositions. In one embodiment of the invention, the nucleic acid may comprise a nucleotide sequence encoding the chimeric hemagglutinin (HA) protein of the recombinant influenza virus of the invention. Methods of nucleic acid vaccination can be carried out by methods known in the art. For example, in U.S. Patent Nos. 6,063,385 and 6,472,375. The nucleic acid may be in the form of a plasmid or gene expression cassette. In one embodiment, the nucleic acid is provided surrounded by liposomes administered to the animal.

In addition, the vaccine of the present invention may further comprise at least one member selected from the group consisting of a solvent, an adjuvant, and an excipient. Examples of the solvent include physiological saline or distilled water. Examples of the immunity enhancer include Freund's incomplete or complete adjuvant, aluminum hydroxide gel, vegetable and mineral oil, etc. Examples of excipients include aluminum phosphate, aluminum hydroxide Side or aluminum potassium sulfate, but are not limited thereto and may further comprise materials used in the manufacture of vaccines well known to those skilled in the art.

The vaccine of the present invention may include, but is not limited to, a recombinant influenza virus comprising a chimeric hemagglutinin gene in a hemagglutination unit (HAU) of 2 ³ to 2 ⁸ HAU. However, if less than the hemagglutination units of vaccine 2 ³ HAU, and may not be able to induce antibody formation for effective administration of the target object may be a non-economic efficiency when compared to in excess of 2 ⁸ HAU.

The vaccine of the present invention may be prepared in oral or parenteral formulations, preferably in the form of a parenteral injection, and may be administered intradermally, intramuscularly, intraperitoneally, nasally or via an eidural route have.

In another aspect, the present invention relates to a method for preventing or treating an influenza virus infectious disease by administering the vaccine to a subject suspected of influenza virus infection.

The term "influenza virus infection disease" in the present invention refers to diseases caused by influenza virus infection such as sinusitis, seizure asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea (Pitkaranta and Hayden, 1998. Ann Med.), The present invention is not limited thereto.

As used herein, the term "individual" refers to all animals, including humans, who are already infected or susceptible to influenza virus. By administering to a subject a composition comprising the extract of the present invention, the disease can be effectively prevented and treated. For example, a composition of the present invention can treat humans infected with various influenza virus subtypes or variant influenza viruses. In addition, the composition of the present invention can treat chickens or pigs infected with various influenza virus subtypes or variant avian influenza viruses. The composition of the present invention can be administered in combination with a conventional therapeutic agent for influenza virus infection.

The term "prophylactic" in the present invention means all actions that inhibit or delay the onset of an influenza virus infection by administration of the composition. The term "treatment" in the present invention means any action that improves or alleviates symptoms due to influenza virus infection by the administration of the composition.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will depend on the species and severity of the subject, age, sex, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration, the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

< Example  1>

Prepare the virus for the experiment

<1-1> A / CK / Korea / 04163/04 ( H9N2 ) And A / MD / Korea / W452 / 2014 ( H5N8 ) Virus preparation

A / CK / Korea / 04163/04 (H9N2) virus was obtained from Chungbuk National University College of Medicine and A / MDk / Korea / W452 / 2014 (H5N8) virus was obtained from Chungbuk National University College of Medicine.

< Example  2>

The Chimeric Hemagglutinin  Production of recombinant viruses containing genes

In order to develop a cross-neutralizing antibody having a cross-protection effect on different subtypes of influenza A virus and different influenza strains of different types of clones, the present inventors firstly applied hemagglutinin The chimeric hemagglutinin gene was isolated from two subtypes (serotype) of HA1 (globular head domain) and HA2 (stalk region) A / PR / 8/19345 (H1N1) or A / Ck / Korea / 04163/04 (H9N2) derived from algae).

<2-1> Chimeric Hemagglutinin  Gene production

First, to prepare a chimeric hemagglutinin gene, the present inventors synthesized an HA1 region (SEQ ID NO: 1) of A / CK / Korea / 04163/04 (H9N2); A chimeric H9 / H5N2 hemagglutinin gene was prepared by fusing the HA2 region (SEQ ID NO: 2) of A / MDk / Korea / W452 / 2014 (H5N8).

