KR101861933B1 - Recombinant avian influenza virus having improved immunogenicity and vaccine composition comprising the same - Google Patents

Abstract

According to one embodiment of the present invention, provided is a recombinant influenza virus comprising H7 hemagglutinin (HA) in which at least two amino acids are glycosylated. The amino acids may be asparagine, and the glycosylation may be N-linked glycosylation (NLG). According to the present invention, immunogenicity and proliferation capability are improved.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to recombinant influenza viruses having improved immunogenicity and vaccine compositions containing the same. BACKGROUND ART < RTI ID = 0.0 >

The present invention relates to a recombinant influenza virus having improved immunogenicity and a vaccine composition comprising the same.

Type A influenza virus (IAV), belonging to Orthomyxoviridae , hosts a variety of animals, including humans. However, interspecies barriers limit the host transmission from existing to new entities.

The difference in the receptor binding specificity of avian and human influenza hemagglutinin (HA) proteins is that hemagglutinin of avian influenza virus binds to α2,3 sialic acid (SA) receptors, whereas human Since influenza virus hemagglutinin binds to the α2,6 SA receptor, avian IAVs are not normally infected by human respiratory cells.

In order to overcome such barriers, avian influenza virus needs to undergo adaptation by continuously infecting new hosts or acquiring specific gene constellations.

In this respect, the human infected avian influenza A (H7N9) virus can be a good example of adaptation and gene mutation acquisition. Before the human infection in 2013, the H7N9 virus underwent numerous adaptations and genetic changes in different animal hosts. The H7N9 avian influenza virus, which has acquired a certain viral fitness, is considered to have a high potential for future pandemics despite the lower case fatality rate (CFR) of the H5N1 highly pathogenic avian influenza virus.

The H7N9 avian influenza virus has a seasonal pattern similar to that of the pandemic influenza virus and has been causing human infections four times to date. However, it is unknown whether the H7N9 virus will cause human infection in the future. Therefore, there is a need for an effective means of protecting people from the potential threats of such viruses.

Among the means for protecting humans from the H7N9 virus, the vaccine may be considered as the best solution. In some studies, clinical trials involving the inclusion of adjuvants in the H7N9 vaccine have been performed. However, the H7N9 vaccine failed to induce HI (Hemagglutination Inhibition) 40 or higher antibody titer, which is the standard of efficacy in influenza vaccine. When the vaccine was administered twice with the immunopotentiating agent, the serum conversion of about 60% seroconvension rate). In other vaccine clinical trials for the H7 subtype, the HI reversal was very low as in the H7N9 vaccine trial.

Therefore, considering the seasonal pattern of H7N9 virus infection in China since 2013, a study that can overcome the low immunogenicity of the H7N9 vaccine before the global spread of H7N9 virus by human liver propagation It must be done.

Previous studies have shown that the introduction of two additional N-linked glycosylation (NLG) in the HA head region of the H1N1 virus can induce broad immunogenicity lost. In addition, two other studies have demonstrated that the immunogenicity of the H5N1 vaccine is affected by NLG.

In connection with the H7 subtype, studies have been reported that the presence of NLG in the amino acid position 141 of hemagglutinin determines the avoidance of the virus from neutralizing antibodies. Considering the importance of functional balance between hemagglutinin and neuraminidase, the variation of the NLG pattern in the hemagglutinin head region has a crucial influence on the infectivity, antigenicity and immunogenicity of the virus, It is possible to change the viral fitness.

However, with respect to the effect of NLG on innate immunity and humoral immunity, NLG introduction in the hemagglutinin head may increase its immunogenicity apart from the infectivity of the virus, It can be a way to develop an effective vaccine candidate.

Accordingly, the present inventors have studied the hemagglutinin NLG pattern variation of the H7 subtype virus to produce an effective H7N9 vaccine. Based on the results of NLG pattern analysis and reverse genetics based on this method, NLG mutant recombinant viruses were prepared and the growth characteristics and immunogenicity of these recombinant viruses were evaluated.

H7N9 vaccine was prepared by the same method as that of seasonal influenza vaccine, and compared with the vaccine produced by NIBSC (National Institutes of Biological Standards and Control) Respectively.

It is an object of the present invention to provide a H7N9 subspecialty recombinant influenza virus having improved immunogenicity and proliferative activity and a vaccine comprising the same.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention, there is provided a recombinant influenza virus comprising two or more amino acids glycosylated H7 hemmaglutinin.

According to one aspect, the amino acid may be an amino acid at positions 141 and 167 of the H7 hemagglutinin.

According to one aspect, the amino acid may be asparagine.

According to one aspect, the glycosylation may be N-linked glycosylation (NLG).

According to one aspect, the H7 hemagglutinin may be derived from avian influenza virus.

According to one aspect, the avian influenza virus may be H7N1, H7N2, H7N3, H7N7 or H7N9 subtype avian influenza virus.

