KR20150118400A - Novel virus vaccine to form cross-protection against multiple subtypes of influenza viruses - Google Patents

Abstract

The present invention relates to a novel influenza virus having an effect in cross immunoreactivity against different subtypes of influenza viruses, a vaccine containing the same as an active ingredient, and a method for preventing or treating influenza virus infection by administering the vaccine into an individual. The recombinant influenza virus according to the present invention contains chimeric hemagglutinin genes manufactured by fusing an HA1 area and an HA2 area of hemagglutinin induced from two different subtypes, thereby showing an effect of the cross immunoactivity against the virus having different sorts of subtypes and clades, and being very usefully used as a purpose of the virus vaccine having a wide neutralization antibody effect.

Description

≪ Desc / Clms Page number 2 > Novel virus vaccines to form cross-protection against multiple influenza viruses that form a cross-immune response against multiple subtypes of influenza viruses.

The present invention relates to a novel influenza virus which is effective for cross-reacting to influenza viruses of different subtypes, a vaccine containing the same as an active ingredient, and a method for preventing or treating an influenza virus infection disease by administering the vaccine to an individual will be.

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, which has various subtypes. As a result, hemagglutinin HA1 region and HA2 region derived from two different subtypes Confirmed that the recombinant influenza virus containing the chimeric hemagglutinin gene prepared by fusion produced cross-reactive immunity effectively against all influenza A viruses having H1N1 serotype, H5N1 serotype and H5N2 serotype, Thereby completing the invention.

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

Therefore, an object of the present invention is to provide a recombinant influenza virus comprising two different subtypes of chimeric hemagglutinin genes.

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

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.

In order to achieve the object of the present invention, the present invention provides recombinant influenza virus (accession number: KCTC12567BP) comprising two different subtypes of chimeric hemagglutinin gene.

In one embodiment of the present invention, the chimeric hemagglutinin gene comprises HA1 and a fusion peptide gene region derived from A / Korea / CJ01 / 09 (H1N1); A / EM / Korea / W150 / 06 (H5N1).

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

In one embodiment of the invention, the virus is derived from A / Puerto Rico / 08/34 (H1N1), with 6 or 7 other genes in addition to the hemagglutinin gene (assuming that the NA gene can also be changed) .

In one embodiment of the present invention, the six or seven genes derived from A / Puerto Rico / 08/34 (H1N1) may comprise polynucleotides of SEQ ID NOS: 4 to 10.

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 can cross-react with all influenza A viruses with H1N1 serotype, H5N1 serotype and H5N2 serotype.

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 recombinant influenza virus of the present invention (accession number: KCTC12567BP) contains hemagglutinin HA1 derived from two different subtypes and chimeric hemagglutinin gene prepared by fusing the fusion peptide region and HA2 region And thus it can be very useful as a viral vaccine having a wide range of neutralizing antibody effects as it exhibits a cross-immune response effect against viruses having various subtypes and clades.

1A is a schematic representation of the hemagglutinin gene structure of a common influenza virus, and FIG. 2B is a schematic representation of the HA1 / HA2 hemagglutinin gene structure of the present invention.
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 diagram illustrating a vaccine schedule in a mouse as an experimental animal to confirm the efficacy of the recombinant influenza virus of the present invention.
FIG. 4 is a graph showing the effect of the recombinant influenza virus vaccine on the ability of the recombinant influenza viruses of the present invention to be tested against the neutralizing antibodies by first or second inoculation of recombinant influenza virus vaccine, followed by the homologous virus maCA04 (H1N1) mouse-adapted CA04 0.0 > 100MLD < _{/ RTI} > ₅₀ for 14 days.
FIG. 5 is a graph showing the results of immunization with recombinant influenza virus vaccine in a dose of 100MLD ₅₀ after the first or second inoculation of mouse recombinant influenza virus vaccine and the homologous virus maCA04 (H1N1) for the ability of the recombinant influenza virus of the present invention to neutralize antibodies Lt; RTI ID = 0.0 > viability < / RTI >
6 is then to the power verification of the neutralizing antibodies in the recombinant virus of the present invention, a mouse, a primary or secondary inoculation the recombinant virus vaccine and then infected with a heterologous virus is highly pathogenic W150 (H5N1) in an amount of 100MLD ₅₀ Lt; RTI ID = 0.0 > 14 < / RTI > days.
7 is then to the power verification of the neutralizing antibodies in the recombinant virus of the present invention, a mouse, a primary or secondary inoculation the recombinant virus vaccine and then infected with a heterologous virus is highly pathogenic W150 (H5N1) in an amount of 100MLD ₅₀ Lt; RTI ID = 0.0 > 14 < / RTI >
Of Figure 8 is 100MLD the to the power verification of the neutralizing antibodies in the recombinant virus of the present invention, a mouse, a primary or secondary inoculation the recombinant virus vaccine and then, the other system (clade) virus is highly pathogenic VN1203 (H5N1) ₅₀ Lt; RTI ID = 0.0 > 14 < / RTI > days after infection.
Of Figure 9 is 100MLD the to the power verification of the neutralizing antibodies in the recombinant virus of the present invention, the recombinant virus vaccine of the mouse primary or secondary immunization, and then other system (clade) virus is highly pathogenic VN1203 (H5N1) ₅₀ Lt; RTI ID = 0.0 > 14 < / RTI > days post infection.
FIG. 10 is a graph showing the results of immunization of recombinant influenza virus vaccine with a 100 MLD ₅₀ amount of ma81 (H5N2), which is a homologous recombinant influenza virus vaccine, in order to test the ability of the recombinant influenza virus of the present invention against neutralizing antibodies Lt; RTI ID = 0.0 > 14 < / RTI > days.
FIG. 11 is a graph showing the results of immunization of recombinant influenza virus vaccine with the recombinant influenza virus vaccine in a dose of 100 MLD ₅₀ after the first or second inoculation of the recombinant influenza virus vaccine and the ma81 (H5N2) Lt; RTI ID = 0.0 > 14 < / RTI >

In one aspect, the present invention relates to a recombinant influenza virus (Accession Number: KCTC12567BP) comprising a chimeric hemagglutinin gene.

"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 HA1 and a fusion peptide gene region derived from A / Korea / CJ01 / 09 (H1N1); A / EM / Korea / W150 / 06 (H5N1).