(5-AGCTATGGCACCAAATAG-3 (SEQ ID NO: 5) recognizing HA1 specifically and Bm HA 1F (5-TAT TCG TCG CAG GGA GCA AAA GCA GGG G-3; A / CK / Korea / 04163/04 (SEQ ID NO: 1) of A / CK / Korea / 04163/04 (H9N2) was constructed using the primer (5-CTATTTGGAGCTATAGCA- (SEQ ID NO: 2) of A / MD / Korea / W452 / 2014 (H5N8) of Bm NS 890R (5-ATA TCG TCT CGT ATT AGT AGA AAC AAG GGT GTT TT- (SEQ ID NO: 5) and Bm NS 890R primer (SEQ ID NO: 8), respectively, and then subjected to fusion and amplification by fusion PCR to obtain the chimeric gene of the present invention No. 3).

<2-2> Chimeric Hemagglutinin  Production of recombinant viruses containing genes

The gene of the present invention (SEQ ID NO: 3) and the gene of A / Ck / Korea / 04163/04 (H9N2) (SEQ ID NO: 4) prepared in Example <2-1> were cloned into the expression vector vPHW2000 (Papers: J Gen Virol, 2013 Jun; 94 (Pt 6): 1230-5.doi: 10.1099 / vir.0.051284-0.Epub 2013 Mar. 13). The expression vectors in which the genes of SEQ ID NO: 3 (HA) and SEQ ID NO: 4 were inserted were all mixed using transfection reagent TransIT-LT1 transfection reagent (Mirus Bio) and left at room temperature for 40 minutes.

The vector mixture was carefully placed in Vero cells (ATCCCCL-81 ™) prepared 24 hours before and after 6 hours, the culture medium was replaced with GIBCO ™ Opti-MEM I Reduced-Serum Medium (1X) liquid. After 36 h of transfection, 1 ml of Opti-MEM I containing 0.2 μg / ml L-1-tosylamido-2-phenylehtyl chlorometyl ketone (TPCK) -trypsin (Sigma-Aldrich) was added to the transfected cells, After the passage of time, the supernatant was inoculated using 10-day-old SPF fertilized eggs. After 48 hours, the recombinant viruses of the present invention were isolated from the fertilized eggs. The thus isolated recombinant virus was named cHA H9 / H5N2, Was used in the experiment.

The recombinant virus of the present invention prepared according to the method described above was deposited on April 15, 2016 with the KCTC of the Korea Research Institute of Bioscience and Biotechnology, and received the deposit number KCTC 13008BP.

< Example  3>

The Chimeric Hemagglutinin  Antigenicity evaluation of recombinant viruses containing genes by mouse animal test

In order to evaluate the antigenicity of the recombinant vaccine candidate main virus cHA H9 / H5N2 developed in the present invention, 10 recombinant viruses (accession number: KCTC 13008BP) prepared in Example 2 were inoculated into 10-day-old SPF fertilized eggs, Afterwards, urine was detected and titers were confirmed by hemagglutinin test with 0.5% turkey RBC. Separated rats were inoculated with formalin at 0.025% concentration, stored at 4 ° C for 72 hours, and completely inactivated, and no proliferative virus was confirmed by inoculation on 10 - day - old SPF fertilized eggs.

Inactivated virus strain in a SW Ti28 swing rotor bucket (Beckman, USA) containing 20% sucrose cushion was settled at 25,000 rpm for 3 hours to isolate the virus purely. The isolated virus was used as a vaccine to measure the amount of protein and make hemagglutinin protein concentration 1.7 μg / 1 ml and 3.5 μg / 1 ml.

According to the vaccination schedule of Fig. 3, the virus solution of the desired concentration was injected into the BALB / C mouse at 5-6 weeks of age using the content of hemagglutinin protein (3.5 g / HA) and the alum as the adjuvant And two times.