According to one aspect, the recombinant influenza virus is selected from the group consisting of NA, Neuraminidase, NP, Nucleoprotein, Matrix protein, NS, Nonstructural protein, PA One or more proteins selected from the group consisting of Polymerase acidic protein (PBI), Polymerase basic protein 1 (PB1), and Polymerase basic protein 2 (PB2).

According to one aspect, the neuraminidase may be derived from the H7N9 subtype of avian influenza virus.

According to one aspect of the present invention, the nucleoprotein (NP), the matrix protein (M), the nonstructural protein (NS), the polymerase acid protein (PA) And Polymerase basic protein 2 (PB2) may be derived from H1N1 subtype avian influenza virus.

According to another embodiment of the present invention, there is provided a vaccine composition comprising the recombinant influenza virus as an active ingredient.

According to one aspect, the vaccine composition may further comprise an adjuvant.

According to one aspect, the immunostimulant is selected from the group consisting of Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), Squalene, Squalane, Aluminum hydroxide ), Aluminum phosphate, and saponin.

According to one aspect, the vaccine composition may further comprise a pharmaceutically acceptable carrier.

According to one aspect, the vaccine composition is capable of cross-reacting with a heterologous influenza virus.

According to one aspect, the vaccine may be one or more selected from the group consisting of subunit vaccines, synthetic vaccines and genetic engineering vaccines.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating an influenza infectious disease comprising the recombinant influenza virus as an active ingredient.

According to one aspect, the influenza infection disease may be at least one selected from the group consisting of sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea.

According to one embodiment of the present invention, there is provided a composition for diagnosing influenza virus infection comprising the recombinant influenza virus or an antigen thereof.

According to another embodiment of the present invention, there is provided an influenza virus infection diagnostic kit comprising the diagnostic composition.

According to another embodiment of the present invention, there is provided a method of providing information on the diagnosis of influenza virus infection, comprising the step of treating the diagnostic composition with a sample.

According to one aspect, the sample may be one or more selected from the group consisting of cells, blood, urine, saliva, and tissue that are expected or infected with the influenza virus.

The recombinant influenza virus of the present invention can glycosylate specific amino acid residues of H7 hemagglutinin (HA) to improve immunogenicity and proliferation of influenza viruses containing the same.

In addition, by using influenza virus with increased immunogenicity as a vaccine, a good immune response can be induced by a small amount.

Figure 1 shows the production and plaque phenotype of the H7 HA NLG mutant virus. Four mutant H7 viruses (rH7 + 141, rH7 + 167, rH7-249 and rH7 + 141 + 167) were constructed by constructing four H7 HA (A / Anhui / 01/2013) NLG mutations ). (A) shows the NLG addition of HA by Western blot analysis using cell lysate of MDCK cells after viral infection. (B) compares the plaque phenotype of each HA NLG mutant virus in MDCK cells. Phosphate buffered saline (PBS) was used as an infected control (MOCK) and parental 7: 1 virus (rH7) was prepared using wild-type H7 HA.
Figure 2 shows the replication rate of the H7 HA NLG mutant virus in mammalian cells. The growth characteristics of four H7 HA NLG mutant viruses were compared in MDCK cells (A), A549 cells (B) and Vero cells (C). The mean values were measured from three independent experiments. The statistical significance of growth differences between viruses was tested by one-way ANOVA and confirmed by Turkey's multiple comparison test. Statistical significance between each of the different cells was as follows: MDCK cells (P &lt;0.05); A549 cells (P &lt;0.01); And Vero cells (P < 0.001). The replication titer of the rH7 virus was used as a control for other viruses. The error bars represent the standard deviation (SD).
Figure 3 shows the immune response of the guinea pig antifouling to each of the H7 HA NLG mutant viruses. The immune response of guinea pig antiserum (A, α-rH7; B, α-rH7 + 141; C, α-rH7 + 167; and D, α-rH7 + 141 + 167) HI and PRNT analysis. The mean values calculated from three independent experiments were expressed as a fold change based on the corresponding response between each virus and the antiserum (for example, the HI or PRNT results of the corresponding reaction between the rH7 virus and the α-rH7 antiserum). The error bars indicate SD.
Figure 4 shows replication of the virus in the lungs of immunized mice after vaccination. After either (A) or (B) the primed-boosted NIBRG-268 or IYv + 141 + 167 vaccine was administered either primed only or differentially immunized with 0.1% Alum immune enhancer , 8 BALB / c mice were infected with 10 ⁶ PFU of NIBRG-268 virus. At 3 and 5 days after infection, the lungs of each mouse were harvested and the titer of the virus was measured by plaque assay in MDCK cells. A control (non-immunized) mice were infected with viruses that had not been vaccinated with six vaccines. Statistical significance of virus titers difference was assessed by Student t-test (*, P <0.05; **, P <0.01; and ***, P <0.001). The error bars represent the standard deviation (SD).

In the following, embodiments will be described in detail with reference to the accompanying drawings. Like reference symbols in the drawings denote like elements.