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 can be obtained from A / Puerto Rico / 08/34 (H1N1), which is currently used as a backbone virus of human vaccine, from 6 or 7 other genes other than the chimeric hemagglutinin gene Quot; can be used.

M (SEQ ID NO: 5), NP (SEQ ID NO: 6), NS (SEQ ID NO: 7), and PA (SEQ ID NO: 7) as seven genes derived from A / Puerto Rico / 08 / (SEQ ID NO: 8), PB1 (SEQ ID NO: 9) and PB2 (SEQ ID NO: 10) genes.

In one embodiment of the present invention, the present inventors have provided the above-mentioned chimeric hemagglutinin gene and seven other genes from A / Puerto Rico / 08/34 (H1N1) and used reverse genetics system To produce the recombinant influenza virus of the present invention, which was named 'cHA H1 / H5N1'. On March 14, 2014, the virus was deposited with KCTC (Korea Research Institute of Bioscience and Biotechnology) and received the deposit number KCTC12567BP.

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 contain the cHA H1 / H5N1 virus (accession number: KCTC12567BP) 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.

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 sequences into viral genomes or specific deletions of viral sequences known to provide toxicity.

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 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, when the hemocyte aggregation unit of the vaccine is less than ²³ HAU, it may not induce effective antibody formation to the subject to be administered, and when it is more than ²⁸ HAU, it may be uneconomical in efficiency.

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 / Korea / CJ01 / 09 ( H1 / N1 ) Virus isolation

The laryngeal secretions collected from patients admitted to the Chungbuk National University Hospital for H1N1 infection were placed in cell culture medium containing 10% Penicillin-Streptomycin (Gibco) antibiotics, and the suspension was prepared by centrifugation. After removal, the supernatant was infected with MDCK (Madin-Darby canine kidney) cells, and then the virus was isolated by culturing in a CO ₂ cell incubator for 48 hours. The isolated virus was identified as a new influenza virus by RT-PCR and gene analysis.

A / Korea / CJ01 / 09 (H1N1) "was named as" A / Korea / Swine Influenza Virus "separated from the above, and it was added to the Korean Collection for Type Cultures 27, from which it was granted the deposit number KCTC 11690 BP.

<1-2> A / EM / Korea / W150 / 06 ( H5N1 ) And A / Puerto Rico / 08/34 ( H1N1 ) Virus preparation

A / Puerto Rico / 08/34 (H1N1) Robert G. Webster, Division of Virology, Department of Infectious Diseases, St. Louis, MO .; Jude Children's Research Hospital, Memphis, TN 38105, and A / EM / Korea / W150 / 06 (H5N1) viruses were isolated from this laboratory. The virus was isolated from wild fowl in 2006 by isolating it as a highly pathogenic virus. The cells were suspended in 10% Penicillin-Streptomycin (Gibco) antibiotic-containing cell culture medium, and the supernatant was infected with MDCK (Madin-Darby canine kidney) cells by centrifugation The virus was isolated by incubating in a CO ₂ cell incubator for 48 hours. The isolated virus was identified as a highly pathogenic H5N1 virus by RT-PCR and genetic analysis. [Note: Youn-Jeong Lee et al, "Highly Pathogenic Avian Influenza Virus (H5N1) in Domestic Poultry and Relationship with Migratory Birds, South Korea "Emerging infectious diseases, (March 2008)]

< 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 hemagglutanin gene was isolated from two subtypes (serotype) of HA1 (globular head domain) and HA2 (stalk region) Rico / 08/34 (H1N1)) to prepare a recombinant virus.

<2-1> Chimeric Hemagglutinin  Gene production

First, the present inventors prepared HA1 and fusion peptide gene regions (SEQ ID NO: 1) of A / Korea / CJ01 / 09 (H1N1) and HA1 and fusion peptide gene regions A chimeric H1 / H5 hemaglutimine gene was prepared by fusing the HA2 Ectodomain gene region (SEQ ID NO: 2) of A / EM / Korea / W150 / 06 (H5N1).

A / Korea / CJ01 / 09 (H1N1) contains a primer (5'-CAT TCC CTG CCA TCC CCC) that specifically recognizes HA1 and Bm HA 1F (5'- TAT TCG TCT CAG GGA GCA AAA GCA GGG G- HA1 and fusion peptide gene region (SEQ ID NO: 1) of A / Korea / CJ01 / 09 (H1N1) was prepared using TTC AAT GAA ACC- 3 'and primers (5'-GGT TTC ATT HA2 Ectodomain of GAA GGG GGA TGG CAG GGA ATG- 3 ') and Bm NS 890R (5'-ATA TCG TCT CGT ATT AGT AGA AAC AAG GGT GTT TT- 3') A / EM / Korea / W150 / 06 (H5N1) Each of the gene fragments was prepared using the gene region (SEQ ID NO: 2), followed by fusion and amplification through PCR to prepare the chimeric gene of the present invention (SEQ ID NO: 3).

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

(SEQ ID NO: 4), M (SEQ ID NO: 5), and NP (SEQ ID NO: 5) of A / Puerto Rico / 08/34 of the present invention prepared in Example <2-1> (SEQ ID NO: 6), NS (SEQ ID NO: 7), PA (SEQ ID NO: 8), PB1 (SEQ ID NO: 9) and PB2 (SEQ ID NO: 10) were used as expression vectors vPHW2000 (using J Gen Virol. 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 NOS: 3 to 10 were respectively inserted were mixed together using transfection reagent TransIT-LT1 transfection reagent (Mirus Bio), and then placed 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 virus of the present invention was isolated from the fertilized eggs. The thus isolated recombinant virus was named 'cHA H1 / H5N1' And used in the following experiment.

The recombinant virus of the present invention prepared according to the method described above was deposited on March 14, 2014 with the KCTC of the Korea Research Institute of Bioscience and Biotechnology, and received the deposit number KCTC12567BP.

< Example  3>

The Chimeric Hemagglutinin  Of a recombinant virus containing the gene Maou Evaluation of antigenicity by animal experiment

In order to evaluate the antigenicity of the recombinant vaccine candidate main virus cHA H1 / H5N1 developed in the present invention, 10 recombinant viruses (accession number: KCTC12567BP) prepared in Example 2 were inoculated into 10 days-old SPF fertilized eggs, Later, urine was detected and titers were confirmed by hemagglutination 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.