Two weeks after the first and second inoculation, serum of the mouse was collected to test the ability of the neutralizing antibody. In order to confirm that the vaccine effect was proved, H9N2 and H5N2, heterozygous H5N8, and H5N1), and then we measured the change in body weight and survival rate. All experiments included two groups inoculated with A / CK / Korea / 04163/04 (H9N2) virus and PBS (control) as controls. After 3, 5, and 7 days of infection, the lung tissue of the mice was collected to measure the titer of the virus. The mice used in this experiment were divided into three groups (3 mice per day for 3, 5, and 7 days: total of 9 mice were used) for collecting lung tissue for each group (the mouse and control groups were the same) Were used.

In addition, chicken (white Leghorn chickens, 6 weeks old, CAVAC Lab. Co., Ltd.) was vaccinated at a concentration of 3.5 ug / 500ul at intervals of 2 weeks once and twice according to the vaccination schedule shown in Fig. 3, Clinical symptoms were monitored up to 14 days after challenge with homozygous (H9N2) and heterozygous (H5N8) virus. Nasal swabs and cloacal swabs on days 1, 3, 5, and 7 after challenge, and virus titers on days 3, 5, and 7 were measured.

<3-1> Measurement of weight and survival rate of mice after vaccination with homologous, heterozygous and heterologous virus infections

In order to examine the effect of the recombinant virus vaccine containing the chimeric hemagglutinin gene of the present invention on the ability to neutralize influenza virus, the recombinant virus vaccine of the present invention prepared in the above procedure was inoculated into a mouse, (H5N2), heterozygous (H5N8, H5N1) viruses were infected at a dose of 10 MLD ₅₀ , and the weight change and survival rate of the mice were measured for 14 days. The results are shown in FIG. 4 and FIG.

As can be seen from FIG. 4, when the mice were inoculated with the mouse-adapted virus ma A / C k / Korea / 04163/04 homozygous virus, the H9N2 vaccine group was monitored for 14 days, After the vaccination of the present invention, a weight change of about 15% occurred until day 14 (FIG. 4A). Survival rates were 100% for both the H9N2 vaccine group and the cHA H9 / H5N2 vaccine group (Fig. 4A). In the case of the heterozygous virus (H5N8), the weight of the H9N2 vaccine was continuously decreased until 14 days after the infection, and all of the mice died. However, in the case of the vaccine according to the present invention, PBS or H9N2 vaccine And the survival rate of 90% was confirmed (FIG. 4B). In addition, when the H9N2 vaccine alone was administered with the A / AB / ma81 / 07 (H5N2) virus, which is another heterotypic virus, the mice lost 20% or more of body weight on the 14th day, , The vaccine according to the present invention shows a recovery of about 15% of the mouse weight by 8 days, but then recovered and 80% survived

<3-2> Homologous and heterologous viruses in chicken and mouse vaccination IgY  or IgG  Antibody Titration

H9N2 and cHA The sera from the first and second inoculations after the H9 / H5N2 vaccine was separated and the IgY antibody titers against homozygous and heterologous viruses were measured by ELISA method and the results are shown in FIG. As shown in FIG. 5, similar antibody titer was observed in the H9N2 and cHA H9 / H5N2 vaccine sera when coated with H9N2 virus. However, in the ELISA test after the heterozygous H5N8 virus coating, And the H9N2 vaccine showed similar antibody titers to the PBS vaccine group.

<3-3> Homozygous and heterologous viruses in chicken vaccination Potency  Measure

H9N2 and cHA H9 / H5N2 vaccine was inoculated into chicken, and then nasal swab, cloacal swab and lung were removed after inoculation with the homozygous (H9N2) and heterozygous (H5N1) virus and the virus titer was measured at 3 and 5 days after infection The results are shown in Figs.

H9N2, and cHA H9 / H5N2 vaccine when vaccinated with homozygous virus in chickens showed lower virus titer than PBS vaccine group (Fig. 6).