Various modifications may be made to the embodiments described below. It is to be understood that the embodiments described below are not intended to limit the embodiments, but include all modifications, equivalents, and alternatives to them.

The terms used in the examples are used only to illustrate specific embodiments and are not intended to limit the embodiments. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises" or "having" and the like refer to the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this embodiment belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

In the following description of the present invention with reference to the accompanying drawings, the same components are denoted by the same reference numerals regardless of the reference numerals, and redundant explanations thereof will be omitted. In the following description of the embodiments, a detailed description of related arts will be omitted if it is determined that the gist of the embodiments may be unnecessarily blurred.

According to one embodiment of the present invention, there is provided a recombinant influenza virus comprising two or more amino acids glycosylated H7 hemmaglutinin.

The subtype of influenza A virus (IAV) is classified according to the antigenicity difference of the surface proteins hemagglutinin (HA) and neuraminidase (NA), and specifically, according to HA and NA They are divided into H subtype and N subtype, respectively.

There are 9 kinds of H type subtypes of influenza A virus from H1 to H16 and N types from N1 to N9, and the recombinant virus is hemagglutinin of H7 subtype, specifically 2 or more amino acids, Gt; H7 hemagglutinin &lt; / RTI &gt;

The term "glycosylation" as used herein refers to a reaction in which a glycosyl group in which a hemiacetal hydroxyl group is removed from a sugar molecule is transferred to a protein. The above-mentioned glycosylation involves the glycosyltransferase specific for each reaction as a catalyst, and the sugar nucleotide is used as the substrate of the feeder. Examples of the receptor include amino acid residues such as serine, threonine, asparagine, and hydroxysine. The complex saccharide can be synthesized by a continuous reaction in which monosaccharide is gradually transferred to the oligosaccharide transferred to such a residue. The complex saccharide may be a constituent material of a living body tissue, particularly, a main component of an extracellular matrix, and plays an important role in cell recognition on other cell surfaces.

In particular, the glycosylated amino acid may be an amino acid at positions 141 and 167 of the H7 hemagglutinin (HA). Specifically, the H7 hemagglutinin may be represented by the amino acid sequence of SEQ ID NO: 1. That is, the amino acid may be asparagine, and the sugar chain thereof may mean N-linked glycosylation.

If NLG addition is not performed at 167 residues of the HA, the immune response can be suppressed because the T-cell epitope of virus (YAEMKWLLSNTDNAAFPQ; located at 155-172 residues of HA) is maintained. The T-cell epitope refers to one of the human-mimetic sequences in the H7N9 genomic segment that inhibits the immune response in the human body.

The NLG addition of 141 residues of H7 hemagglutinin alone may reduce HA binding to SA receptors. On the other hand, the addition of double NLGs of 141 residues and 167 residues can enhance the replication of virus (Fig. 2) and induce a broader antibody response (Fig. 3).

As used herein, the term " N-Linked Glycosylation "(NLG) is one of the protein variants after translation and refers to three sequential amino acid sequences in the protein capable of attaching to polysaccharides, Quot; means that the glycan is conjugated to the asparagine (Asn, N) residue when it is composed of "NXS / T (X is all amino acids except proline). Hereinafter, "NLG addition" means the glycosylation of non-glycosylated amino acid, and "NLG elimination" means non-glycosylation of glycosylated amino acid.

Changes in NLG status around the HA head region of influenza viruses can affect the binding of the virus to sialic acid (SA) receptors and consequently can alter the infectivity of the virus. In addition, NLG changes can often cause immune avoidance, masking the specific antigen site and concealing the virus itself from recognition of the host immune system.

It has been reported that the NLG status change around the HA head region of the H1N1 virus, which was prevalent in 1918 and 2009, specifically the addition of NLG provides a cross-over immune response against the H1N1 virus.

Similarly, studies have shown that when NLG is added to the HA head region of the H1N1 virus that was prevalent in 2009, extensive HI and PRNT responses to various NLG mutations with different amino acid mutations are observed.

In this regard, the present inventors tried to verify the vaccine efficacy by inducing NLG addition and NLG elimination mutations in specific amino acids of H7 hemagglutinin derived from avian influenza virus, which is likely to be infected with human.

In this case, the avian influenza virus may be H7N1, H7N2, H7N3, H7N7 or H7N9 subtype influenza virus having H7 subtype hemagglutinin, preferably H7N9 subtype avian influenza virus, more specifically A / Anhui / 01/2013. The H7 hemagglutinin represented by the amino acid sequence of SEQ ID NO: 1 can be hemagglutinin of A / Anhui / 01/2013.

For the production of the recombinant influenza virus, we used six internal genes of A / Puerto Rico / 8/34 as a backbone, double NLG added HA (HA + 141 + 167) and H7N9 A / Anhui / 01 / 2013 virus, the H7N9 vaccine (IYv + 141 + 167) was prepared.