The inactivated virus strain was placed in a SW28 swing rotor bucket (Beckman, USA) containing 20% sucrose cushion and 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.

Antigenicity was evaluated by primary intramuscular injection of the desired concentration of virus solution according to the content of hemagglutinin protein (1.7 μg / 0.1 ml, 3.5 μg / 0.1 ml) in 4-week-old BALB / C mice, The second vaccination was carried out in the same way at the time of elapse.

Two weeks after the first and second inoculation, the serum of the mouse was collected to test the ability of the neutralizing antibody. To confirm whether the vaccine effect was proved, the mice were infected with homologous, heterologous, And survival rate were measured. All experiments included two groups inoculated with the A / Korea / CJ01 / 09 (H1N1) virus and PBS in the control group. 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.

The homozygous, heterozygous and other clade viruses used to test the ability of neutralizing antibodies Abbreviation of virus Full name of the virus and where to buy it maCA04 (H1N1) A / California / 04/09 (H1N1); mouse-adaptation strain; ID Bio Co., Ltd., Chungbuk National University W150 (H5N1) A / EM / Korea / W150 / 06 (H5N1); Bio Reader, VN1203 (H5N1) A / Viet Nam / 1203/2004 (H5N1); Bioreaders, International Vaccine Institute ma81 (H5N2) A / aquatic bird / Korea / MA81 / 2007 (H5N2); Bioreaders, ID Bio Co., Ltd., Chungbuk National University

<3-1> Measurement of the weight and survival rate of mice after vaccination with allogeneic 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 above was inoculated into a mouse, MaCA04 (H1N1) was infected at a dose of 100MLD ₅₀ , and the weight change and survival rate of the mice were measured for 14 days.

As a result, as shown in FIG. 4, in the experimental group in which the first and second vaccines were performed with the recombinant viruses of the present invention, slight weight changes occurred until day 7 after infection with the homologous virus, but at 14 days, We were able to confirm that weight recovery was achieved in mice. Further, as shown in FIG. 5, it was possible to monitor that the survival rate was 100%.

<3-2> Measurement of weight and survival rate of mice after vaccination with heterologous virus infection

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, Weight change and survival rate of mice were measured for 14 days after infection with 100MLD ₅₀ of highly pathogenic W150 (H5N1).

As a result, in the experimental group in which the first vaccine was performed with the recombinant virus of the present invention, when the infection of the virus occurred, the body weight began to decrease from the 2nd day onwards and died on the 10th day (see FIG. 6A) 20% of the body weight of the first day was decreased, and it recovered to 90% at the end of 14 days (see FIG. 6B).

In addition, as shown in FIG. 7, the survival rate of the vaccine with high hemagglutinin content is 100% at the time of virus infection after the second vaccination, while the survival rate of the vaccine with low hemagglutinin content is as low as 50% .

<3-3> Other system after vaccination ( clade ) Measuring the weight and survival rate of mice with viral infection

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, (VN / 1203 (H5N1), a clade virus, was infected at a dose of 100MLD ₅₀ , and the weight change and survival rate of the mice were measured for 14 days.

As a result, as shown in Fig. 8, in the experimental group in which the first vaccine was performed with the recombinant virus of the present invention, virus infection occurred after the first vaccination, the body weight started to decrease from the second day, and the body weight was restored after 12 days Respectively.

In addition, as shown in FIG. 9, in the experimental group subjected to the first vaccination, all mice died at 12 days after the infection, but mice were 100% survived in the second vaccination group.

<3-4> Measurement of weight and survival rate of mice after vaccination with heterologous virus infection

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, Ruti NIN and New Lamy is to measure the weight change and the survival of the kinase gene is both mice 14 days after infection, the virus is another variant ma81 (H5N2) in the amount of 100MLD _50.

As a result, all of the body weight of the control group was decreased after the first vaccination, and all of the mice died on the 7th day of infection (see FIGS. 10A and 11A). On the other hand, in the experimental group in which the first vaccine was performed with the recombinant virus of the present invention, it was confirmed that mice were 100% survived on the 7th day of infection (see FIG. 11A).

Also, after the second vaccination, rapid weight change was observed in the vaccine group with high hemagglutinin content of the control group A / Korea / CJ01 / 09 (H1N1) virus, but recovered from day 8, And 30%, respectively. On the other hand, in the experimental group in which the second vaccine was performed with the recombinant virus of the present invention, no significant change in body weight and survival rate with time elapsed after infection were observed, and it was confirmed that the recombinant virus vaccine of the present invention was excellent in efficacy (FIGS. 10B and 10B) See FIG. 11B).

&Lt; 3-4 > Vaccination of various virus infections after vaccination Potency  Measure

The mice were inoculated with maCA04 (H1N1), W150 (H5N1; clade 2.2), VN / 1203 (H5N1; clade 1) and ma81 (H5N2) , And 7 days later, mouse lung tissue was collected to measure the titer of the virus. Mice were anesthetized by intraperitoneal anesthesia with 30 ~ 40 mg / kg of body weight per mouse and then infected with ₃₀ ml of 100MLD ₅₀ by intranasal inoculation. MLD ₅₀ represents the titer of a virus that can kill 50% of the mice, and in this experiment infected 100 times the amount of MLD ₅₀ . In order to measure virus titers, 3 mice were collected from control and experimental groups at 3, 5, and 7 days after viral infection.

The measured virus titers are shown in Table 2 below.