In addition, the H9N2 vaccine did not protect against heterologous virus when the viral titers were measured in chicken organs, nasal swab, and cloacal swab at 3,5 days after the inoculation with the heterozygous H5N8 virus. On day 5 of vaccination, all chickens died and virus titers could not be measured. However, in the case of cHA H9 / H5N2 vaccine, virus titers were observed in organ and swab samples on the 3rd day after challenge, (FIG. 8), it was confirmed that the vaccine of the present invention exhibited the effect of vaccine against not only the homologous virus H9N2 but also the heterozygous virus H5N8.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Korea Biotechnology Research Institute KCTC13008BP 20160415

<110> I.D.Bio. <120> RECOMBINANT INFLUENZA VIRUS TO FORM CROSS-PROTECTION AGAINST          MULTIPLE SUBTYPES H9 AND H5 OF INFLUENZA VIRUSES AND VACCINE          COMPRISING THE SAME <130> PN1604-114 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 1014 <212> DNA <213> Influenzae virus <400> 1 atggaagtaa tagcactaat agctatactg gtagtgacag gaacgagcaa tgctgataaa 60 atttgcattg gataccagtc aacaaactcc acagaaactg ttgatacact agtagaaacc 120 aatgtccctg tgacacatgc caaagaattg cttcacacag agcacaatgg aatgttatgt 180 gcaacaaatc aggggcatcc tcttaaccta gacacctgca ccgttgaagg gttggtgtat 240 ggcaatcctt cctgtgattt gctactggga ggaaaagaat ggtcttacat tgtcgaaaga 300 tcatcagcca ttaatggggt gtgttaccct ggaagggtag agaatctgga agaactaaga 360 tcttttttca gttctgctca ctcctacaaa agactcctgc tcttcccaga ccacacttgg 420 aatgtgactt acactggaac aagcaaagca tgctcaggtt cattctacag aagtatgaga 480 tggctgacac acaagagcaa ttcttaccct attcaagacg ctcaatatac caacgattgg 540 ggaaagaata ttctcttcat gtggggcata caccattcac ctactgatac tgagcaaatg 600 aatctataca aaaaagctga tacaacaacg agcataacaa cggaagatat caatcggact 660 ttcaaaccag tgataggacc aaggcccctt gtcaatggtc aacaagggag gattgattat 720 tattggtcag tactaaagcc aggccagaca ttgcgaataa gatccaatgg aaatctaatt 780 gctccatggt atggacacac tctttcagga gaaagccatg gaagaatcct gaagaccgat 840 ttaaatagtg gcaactgcgt agtacagtgc caaactgaga aaggtggttt gaatacgacc 900 ttgccattcc acaatgtcag caagtatgca tttgggaact gccccaaata tgttggagtg 960 aagagtctca aactggcagt tggtctaaga aatgtgcctg ctgcatcagg taga 1014 <210> 2 <211> 669 <212> DNA <213> Influenzae virus <400> 2 ggactatttg gagctatagc agggtttata gagggaggat ggcagggaat ggtagacggt 60 tggtatgggt accaccatag caatgagcag gggagtgggt acgctgcaga caaagaatcc 120 acccaaaagg caatagatgg agttaccaat aaggtcaact caatcattga caaaatgaac 180 actcaatttg aggccgttgg aagggaattt aataacttag aaaggagaat agagaattta 240 aacaagaaaa tggaagacgg attcctagat gtctggactt ataatgctga acttttagtt 300 ctcatggaaa atgagagaac tctagatttc catgactcaa atgtcaagaa cctttacgac 360 aaagtccgac tacagcttag ggataatgca aaggagctgg gtaatggttg tttcgagttc 420 tatcacaaat gtgataaaga atgtatggaa agcgtaagaa atgggacgta tgactaccct 480 aagtattcag aagaagcaag attaaaaaga gaagaaataa gcggagtgaa attagaatca 540 ataggaactt accaaatact gtcaatttat tcaacagtgg cgagttccct agcactggca 600 atcatagtgg ctggtctatc tttatggatg tgctctaatg ggtcgctaca atgcagaatt 660 tgcatctaa 669 <210> 3 <211> 1683 <212> DNA <213> Influenzae virus <400> 3 atggaagtaa tagcactaat agctatactg gtagtgacag gaacgagcaa tgctgataaa 60 atttgcattg gataccagtc aacaaactcc acagaaactg ttgatacact agtagaaacc 120 aatgtccctg tgacacatgc caaagaattg cttcacacag agcacaatgg aatgttatgt 180 gcaacaaatc aggggcatcc tcttaaccta gacacctgca ccgttgaagg gttggtgtat 240 ggcaatcctt cctgtgattt gctactggga ggaaaagaat ggtcttacat tgtcgaaaga 300 tcatcagcca ttaatggggt gtgttaccct ggaagggtag agaatctgga agaactaaga 360 tcttttttca gttctgctca ctcctacaaa agactcctgc tcttcccaga ccacacttgg 420 aatgtgactt acactggaac aagcaaagca tgctcaggtt cattctacag aagtatgaga 480 tggctgacac acaagagcaa ttcttaccct