The six internal genes of A / Puerto Rico / 8/34 are composed of polymerase acidic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and nucleocapsid protein NP, Nucleoprotein), Matrix protein (M, Matrix protein), and Nonstructural protein (NS).

Thus, the recombinant influenza virus may be selected from the group consisting of NA, Neuraminidase, NP, Nucleoprotein, Matrix protein, Nonstructural protein (NS), Polymerase acid protein, PB1 (Polymerase basic protein 1), and PB2 (Polymerase basic protein 2).

At this time, the neuraminidase can be derived from H7N9 subtype avian influenza virus, specifically A / Anhui / 01/2013. Specifically, the neuraminidase can be represented by the amino acid sequence of SEQ ID NO: 2.

In addition, the nucleoprotein (NP), matrix protein (M), nonstructural protein (NS), polymerase subunit A (PA), polymerase subunit B1 subunit B2) may be derived from the H1N1 subtype of avian influenza virus, specifically A / Puerto Rico / 8/34.

Specifically, the matrix protein (M) encoding gene expresses M1 and M2 proteins, and the non-expressed protein (NS) encoding gene can express NS1 and NS2 proteins. Accordingly, NP, M1, M2, NS1, NS2, PA, PB1 and PB2 may be represented by the amino acid sequences of SEQ ID NOS: 3 to 10, respectively.

Various assays for the IYv + 141 + 167 vaccine showed that HI titers could be induced higher than NIBRG-268 vaccine regardless of the addition of immunostimulants (Table 2). In addition, the IYv + 141 + 167 vaccine effectively protects the mouse from infection with the virus (Fig. 4), and can be used for 3 days and 5 days after the priming-boosting of the vaccine containing the immunostimulant At this point in time, the virus can be killed in the lungs of mice (Figure 4B).

The addition of double NLG in the HA head region improves the proliferative activity of H7N9 virus, thereby increasing the yield of vaccine production using the same, and can impart excellent immunogenicity and cross immunogenicity to the virus vaccine. In addition, the recombinant influenza virus can maintain a balance between infectivity and immunogenicity and can be used as a highly efficient attenuated vaccine.

Accordingly, another embodiment of the present invention provides a vaccine composition comprising the recombinant influenza virus as an active ingredient. The vaccine may itself be a Sadox vaccine.

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

In addition, since the vaccine composition contains the recombinant influenza virus as an active ingredient, it can exert excellent immunogenicity on its own, but may further include an adjuvant to further enhance its effect.

The immune enhancer may be selected from the group consisting of complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), Squalene, Squalane, aluminum hydroxide, aluminum phosphate Aluminum phosphate, and saponin. Preferably, aluminum hydroxide and aluminum phosphate are mixed with gel (Alum), but the present invention is not limited thereto.

Further, the vaccine composition may further comprise not only the immunostimulant but also a pharmaceutically acceptable carrier. The carrier is used to formulate the vaccine composition, and can usually include diluents, excipients, stabilizers, preservatives and the like.

The diluent may be a non-aqueous solvent such as propylene glycol, polyethylene glycol, vegetable oil such as olive oil or peanut oil, or water (preferably 0.05 M phosphate buffer) containing saline (preferably 0.8% saline) , And the like, but the present invention is not limited thereto.

Examples of the excipient include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skimmed milk, glycerol, , Ethanol, and the like, but are not limited thereto.

Examples of the stabilizer include proteins such as carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate and glucose and animal, vegetable or microbial proteins such as milk powder, serum albumin and casein. no.

Examples of the preservative include, but are not limited to, thimerosal, mertiolate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, polyamicin B and the like.

On the other hand, the vaccine composition can cross-react with recombinant influenza virus and xenogeneic influenza virus contained therein. For example, if the vaccine composition comprises an A / Anhui / 1/2013 virus-based recombinant influenza virus subtype of H7N9, the inoculation of the vaccine composition can be achieved with other H7N9 subtypes of A / Shanghai / 2/2013 virus and heterologous influenza virus &Lt; / RTI &gt;

The vaccine may be at least one selected from the group consisting of subunit vaccines, synthetic vaccines and genetic engineering vaccines.

The subunit vaccine is a vaccine prepared by extracting only an antigen component that can cause an immune function among the constituents of virus, and can minimize side effects by inducing immune formation only at an antigenic site necessary for viral defense. For example, HA protein and / or NA protein of avian influenza virus can be extracted and used.

The synthetic vaccine may be prepared by chemically synthesizing only an antigen or an antigenic determinant of a virus or a vaccine containing a peptide produced by recombinant DNA technology, for example, by using HA protein and / or NA protein of avian influenza virus .

The genetic engineering vaccine may be modified or deleted of a specific gene causing virulence of the virus.

The vaccine may also be used as a mixed or complex vaccine, which can be mixed with inactivated strains or antigens to prevent other diseases, including avian influenza, together. The combined vaccine means a vaccine in which different virus vaccines are used together, and the combined vaccine means a vaccine in which a virus vaccine and a bacterial vaccine are used together.