After the recombinant vaccination of the present invention, the virus titer challenge virus and HA content A / Korea / CJ01 / 09 (H1N1) vaccination The recombinant virus vaccine of the present invention Days post infection Days post infection 3 days 5 days 7 days 3 days 5 days 7 days Primary vaccine group 1.7 μg HA maCA04 (H1N1) 3.8 2.8 1.3 4.3 3.8 1.0 W150 (H5N1) 5.5 4.5 4.5 5.5 5.5 4.5 VN1203 (H5N1) 5.5 5.5 4.5 4.5 5.5 5.5 ma81 (H5N2) 8.5 4.5 4.5 6.5 4.5 2.5 3.5 μg HA maCA04 (H1N1) 3.6 1.7 0.7 4.0 3.8 1.4 W150 (H5N1) 4.5 5.5 3.5 4.5 3.5 2.5 VN1203 (H5N1) 4.5 5.5 5.5 4.5 4.5 3.5 ma81 (H5N2) 5.5 4.5 1.5 4.5 4.5 0.7 Secondary vaccine group 1.7 μg HA maCA04 (H1N1) 2.8 1.7 0.7 4.3 2.8 0.7 W150 (H5N1) 3.5 5.5 3.5 2.5 2.5 0.7 VN1203 (H5N1) 3.5 4.5 5.5 4.5 2.5 0.7 ma81 (H5N2) 3.5 6.5 5.5 5.5 5.5 0.7 3.5 μg HA maCA04 (H1N1) 1.7 1.7 0.7 3.0 1.7 0.7 W150 (H5N1) 3.5 2.5 0.7 2.5 1.7 0.7 VN1203 (H5N1) 3.5 4.5 4.5 2.5 1.3 0.7 ma81 (H5N2) 4.3 2.8 1.7 2.5 2.5 0.7

mock-vaccination group Days post infection 3 days 5 days 7 days maCA04 (H1N1) 3.3 3.3 1.7 W150 (H5N1) 4.7 5.3 4 VN1203 (H5N1) 4.3 4.5 6.3 ma81 (H5N2) 5.5 7.5 4.5