attcaagacg ctcaatatac caacgattgg 540 ggaaagaata ttctcttcat gtggggcata caccattcac ctactgatac tgagcaaatg 600 aatctataca aaaaagctga tacaacaacg agcataacaa cggaagatat caatcggact 660 ttcaaaccag tgataggacc aaggcccctt gtcaatggtc aacaagggag gattgattat 720 tattggtcag tactaaagcc aggccagaca ttgcgaataa gatccaatgg aaatctaatt 780 gctccatggt atggacacac tctttcagga gaaagccatg gaagaatcct gaagaccgat 840 ttaaatagtg gcaactgcgt agtacagtgc caaactgaga aaggtggttt gaatacgacc 900 ttgccattcc acaatgtcag caagtatgca tttgggaact gccccaaata tgttggagtg 960 aagagtctca aactggcagt tggtctaaga aatgtgcctg ctgcatcagg tagaggacta 1020 tttggagcta tagcagggtt tatagaggga ggatggcagg gaatggtaga cggttggtat 1080 gggtaccacc atagcaatga gcaggggagt gggtacgctg cagacaaaga atccacccaa 1140 aaggcaatag atggagttac caataaggtc aactcaatca ttgacaaaat gaacactcaa 1200 tttgaggccg ttggaaggga atttaataac ttagaaagga gaatagagaa tttaaacaag 1260 aaaatggaag acggattcct agatgtctgg acttataatg ctgaactttt agttctcatg 1320 gaaaatgaga gaactctaga tttccatgac tcaaatgtca agaaccttta cgacaaagtc 1380 cgactacagc ttagggataa tgcaaaggag ctgggtaatg gttgtttcga gttctatcac 1440 aaatgtgata aagaatgtat ggaaagcgta agaaatggga cgtatgacta ccctaagtat 1500 tcagaagaag caagattaaa aagagaagaa ataagcggag tgaaattaga atcaatagga 1560 acttaccaaa tactgtcaat ttattcaaca gtggcgagtt ccctagcact ggcaatcata 1620 gtggctggtc tatctttatg gatgtgctct aatgggtcgc tacaatgcag aatttgcatc 1680 taa 1683 <210> 4 <211> 1640 <212> DNA <213> Influenzae virus <400> 4 atggaagtaa tagcactaat agctatactg ggagtgacgg gaacgagcaa tgctgataaa 60 atttgcattg gataccagtc aacaaactcc acagaaactg ttgatacact agtagaaacc 120 aatgtccctg tgacacatgc caaagaactg cttcacacag agcacaatgg aatgttatgt 180 gcaacaaatc aggggcatcc tcttatccta gacacttgca ccgttgaagg gttggtgtat 240 ggcaatcctt cctgtgattt gctactggga ggaaaagaat ggtcttacat tgtcgaaaga 300 tcatcagcca ttaatgggat gtgttaccct ggaagggtag agaatccgga agaactaaaa 360 tcttttttca gttctgctcg ctcccacaaa agactcctgc tctttccaga ccgcacttgg 420 aatgtgactt actctggaac aagcaaagca tgctcaggtt cattctacag aagtatgaga 480 tggctgacac acaagagcaa ttcttaccct attcaagacg ctcaatatac caacgattgg 540 ggaaagaata ttctcttcat gtggggcata caccatccac ctactgatac tgagcaaatg 600 aatctataca aaaaagctga tacaacaacg agcataacaa cggaagatat caatcggact 660 ttcaaaccag tgataggacc aaggcccctt gtcaatggtc aacaagggag gattgattat 720 tattggtcag tactaaagcc aggccagaca ttgcgaataa gatccaatgg aaatctaatt 780 gctccatggt atggacacat tctttcagga gaaagccatg gaagaatcct gaagaccgat 840 ttaaatagtg gcaactgcgt agtacagtgc caaactgaga aaggtggttt gaatacgaca 900 ttgccattcc acaatgtcag caagtatgca tttgggaact gccccaaata tgttggagtg 960 aagagtctca aactggcagt tggtctaaga aatgtgcctg ctgcatcagg tagagggcta 1020 tttggtgcca tagctggatt catagaagga ggttggccag ggctagttgc aggctggtac 1080 gggtttcagc attcaaatga tcaaggagtt ggaatggccg cagacagaga gtcaactcaa 1140 aaagcagtcg acaaaataac atccaaagta aataacataa tcgataagat gaacaagcag 1200 tatgaaatca ttgatcatga gttcaatgaa attgaagcca gactcaatat gatcaacaat 1260 aagattgatg accaaataca agacatctgg gcgtataatg cagaattact agtactgctt 1320 gaaaatcaaa aaacacttga tgagcatgat gcaaatgtta acaatctgta caataaggtg 1380 aagagagcat tggggtcaaa tgcaatagag gatgggaagg ggtgcttcga gttgtatcac 1440 aaatgtgatg atcaatgcat ggaaacaatt agaaacggga cttatgacat gataaagtac 1500 aaagaagaat caaaactaga aaggcagaaa atagaagggg tgaaactgga gtttgaagga 1560 acttacaaga ttcttactat ttattcgact gtcgcctcat ctcttgtgct tgcaataggg 1620 tttgctgcct tcatgttctg 1640 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tattcgtctc agggagcaaa agcagggg 28 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 agctatggca ccaaatag 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ctatttggag ctatagca 18 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 atatcgtctc gtattagtag aaacaagggt gtttt 35