The vaccine may be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, ocular or intradermal routes.

Parenteral administration refers to a mode of administration which includes intravenous, intramuscular, intraperitoneal, intrasternal, percutaneous and intramuscular injection and infusion. The parenteral administration of the vaccine is preferably carried out under the desired purity in the form of a pharmaceutically acceptable carrier, that is, a unit dose formulation which is non-toxic to the receptor at the concentration and dose used and which can be miscible with other pharmaceutical ingredients Lt; / RTI &gt;

The formulation of the vaccine is applicable to any formulation, and the prepared formulation can be used for oral, parenteral, and application. The formulations of the vaccine may be, but are not limited to, tablets, capsules, soft capsules, liquids, granules, pills or injectable forms (solutions, suspensions or emulsions).

Based on the above-mentioned pharmacological effects, another embodiment of the present invention provides a pharmaceutical composition comprising the recombinant influenza virus as an active ingredient, for the prophylaxis or treatment of influenza infectious diseases. The above-mentioned recombinant influenza viruses and other components that can be additionally contained are as described above.

The influenza infectious disease is a disease caused by influenza infection, and may be one or more selected from the group consisting of sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea.

The term "prophylactic, " as used herein, refers to any act that inhibits or delay the onset of an influenza virus infection by administration of the composition. As used herein, the term "treatment" refers to any action that improves or alleviates symptoms due to influenza virus infection by administration of the composition.

The pharmaceutical composition may be administered in a pharmaceutically effective amount. As used herein, 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 vary depending on the species and severity, age, sex, The type of virus, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. 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 side effects, which can be easily determined by a person skilled in the art.

Also, an embodiment of the present invention provides a composition for diagnosing influenza virus infection comprising the recombinant influenza virus or an antigen thereof, or an influenza virus diagnostic kit.

Further, another embodiment of the present invention provides a method for providing information on the diagnosis of influenza virus infection, comprising the step of treating the diagnostic composition with a sample. At this time, the sample may be at least one selected from the group consisting of infected cells, blood, urine, saliva and tissues, which are expected to be infected with influenza virus, but are not limited thereto.

The influenza virus or antigen thereof may be used not only for the elimination of influenza viruses in infected or infected cells through an antigen-antibody complex reaction, but also for specifically detecting influenza viruses.

The diagnostic composition and the diagnostic kit may include not only the influenza virus but also tools, reagents and the like commonly used in the art used for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling agents capable of generating detectable signals, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine But are not limited to, resins, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

Antigen-antibody complex formation can be induced by immunofluorescent staining, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, (Complement Fixation Assay), FACS, and a protein chip. However, the present invention is not limited thereto.

Labels that enable qualitative or quantitative measurement of the formation of antigen-antibody complexes include, but are not limited to, enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes. Enzymes that can be used as detection labels include, but are not limited to,? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, A prodrug thereof, a prodrug thereof, a prodrug, a hexose kinase, a GDPase, an RNase, a glucose oxidase and a luciferase, a phosphofructokutase, a phosphoenolpyruvate carboxylase, an aspartate aminotransferase, a phosphoenolpyruvate decarboxylase, But are not limited thereto. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine and the like. The ligand includes, but is not limited to, a biotin derivative. Emitters include, but are not limited to, acridinium ester, luciferin, luciferase, and the like.

The fine particles include, but are not limited to, colloidal gold and colored latex. The redox molecule includes, but is not limited to, ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone and the like. The radioactive isotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁶ Cl, ⁵¹ Cr, ⁵⁶ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re.

Hereinafter, the experimental materials and methods used in the embodiments of the present invention will be described in detail.

Virus and cell preparation

The A / Korea / 01/2009 (H1N1) virus provided by the CDC was used. After ensuring pure isolates through plaque assay in MDCK cells, each virus was propagated in a fertile chicken egg for 45-72 hours. MDCK cells, 293T cells (human embryonic kidney), A549 cells (human lung epithelial), and Vero cells (African green monkey kidney) were obtained from the American Type Culture Collection (ATCC). MDCK cells were maintained in EMEM medium containing 10% FBS and antibiotics and 293T, A549 and Vero cells in DEME medium containing 10% FBS and antibiotics at 37 ° C and 5% CO ₂ , respectively.

HA of the H7 subtype virus NLG  Pattern analysis

The entire 676 HA nucleotide sequence of the H7 subtype virus (H7N1 (n = 82); H7N2 (n = 142); H7N3 (n = 207); H7N7 (n = 145); and H7N9 NLG pattern analysis was performed (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). Subsequently, the entire HA sequence was converted into a set of amino acid sequences using AliView (v1.17.1), and the N-GlycoSite website (http://www.hiv.lanl.gov/content/sequence/GLYCOSITE/glycosite.html) Respectively.