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 KCTC12567BP 20140314

<110> I.D.BIO. <120> Novel virus vaccine to form cross-protection against multiple          subtypes of influenza viruses <130> PN1312-424 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 1068 <212> DNA <213> plynucelotide sequence of A / Korea / CJ01 / 09 (H1N1) derived HA1 and fusion peptide gene region <400> 1 atgaaggcaa tactagtagt tctgctatat acatttgcaa ccgcaaatgc agacacatta 60 tgtataggtt atcatgcaaa caattcaaca gacactgtag acacagtact agaaaagaat 120 gtaacagtaa cacactctgt taaccttcta gaagacaagc ataacgggaa actatgcaaa 180 ctaagagggg tagccccatt gcatttgggt aaatgtaaca ttgctggctg gatcctggga 240 aatccagagt gtgaatcact ctccacagca agctcatggt cctacattgt ggaaacatct 300 agttcagaca atggaacgtg ttacccagga gatttcatcg attatgagga gctaagagag 360 caattgagct cagtgtcatc atttgaaagg tttgagatat tccccaagac aagttcatgg 420 cccaatcatg actcgaacaa aggtgtaacg gcagcatgtc ctcatgctgg agcaaaaagc 480 ttctacaaaa atttaatatg gctagttaaa aaaggaaatt catacccaaa gctcagcaaa 540 tcctacatta atgataaagg gaaagaagtc ctcgtgctat ggggcattca ccatccatct 600 actagtgctg accaacaaag tctctatcag aatgcagatg catatgtttt tgtggggaca 660 tcaagataca gcaagaagtt caagccggaa atagcaataa gacccaaagt gagggatcaa 720 gaagggagaa tgaactatta ctggacacta gtagagccgg gagacaaaat aacattcgaa 780 gcaactggaa atctagtggt accgagatat gcattcgcaa tggaaagaaa tgctggatct 840 ggtattatca tttcagatac accagtccac gattgcaata caacttgtca gacacccaag 900 ggtgctataa acaccagcct cccatttcag aatatacatc cgatcacaat tggaaaatgt 960 ccaaaatatg taaaaagcac aaaattgaga ctggccacag gattgaggaa tgtcccgtct 1020 attcaatcta gaggcctatt tggggccatt gccggtttca ttgaaggg 1068 <210> 2 <211> 633 <212> DNA <213> polynucletide sequence of A / EM / Korea / W150 / 06 (H5N1) derived HA2 ectodomain gene region <400> 2 ggatggcagg gaatggtaga tggttggtat ggataccacc atagcaacga gcaggggagt 60 gggtacgctg cagacaaaga atccactcaa aaggcaatag atggagtcac caataaagtc 120 aactcgatca ttgacaaaat gaacactcaa tttgaggccg ttggaaggga atttaataac 180 ttagaaagga gaatagagaa tttaaacaag aagatggaag acggatttct agatgtctgg 240 acttataatg ctgaacttct ggttctcatg gaaaatgaga gaactctaga ctttcatgac 300 tcaaatgtca agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag 360 cttggtaacg gttgtttcga gttctatcac agatgtgata atgaatgtat ggaaagtgta 420 agaaacggaa cgtatgacta cccgcagtat tcagaagaat caagattaaa aagagaggaa 480 ataagtggag taaaattgga atcaatagga acttatcaaa tactgtcaat ttattcaaca 540 gtggcgagct ccctagcact ggcaatcatg gtggctggtc tatctttatg gatgtgttcc 600 aatggatcgt tacaatgcag aatttgcatt taa 633 <210> 3 <211> 1699 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide sequence of chimeric hemagglutinin gene <400> 3 atgaaggcaa tactagtagt tctgctatat acatttgcaa ccgcaaatgc agacacatta 60 tgtataggtt atcatgcaaa caattcaaca gacactgtag acacagtact agaaaagaat 120 gtaacagtaa cacactctgt taaccttcta gaagacaagc ataacgggaa actatgcaaa 180 ctaagagggg tagccccatt gcatttgggt aaatgtaaca ttgctggctg gatcctggga 240 aatccagagt gtgaatcact ctccacagca agctcatggt cctacattgt ggaaacatct 300 agttcagaca atggaacgtg ttacccagga gatttcatcg attatgagga gctaagagag 360 caattgagct cagtgtcatc atttgaaagg tttgagatat tccccaagac aagttcatgg 420 cccaatcatg actcgaacaa aggtgtaacg gcagcatgtc ctcatgctgg agcaaaaagc 480 ttctacaaaa atttaatatg gctagttaaa aaaggaaatt catacccaaa gctcagcaaa 540 tcctacatta atgataaagg gaaagaagtc ctcgtgctat ggggcattca ccatccatct 600 actagtgctg accaacaaag tctctatcag aatgcagatg catatgtttt tgtggggaca 660 tcaagataca gcaagaagtt caagccggaa atagcaataa gacccaaagt gagggatcaa 720 gaagggagaa tgaactatta ctggacacta gtagagccgg gagacaaaat aacattcgaa 780 gcaactggaa atctagtggt accgagatat gcattcgcaa tggaaagaaa tgctggatct 840 ggtattatca tttcagatac accagtccac gattgcaata caacttgtca gacacccaag 900 ggtgctataa acaccagcct cccatttcag aatatacatc cgatcacaat tggaaaatgt 960 ccaaaatatg taaaaagcac aaaattgaga ctggccacag gattgaggaa tgtcccgtct 1020 attcaatcta gaggcctatt tggggccatt gccggtttca ttgaaggggg atggcaggga 1080 atggtagatg gttggtatgg ataccaccat agcaacgagc aggggagtgg gtacgctgca 1140 gacaaagaat ccactcaaaa ggcaatagat ggagtcacca ataaagtcaa ctcgatcatt 1200 gacaaaatga acactcaatt tgaggccgtt ggaagggaat ttaataactt agaaaggaga 1260 atagagaatt taaacaagaa gatggaagac ggatttctag atgtctggac ttataatgct 1320 gaacttctgg ttctcatgga aaatgagaga actctagact ttcatgactc aaatgtcaaa 1380 cctttacgac aaggtccgac tacagcttag ggataatgca aaggagcttg gtaacggttg 1440 tttcgagttc tatcacagat gtgataatga atgtatggaa agtgtaagaa acggaacgta 1500 tgactacccg cagtattcag aagaatcaag attaaaaaga gaggaaataa gtggagtaaa 1560 attggaatca ataggaactt atcaaatact gtcaatttat tcaacagtgg cgagctccct 1620 agcactggca atcatggtgg ctggtctatc tttatggatg tgttccaatg gatcgttaca 1680 atgcagaatt tgcatttaa 1699 <210> 4 <211> 1373 <212> DNA <213> Polynucleotide sequence of A / Puerto Rico / 08/34 (H1N1) derived NA gene <400> 4 atgaatccaa atcagaaaat aataaccatt ggatcaatct gtctggtagt cggactaatt 60 agcctaatat tgcaaatagg gaatataatc tcaatatgga ttagccattc aattcaaact 120 ggaagtcaaa accatactgg aatatgcaac caaaacatca ttacctataa aaatagcacc 180 tgggtaaagg acacaacttc agtgatatta accggcaatt catctctttg tcccatccgt 240 gggtgggcta tatacagcaa agacaatagc ataagaattg gttccaaagg agacgttttt 300 gtcataagag agccctttat ttcatgttct cacttggaat gcaggacctt ttttctgacc 360 caaggtgcct tactgaatga caagcattca agtgggactg ttaaggacag aagcccttat 420 agggccttaa tgagctgccc tgtcggtgaa gctccgtccc cgtacaattc aagatttgaa 480 tcggttgctt ggtcagcaag tgcatgtcat gatggcatgg gctggctaac aatcggaatt 540 tcaggtccag ataatggagc agtggctgta ttaaaataca acggcataat aactgaaacc 600 ataaaaagtt ggaggaagaa aatattgagg acacaagagt ctgaatgtgc ctgtgtaaat 660 ggttcatgtt ttactataat gactgatggc ccgagtgatg ggctggcctc gtacaaaatt 720 ttcaagatcg aaaaggggaa ggttactaaa tcaatagagt tgaatgcacc taattctcac 780 tatgaggaat gttcctgtta ccctgatacc ggcaaagtga tgtgtgtgtg cagagacaat 840 tggcatggtt cgaaccggcc atgggtgtct ttcgatcaaa acctggatta tcaaatagga 900 tacatctgca gtggggtttt cggtgacaac ccgcgtcccg aagatggaac aggcagctgt 960 ggtccagtgt atgttgatgg agcaaacgga gtaaagggat tttcatatag gtatggtaat 1020 ggtgtttgga taggaaggac caaaagtcac agttccagac atgggtttga gatgatttgg 1080 gatcctaatg gatggacaga gactgatagt aagttctctg tgaggcaaga tgttgtggca 1140 atgactgatt ggtcagggta tagcggaagt ttcgttcaac atcctgagct gacagggcta 1200 gactgtatga ggccgtgctt ctgggttgaa ttaatcaggg gacgacctaa ggagggaaca 1260 atctggacta gtgcgagcag catttctttt tgtggcgtga atagtgatac tgtagattgg 1320 tcttggccag acggtgctga