Claims (9)

H9 and H5 Recombinant influenza virus comprising accessory subtype chimeric hemagglutinin gene (Accession No. KCTC 13008BP). The method according to claim 1,
The chimeric hemagglutinin gene comprises an HA1 spherical head domain derived from A / CK / Korea / 04163/04 (H9N2); A recombinant influenza virus characterized in that the HA2 gene derived from A / MD / Korea / W452 / 2014 (H5N8) is fused. The method according to claim 1,
Wherein the chimeric hemagglutinin gene comprises the polynucleotide of SEQ ID NO: 3. An influenza virus vaccine comprising the virus of claim 1 as an active ingredient. 5. The method of claim 4,
Wherein the vaccine is an influenza virus vaccine characterized in that the virus is an attenuated virulent virally vaccine or a vaccine. 5. The method of claim 4,
Characterized in that said virus forms a cross-immune response against both influenza A viruses with H9 serotype and H5 serotype. A method for preventing or treating an influenza virus infection disease by administering the vaccine of claim 4 to an individual other than a human. A diagnostic composition for influenza virus comprising the virus of claim 1 or an antigen thereof. (Accession No .: KCTC 13008BP), characterized in that it detects an influenza virus (Accession No: KCTC 13008BP) in the infected or infected cells through an antigen-antibody reaction using the virus of claim 1 or an antigen thereof Way.

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