Plasmid based Reverse genetics

The HA gene and NA gene of A / Anhui / 01/2013 were synthesized (Cosmogenetech, Seoul, Republic of Korea) and NLG transformation was performed. As described above, four HA NLG mutant plasmids of A / Anhui / 01/2013 were cloned into an ambisense pDZ plasmid to construct a plasmid for reverse genetics, and this plasmid was used as an A / Korea / 01/2009 backbone. Were used to produce 7: 1 recombinant virus. Eight plasmids were transfected into 293T / MDCK cells for the construction of specific HA NLG mutant viruses. After 48-72 hours, a hemagglutination assay was performed on the supernatant using 0.5% (v / v) tRBC (turkey RBC), and the hemagglutination assay was performed using a plaque assay Positive samples were inoculated into MDCK cells for isolation. Then, the purified virus was inoculated on fertilized eggs of 10 days of age to proliferate. The gene sequences of the rescued recombinant viruses were confirmed by reverse transcription PCR.

Western Blat  analysis

In order to confirm the expression of NLG introduced into HA of each recombinant virus, MDCK cells were inoculated with recombinant virus (MOI, Multiplicity of Infection) of 3 recombinant viruses. After 16 hours, RIPA buffer (Sigma, St. Louis, MO) was treated for SDS-PAGE to obtain cell lysate. HA in cell lysates was first detected by guinea pig polyclonal antibody against rH7 virus and secondary detected by anti-guinea pig IgG-HRP antibody (KPL, Gaithersburg, MA). As a control, β-actin was detected using a monoclonal antibody produced in mouse.

Intracellular NLG  Analysis of growth characteristics of mutant viruses

To analyze the growth characteristics of the recombinant virus, MDCK, A549 and Vero cells were inoculated with 0.001 MOI of virus. After 1 hour, each cell was washed and then cultured in a medium containing 0.3% BSA and trypsin (1 μg / ml) treated with TPCK (tosyl phenylalanyl chloromethyl ketone). Then, the supernatant was collected at specific time intervals and the virus titer was measured by the plaque assay in the MDCK cells.

Hemagglutination inhibition test (HI, Hemagglutination  Inhibition Analysis

At weekly intervals, 10 ⁵ PFU (Plaque Forming Unit) virus was infected into the nasal cavity with guinea pigs. Whole blood was collected from guinea pigs and serum was obtained. The serum was treated with neuraminidase (NA), a receptor destroying enzyme, Vibrio cholerae , to remove non-specific factors. HI analysis was performed by reacting 4 HAU (Hemagglutination Unit) diluted virus with 2-fold diluted serum in 96-well plate for 1 hour at 37 ° C, adding 50 μl of 0.5% tRBC to each well, And the HI titer was measured after the lapse of time. As a control, PBS was used.

Plaque reduction Neutralization test ( PRNT , A plaque-reduction neutralization test)

For the determination of neutralizing antibody titers, 100 PFU of virus was reacted with guinea pig antiserum twice diluted at 37 ° C. After 1 hour, plaque assay was performed by inoculating each virus-serum reaction into MDCK cells. Thereafter, the PRNT titer was determined based on the maximum dilution factor that showed a virus plaque reduction of 50% or more as compared with the PBS control group.

Vaccine preparation

Using the HA of the A / Anhui / 01/2013 and the native NA genes of the NLGs, the six internal gene backbones of the A / Puerto Rico / 8/34 (H1N1) vaccine donor The H7N9 vaccine candidate host virus was developed. After the virus was propagated in fertilized eggs, allantoic fluid was collected and differential centrifugation of a sucrose density gradient was performed. Then, the vaccine candidate main virus was concentrated and purified using Triton ^占 X-100 (AMRESCO, Cleveland, OH) and deactivated with formaldehyde. Then, the HA content of the vaccine was measured using the SRID method. The NIBRG-268 vaccine virus was also treated according to the same procedure as above.

Animal experiment

The vaccine (15 μg) was intramuscularly immunized with female BALB / c mice at 5 weeks of age at intervals of 2 weeks, with or without the addition of 0.1% Alum immune enhancer. Then, mouse serum was collected and treated with RDE to perform HI and PRNT assays. For the challenge, 8 mice immunized per group were intranasally infected with 10 ⁶ PFU of the NIBRG-268 virus and the mice were harvested at 3 and 5 days post-infection . Mock-vaccinated mice were treated with PBS.

Statistical analysis

Different replication characteristics of intracellular viruses were verified via one-way ANOVA and confirmed through the Tukey's multiple comparison test. The statistical significance of differences in viral titers to the lungs of mice was assessed using Student t test.

Example  1: H7 subtype of HA virus NLG  Pattern and NLG  Mutant virus production

HA NLG patterns of H7 subtype viruses (H7N1, H7N2, H7N3, H7N7, and H7N9) were examined using HA gene sequences downloaded from the National Biotechnology Information Center (NCBI, Bethesda, MD) . Of the total 676 HA sequences, eight potential glycosides corresponding to the amino acid residues at positions 30, 46, 141, 167, 249, 336, 439, and 511 of HA protein (HA numbering reference to the H7N9 virus) (Table 1).