gttgccattc agcattgaca agtagtctgt tca 1373 <210> 5 <211> 1000 <212> DNA <213> polynucleotide sequence of A / Puerto Rico / 08/34 (H1N1) derived M gene <400> 5 gtagatattg aaagatgagt cttctaaccg aggtcgaaac gtacgtactc tctatcatcc 60 cgtcaggccc cctcaaagcc gagatcgcac agagacttga agatgtcttt gcagggaaga 120 acaccgatct tgaggttctc atggaatggc taaagacaag accaatcctg tcacctctga 180 ctaaggggat tttaggattt gtgttcacgc tcaccgtgcc cagtgagcga ggactgcagc 240 gtagacgctt tgtccaaaat gcccttaatg ggaacgggga tccaaataac atggacaaag 300 cagttaaact gtataggaag ctcaagaggg agataacatt ccatggggcc aaagaaatct 360 cactcagtta ttctgctggt gcacttgcca gttgtatggg cctcatatac aacaggatgg 420 gggctgtgac cactgaagtg gcatttggcc tggtatgtgc aacctgtgaa cagattgctg 480 actcccagca tcggtctcat aggcaaatgg tgrcaacaac caatccacta atcagacatg 540 agaacagaat ggttttagcc agcactacag ctaaggctat ggagcaaatg gctggatcga 600 gtgagcaagc agcagaggcc atggaggttg ctattcgggc taggcaaatg gtgcaggcaa 660 tgagaaccat tgggactcat cctagctcca gtgctggtct gaaaaatgat cttcttgaaa 720 atttgcaggc ctatcagaaa cgaatggggg tgcagatgca acggttcaag tgatcctctc 780 attattgcct caartatcat tgggatcttg cacttgayat tgtggattct tgatcgtctt 840 tttttcaaat gcatttaccg tctctttaaa tacggtttga aaagagggcc ttctacggaa 900 ggagtgccaa agtctatgag ggaagaatat caaaaggaac agcagagtgc tgtggatgct 960 gacgatggtc attttgtcag catagagctg gagtaaaaaa 1000 <210> 6 <211> 1501 <212> DNA <213> polynucleotide sequence of A / Puerto Rico / 08/34 (H1N1) derived NP gene <400> 6 atggcgtccc aaggcaccaa acggtcttac gaacagatgg agactgatgg agaacgccag 60 aatgccactg aaatcagagc atccgtcgga aaaatgattg gtggaattgg acgattctac 120 atccaaatgt gcaccgaact caaactcagt gattatgagg gacggttgat ccaaaacagc 180 ttaacaatag agagaatggt gctctctgct tttgacgaaa ggagaaataa atacctggaa 240 gaacatccca gtgcggggaa agatcctaag aaaactggag gacctatata caggagagta 300 aacggaaagt ggatgagaga actcatcctt tatgacaaag aagaaataag gcgaatctgg 360 cgccaagcta ataatggtga cgatgcaacg gctggtctga ctcacatgat gatctggcat 420 tccaatttga atgatgcaac ttatcagagg acaagagctc ttgttcgcac cggaatggat 480 cccaggatgt gctctctgat gcaaggttca actctcccta ggaggtctgg agccgcaggt 540 gctgcagtca aaggagttgg aacaatggtg atggaattgg tcaggatgat caaacgtggg 600 atcaatgatc ggaacttctg gaggggtgag aatggacgaa aaacaagaat tgcttatgaa 660 agaatgtgca acattctcaa agggaaattt caaactgctg cacaaaaagc aatgatggat 720 caagtgagag agagccggaa cccagggaat gctgagttcg aagatctcac ttttctagca 780 cggtctgcac tcatattgag agggtcggtt gctcacaagt cctgcctgcc tgcctgtgtg 840 tatggacctg ccgtagccag tgggtacgac tttgaaagag agggatactc tctagtcgga 900 atagaccctt tcagactgct tcaaaacagc caagtgtaca gcctaatcag accaaatgag 960 aatccagcac acaagagtca actggtgtgg atggcatgcc attctgccgc atttgaagat 1020 ctaagagtat taagcttcat caaagggacg aaggtgctcc caagagggaa gctttccact 1080 agaggagttc aaattgcttc caatgaaaat atggagacta tggaatcaag tacacttgaa 1140 ctgagaagca ggtactgggc cataaggacc agaagtggag gaaacaccaa tcaacagagg 1200 gcatctgcgg gccaaatcag catacaacct acgttctcag tacagagaaa tctccctttt 1260 gacagaacaa ccattatggc agcattcaat gggaatacag agggaagaac atctgacatg 1320 aggaccgaaa tcataaggat gatggaaagt gcaagaccag aagatgtgtc tttccagggg 1380 cggggagtct tcgagctctc ggacgaaaag gcagcgagcc cgatcgtgcc ttcctttgac 1440 atgagtaatg aaggatctta tttcttcgga gacaatgcag aggagtacga caattaaaga 1500 a 1501 <210> 7 <211> 848 <212> DNA <213> polynucleotide sequence of A / Puerto Rico / 08/34 (H1N1) derived NS gene <400> 7 atggattcca acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa 60 cgagttgcag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagatcag 120 aaatccctaa gaggaagggg cagcactctc ggtctggaca tcgagacagc cacacgtgct 180 ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc 240 atggcctctg tacctgcgtc gcgttaccta actgacatga ctcttgagga aatgtcaagg 300 gactggtcca tgctcatacc caagcagaaa gtggcaggcc ctctttgtat cagaatggac 360 caggcgatca tggataagaa catcatactg aaagcgaact tcagtgtgat ttttgaccgg 420 ctggagactc taatattgct aagggctttc accgaagagg gagcaattgt tggcgaaatt 480 tcaccattgc cttctcttcc aggacatact gctgaggatg tcaaaaatgc agttggagtc 540 ctcatcggag gacttgaatg gaatgataac acagttcgag tctctgaaac tctacagaga 600 ttcgcttgga gaagcagtaa tgagaatggg agacctccac tcactccaaa acagaaacga 660 gaaatggcgg gaacaattag gtcagaagtt tgaagaaata agatggttga ttgaagaagt 720 gagacacaaa ctgaagataa cagagaatag ttttgagcaa ataacattta tgcaagcctt 780 acatctattg cttgaagtgg agcaagagat aagaactttc tcgtttcagc ttatttagta 840 ataaaaaa 848 <210> 8 <211> 2193 <212> DNA <213> polynucleotide sequence of A / Puerto Rico / 08/34 (H1N1) derived PA gene <400> 8 atggaagatt ttgtgcgaca atgcttcaat ccgatgattg tcgagcttgc ggaaaaaaca 60 atgaaagagt atggggagga cctgaaaatc gaaacaaaca aatttgcagc aatatgcact 120 cacttggaag tatgcttcat gtattcagat tttcacttca tcaatgagca aggcgagtca 180 ataatcgtag aacttggtga tccaaatgca cttttgaagc acagatttga aataatcgag 240 ggaagagatc gcacaatggc ctggacagta gtaaacagta tttgcaacac tacaggggct 300 gagaaaccaa agtttctacc agatttgtat gattataagg agaatagatt catcgaaatt 360 ggagtaacaa ggagagaagt tcacatatac tatctggaaa aggccaataa aattaaatct 420 gagaaaacac acatccacat tttctcgttc actggggaag aaatggccac aaaggcagac 480 tacactctcg atgaagaaag cagggctagg atcaaaacca gactattcac cataagacaa 540 gaaatggcca gcagaggcct ctgggattcc tttcgtcagt ccgagagag agaagagaca 600 attgaagaaa ggtttgaaat cacaggaaca atgcgcaagc ttgccgacca aagtctcccg 660 ccgaacttct ccagccttga aaattttaga gcctatgtgg atggattcga accgaacggc 720 tacattgagg gcaagctgtc tcaaatgtcc aaagaagtaa atgctagaat tgaacctttt 780 ttgaaaacaa caccacgacc acttagactt ccgaatgggc ctccctgttc tcagcggtcc 840 aaattcctgc tgatggatgc cttaaaatta agcattgagg acccaagtca tgaaggagag 900 ggaataccgc tatatgatgc aatcaaatgc atgagaacat tctttggatg gaaggaaccc 960 aatgttgtta aaccacacga aaagggaata aatccaaatt atcttctgtc atggaagcaa 1020 gtactggcag aactgcagga cattgagaat gaggagaaaa ttccaaagac taaaaatatg 1080 aagaaaacaa gtcagctaaa gtgggcactt ggtgagaaca tggcaccaga aaaggtagac 1140 tttgacgact gtaaagatgt aggtgatttg aagcaatatg atagtgatga accagaattg 1200 aggtcgcttg caagttggat tcagaatgag tttaacaagg catgcgaact gacagattca 1260 agctggatag agctcgatga gattggagaa gatgtggctc caattgaaca cattgcaagc 1320 atgagaagga attatttcac atcagaggtg tctcactgca gagccacaga atacataatg 1380 aagggggtgt acatcaatac tgccttgctt aatgcatctt gtgcagcaat ggatgatttc 1440 caattaattc caatgataag caagtgtaga actaaggagg gaaggcgaaa gaccaacttg 1500 tatggtttca tcataaaagg aagatcccac ttaaggaatg acaccgacgt ggtaaacttt 1560 gtgagcatgg agttttctct cactgaccca agacttgaac cacataaatg ggagaagtac 1620 