Only HA of the H7N7 subtype virus used all of the above 8 glycosylation sites. Of the four amino acid residues (141, 167, 249 and 336) located around the HA globular head region, the H7N1, H7N2, H7N3 and H7N9 subtypes do not contain NLG at the 336 residue whereas the N7N9 subtype Did not contain NLG in both 167 residues and 336 residues.

In addition, less than 4% of the H7N9 viruses use NLG of 141 residues of the HA protein (Table 1). In order to analyze the effect of NLG on the replication and immunogenicity of the H7 subtype virus, NLG mutations around the HA head region were analyzed, and genetic analysis of A / Korea / 01/2009 (H1N1) using plasmid-based reverse genetics Four mutant viruses were constructed using HA from A / Anhui / 01/2013 (H7N9), which mutated NLG based on the backbone: residues 141 (rH7 + 141) or 167 (rH7 + 167) Two mutations in single NLG-added; Double NLG-added mutants at residues 141 and 167 (rH7 + 141 + 167); And a single NLG-deleted mutation at residue 249 (rH7-249).

In addition, single NLG-added mutations at residue 336 were not rescued from three independent tests and were not included in this study. Wild type HA of A / Anhui / 01/2013 was used to generate parental HA recombinant virus (rH7) as a control.

Table 1 below shows the frequency of HA NLG in selected H7 subtypes. In Table 1, the HA residue number is indicated according to the H7N9 HA numbering standard.

Subtype % NLG at HA residue (the number of sequences) # of sequences 30 46 141 167 249 336 439 511 H7N1 98.78 (81) 100 (82) 25.61 (21) 24.39 (20) 100 (82) - 100 (82) 98.78 (81) 82 H7N2 100 (142) 100 (142) 4.93 (7) 5.63 (8) 99.3 (141) - 100 (142) 100 (142) 142 H7N3 100 (207) 97.1 (201) 8.21 (17) 5.8 (12) 100 (207) - -99.52 (206) 100 (207) 207 H7N7 96.55 (140) 100 (145) 7.59 (11) 2.07 (3) 100 (145) 10.34 (15) 100 (145) 100 (145) 145 H7N9 100 (51) 100 (51) 3.92 (2) - 100 (51) - 100 (51) 100 (51) 51 Total 98.96 (669) 99.11 (670) 9.32 (63) 7.1 (48) 99.85 (675) 2.22 (15) 99.85 (675) 99.85 (675) 676

Example  2: H7 HA in mammalian cells NLG  Analysis of plaque phenotype and replication characteristics of mutant viruses

In order to confirm the change of NLG state in the HA protein of the rescued mutant virus according to Example 1, MDCK (Madin-Darby canine kidney) was infected with each virus and the cell lysate Western blotting was performed. In the Western blot analysis, single NLG-added mutant viruses (rH7 + 141 and rH7 + 167) showed lower mobility than the parental rH7 virus, meaning that they were NLG-added to each particular residue.

Also, in the same analysis as above, it was confirmed that the mobility of the double NLG-added mutant (rH7 + 141 + 167) HA was lowest (Fig. 1A). However, HA of rH7-249 was not observed in the same Western blot analysis.

Next, the plaque phenotype of the NLG mutant virus was analyzed in MDCK cells. Compared to the parental rH7 virus, the rH7 + 141, rH7 + 167, and rH7 + 141 + 167 viruses generated plaques of similar or somewhat increased size, whereas the plaques of rH7-249 significantly decreased in size (Fig. 1B). In relation, increased plaque sizes of the NLG-added viruses (rH7 + 141, rH7 + 167, and rH7 + 141 + 167) were found to be associated with increased replication in MDCK, A549, and Vero cells 2).

Of the three viruses, the rH7 + 167 and rH7 + 141 + 167 viruses showed an improved replication rate in all cells compared to the rH7 + 141 virus (Figs. 2A to C) Showed the highest replication rate in the cells as compared to the wild type virus (Fig. 2A).

However, the NLG-depleted rH7-249 virus did not show an improved replication rate in the cells (Figs. 2A-C), which was analyzed to be similar to the reduction of the virus plaque size (Fig. 1B). However, the NLG mutant viruses did not show any lethal pathogenicity in mice.

Taken together, all these results indicate that the three HA NLG-added mutant viruses have high potential as a high yield vaccine candidate host virus compared to the NLG-deleted mutant virus.

Example  3: H7 HA NLG  Measurement of immunogenicity of mutant viruses

In order to analyze the potential of the mutant viruses prepared according to Example 1 as vaccine candidate host viruses, the immunogenic properties of each NLG mutant virus were measured. However, the rH-249 virus did not show sufficient hemagglutination reaction titer for HI analysis and was excluded from this serological analysis.