tgtgttcttg agataggaga tatgcttata agaagtgcca taggccaggt ttcaaggccc 1680 atgtcttgt atgtgagaac aaatggaacc tcaaaaatta aaatgaaatg gggaatggag 1740 atgaggcgtt gcctcctcca gtcacttcaa caaattgaga gtatgattga agctgagtcc 1800 tctgtcaaag agaaagacat gaccaaagag ttctttgaga acaaatcaga aacatggccc 1860 attggagagt cccccaaagg agtggaggaa agttccattg ggaaggtctg caggacttta 1920 ttagcaaagt cggtattcaa cagcttgtat gcatctccac aactagaagg attttcagct 1980 gaatcaagaa aactgcttct tatcgttcag gctcttaggg acaacctgga acctgggacc 2040 tttgatcttg gggggctata tgaagcaatt gaggagtgcc tgattaatga tccctgggtt 2100 ttgcttaatg cttcttggtt caactccttc cttacacatg cattgagtta gttgtggcag 2160 tgctactatt tgctatccat actgtccaaa aaa 2193 <210> 9 <211> 2300 <212> DNA <213> polynucleotide sequence of A / Puerto Rico / 08/34 (H1N1) derived PB1 gene <400> 9 atggatgtca atccgacctt acttttctta aaagtgccag cacaaaatgc tataagcaca 60 actttccctt atactggaga ccctccttac agccatggga caggaacagg atacaccatg 120 gatactgtca acaggacaca tcagtactca gaaaagggaa gatggacaac aaacaccgaa 180 actggagcac cgcaactcaa cccgattgat gggccactgc cagaagacaa tgaaccaagt 240 ggttatgccc aaacagattg tgtattggag gcgatggctt tccttgagga atcccatcct 300 ggtatttttg aaaactcgtg tattgaaacg atggaggttg ttcagcaaac acgagtagac 360 aagctgacac aaggccgaca gacctatgac tggactctaa atagaaacca acctgctgca 420 acagcattgg ccaacacaat agaagtgttc agatcaaatg gcctcacggc caatgagtct 480 ggaaggctca tagacttcct taaggatgta atggagtcaa tgaacaaaga agaaatgggg 540 atcacaactc attttcagag aaagagacgg gtgagagaca atatgactaa gaaaatgata 600 acacagagaa caatgggtaa aaagaagcag agattgaaca aaaggagtta tctaattaga 660 gcattgaccc tgaacacaat gaccaaagat gctgagagag ggaagctaaa acggagagca 720 attgcaaccc cagggatgca aataaggggg tttgtatact ttgttgagac actggcaagg 780 agtatatgtg agaaacttga acaatcaggg ttgccagttg gaggcaatga gaagaaagca 840 aagttggcaa atgttgtaag gaagatgatg accaattctc aggacaccga actttctttc 900 accatcactg gagataacac caaatggaac gaaaatcaga atcctcggat gtttttggcc 960 atgatcacat atatgaccag aaatcagccc gaatggttca gaaatgttct aagtattgct 1020 ccaataatgt tctcaaacaa aatggcgaga ctgggaaaag ggtatatgtt tgagagcaag 1080 agtatgaaac ttagaactca aatacctgca gaaatgctag caagcatcga tttgaaatat 1140 ttcaatgatt caacaagaaa gaagattgaa aaaatccgac cgctcttaat agaggggact 1200 gcatcattga gccctggaat gatgatgggc atgttcaata tgttaagcac tgtattaggc 1260 gtctccatcc tgaatcttgg acaaaagaga tacaccaaga ctacttactg gtgggatggt 1320 cttcaatcct ctgacgattt tgctctgatt gtgaatgcac ccaatcatga agggattcaa 1380 gccggagtcg acaggtttta tcgaacctgt aagctacttg gaatcaatat gagcaagaaa 1440 aagtcttaca taaacagaac aggtacattt gaattcacaa gttttttcta tcgttatggg 1500 tttgttgcca atttcagcat ggagcttccc agttttgggg tgtctgggat caacgagtca 1560 gcggacatga gtattggagt tactgtcatc aaaaacaata tgataaacaa tgatcttggt 1620 ccagcaacag ctcaaatggc ccttcagttg ttcatcaaag attacaggta cacgtaccga 1680 tgccatagag gtgacacaca aatacaaacc cgaagatcat ttgaaataaa gaaactgtgg 1740 gagcaaaccc gttccaaagc tggactgctg gtctccgacg gaggcccaaa tttatacaac 1800 attagaaatc tccacattcc tgaagtctgc ctaaaatggg aattgatgga tgaggattac 1860 caggggcgtt tatgcaaccc actgaaccca tttgtcagcc ataaagaaat tgaatcaatg 1920 aacaatgcag tgatgatgcc agcacatggt ccagccaaaa acatggagta tgatgctgtt 1980 gcaacaacac actcctggat ccccaaaaga aatcgatcca tcttgaatac aagtcaaaga 2040 ggagtacttg aggatgaaca aatgtaccaa aggtgctgca atttatttga aaaattcttc 2100 cccagcagtt catacagaag accagtcggg atatccagta tggtggaggc tatggtttcc 2160 agagcccgaa ttgatgcacg gattgatttc gaatctggaa ggataaagaa agaagagttc 2220 actgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa atagtgaatt 2280 tagcttgtcc ttcatgaaaa 2300 <210> 10 <211> 2280 <212> DNA <213> polynucleotide sequence of A / Puerto Rico / 08/34 (H1N1) derived PB2 gene <400> 10 cgagatagc acaaaaacca ccgtggacca tatggccata atcaagaagt acacatcagg aagacaggag 120 aagaacccag cacttaggat gaaatggatg atggcaatga aatatccaat tacagcagac 180 aagaggataa cggaaatgat tcctgagaga aatgagcaag gacaaacttt atggagtaaa 240 atgaatgatg ccggatcaga ccgagtgatg gtatcacctc tggctgtgac atggtggaat 300 aggaatggac caataacaaa tacagttcat tatccaaaaa tctacaaaac ttattttgaa 360 agagtcgaaa ggctaaagca tggaaccttt ggccctgtcc attttagaaa ccaagtcaaa 420 atacgtcgga gagttgacat aaatcctggt catgcagatc tcagtgccaa ggaggcacag 480 gatgtaatca tggaagttgt tttccctaac gaagtgggag ccaggatact aacatcggaa 540 tcgcaactaa cgataaccaa agagaagaaa gaagaactcc aggattgcaa aatttctcct 600 ttgatggttg catacatgtt ggagagagaa ctggtccgca aaacgagatt cctcccagtg 660 gctggtggaa caagcagtgt gtacattgaa gtgttgcatt tgactcaagg aacatgctgg 720 gaacagatgt atactccagg aggggaagtg aggaatgatg atgttgatca aagcttgatt 780 attgctgcta ggaacatagt gagaagagct gcagtatcag cagatccact agcatcttta 840 ttggagatgt gccacagcac acagattggt ggaattagga tggtagacat ccttaggcag 900 aacccaacag aagagcaagc cgtggatata tgcaaggctg caatgggact gagaattagc 960 tcatccttca gttttggtgg attcacattt aagagaacaa gcggatcatc agtcaagaga 1020 gaggaagagg tgcttacggg caatcttcaa acattgaaga taagagtgca tgagggatat 1080 gaagagttca caatggttgg gagaagagca acagccatac tcagaaaagc aaccaggaga 1140 ttgattcagc tgatagtgag tgggagagac gaacagtcga ttgccgaagc aataattgtg 1200 gccatggtat tttcacaaga ggattgtatg ataaaagcag tcagaggtga tctgaatttc 1260 gtcaataggg cgaatcaacg attgaatcct atgcatcaac ttttaagaca ttttcagaag 1320 gatgcgaaag tgctttttca aaattgggga gttgaaccta tcgacaatgt gatgggaatg 1380 attgggatat tgcccgacat gactccaagc atcgagatgt caatgagagg agtgagaatc 1440 agcaaaatgg gtgtagatga gtactccagc acggagaggg tagtggtgag cattgaccgt 1500 tttttgagaa tccgggacca acgaggaaat gtactactgt ctcccgagga ggtcagtgaa 1560 acacagggaa cagagaaact gacaataact tactcatcgt caatgatgtg ggagattaat 1620 ggtcctgaat cagtgttggt caatacctat caatggatca tcagaaactg ggaaactgtt 1680 aaaattcagt ggtcccagaa ccctacaatg ctatacaata aaatggaatt tgaaccattt 1740 cagtctttag tacctaaggc cattagaggc caatacagtg ggtttgtaag aactctgttc 1800 caacaaatga gggatgtgct tgggacattt gataccgcac agataataaa acttcttccc 1860 ttcgcagccg ctccaccaaa gcaaagtaga atgcagttct cctcatttac tgtgaatgtg 1920 aggggatcag gaatgagaat acttgtaagg ggcaattctc ctgtattcaa ctataacaag 1980 gccacgaaga gactcacagt tctcggaaag gatgctggca ctttaactga agacccagat 2040 gaaggcacag ctggagtgga gtccgctgtt ctgaggggat tcctcattct gggcaaagaa 2100 gacaagagat atgggccagc actaagcatc aatgaactga gcaaccttgc gaaaggagag 2160 aaggctaatg tgctaattgg gcaaggagac gtggtgttgg taatgaaacg gaaacgggac 2220 tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc catcaattag 2280                                                                         2280

Claims (9)

Recombinant influenza virus containing two different subtypes of chimeric hemagglutinin gene (Accession No. KCTC12567BP). The method according to claim 1,
The chimeric hemagglutinin gene comprises HA1 and a fusion peptide gene region derived from A / Korea / CJ01 / 09 (H1N1); A / EM / Korea / W150 / 06 (H5N1) derived recombinant influenza virus. The method according to claim 1,
Wherein the chimeric hemagglutinin gene comprises the polynucleotide of SEQ ID NO: 3. The method according to claim 1,
Wherein said virus is derived from A / Puerto Rico / 08/34 (H1N1) in addition to the hemagglutinin gene. 5. The method of claim 4,
Wherein the seven genes derived from the A / Puerto Rico / 08/34 (H1N1) are composed of the polynucleotides of SEQ ID NOS: 4 to 10. An influenza virus vaccine comprising the virus according to any one of claims 1 to 5 as an active ingredient. The method according to claim 6,
Wherein the vaccine is an influenza virus vaccine characterized in that the virus is an attenuated virulent vaccine, a sadox vaccine or a subunit vaccine (immunogenic fragment). The method according to claim 6,
Wherein said virus forms a cross-immune response against both influenza A viruses with H1N1 serotype, H5N1 serotype and H5N2 serotype. A method for preventing or treating an influenza virus infection disease by administering the vaccine of claim 6 to an individual other than a human.

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