Each virus was infected with guinea pigs through the nasal passages, and serum was obtained and HI analysis was conducted using the virus. As a result, rH7 and rH7 + 167 sera showed relatively low immunogenicity against NLG added virus at 141 residues (Fig. 3A and C). However, serum of rH7 + 141 showed excellent immunogenicity against all three NLG-added viruses (Fig. 3B).

The most extensive HI reactivity was identified in the double NLG-added rH7 + 141 + 167 virus. The HI level of the rH7 + 141 + 167 serum was 1.5 times higher than all the viruses used in the experiment, and 4 times higher than that of the MoH7 virus (FIG. 3D). As in the HI analysis, the PRNT titers of each serum were analyzed for all viruses (Figures 3A-D).

As a result of the analysis, the rH7 + 141 + 167 virus showed the broadest immunogenicity as in the HI analysis, which means that it can be a good candidate for the vaccine virus against the H7 subtype virus.

Example  4: duplex NLG - containing HA added H7N9  Cross-immunogenicity of vaccine and Defense  analysis

To confirm the cross-immunogenicity of dual NLG-added HA as an effective H7N9 vaccine in experimental animals, a double NLG-added HA (IYv + 141 + 167) with PR8 (A / Puerto Rico / 8/34) A H7N9 vaccine candidate strain containing the NA gene of A / Anhui / 01/2013 was prepared.

The vaccine was formulated in the same manner as in the preparation of influenza vaccine for influenza. Muscle inoculation was performed with different 0.1% Alum immunostimulant and number of inoculations. A H7N9 vaccine (NIBRG-268) containing the HA and NA genes from A / Anhui / 01/2013 was used as a control.

Table 2 below shows the HI titer of the antiserum obtained from immunized mice by performing only the first inoculation or up to the second inoculation.

Virus Single immunization [GMT (95% CI)] NIBRG-268 IYv + 141 + 167 Only 15ug HA 0.1% Alum + 15μg HA Only 15ug HA 0.1% Alum + 15μg HA Anhui 0 0 24.38 (17.83 - 33.33) 36.23 (20.35 to 64.5) Shanghai 0 0 13.46 (9.55 to 18.96) 20 (13.81-28.96) rH7 + 141 + 167 0 0 24.28 (16.46 ~ 32.3) 35.64 (20.61 to 61.62) Virus Double immunization [GMT (95% CI)] NIBRG-268 IYv + 141 + 167 Only 15ug HA 0.1% Alum + 15μg HA Only 15ug HA 0.1% Alum + 15μg HA Anhui 29.72 (17.95-49.22) 56.57 (37.98-84.26) 97.52 (53-179.3) 262.5 (161.7 to 426.2) Shanghai 22.08 (12.4-39.31) 50.4 (27.82 to 91.28) 97.52 (53-179.4) 262.5 (161.7 to 426.2) rH7 + 141 + 167 12.6 (4.66 ~ 34.05) 25.2 (6.324 to 68.1) 63.5 (26.31 to 153.2) 211.1 (131.8 ~ 338.2)

As shown in Table 2 above, HI analysis showed that NIBRG-268 did not induce HI-reactive antibodies in the mice in which only the first inoculation was performed and the mice supplemented with the immunostimulants. On the other hand, low immunogenicity of NIBRG-268 was observed in mice administered to the second inoculation.

However, the IYv + 141 + 167 vaccine showed HI titer for each mouse with different conditions. Single immunization showed HI titers of 20-36.23 and 13.46-24.38, respectively, for the three H7N9 viruses with or without immunostimulants (Table 2). In the case of double immunization, immunized mice showed HI titer (63.5-97.52) increased by 2-fold or 3-fold compared to single immunized mice without immunopotentiating agent , And increased HI titer (211.1-262.5) when supplemented with an immunostimulant.

In particular, NIBRG-268, which does not contain an immunostimulant, in the mice up to the second inoculation shows a HI titer of less than 40, indicating that it can not form an antibody suitable for the criteria required as a vaccine. On the other hand, the IYv + 141 + 167 vaccine showed an HI titer of 40 or more without an immunostimulant, indicating excellent antibody-forming ability. These results indicate that the IYv + 141 + 167 vaccine exhibits better immunogenicity than NIBRG-268.

In addition, referring to the experimental results on the A / Shanghai / 2/2013 virus shown in Table 2, it was confirmed that the second vaccination of IYv + 141 + 167 vaccine showed HI titer equivalent to that of A / Anhui / 01/2013 virus This confirms that the IYv + 141 + 167 vaccine possesses cross-immunogenicity that may also have vaccine efficacy against other influenza viruses.

On the other hand, it was confirmed that the increase in HI titer in the mouse immunized with the IYv + 141 + 167 vaccine was consistent with the decrease in the virus titer confirmed in the lung of an attacked mouse (FIG. 4). According to the above HI results, the mice immunized with the IYv + 141 + 167 vaccine showed low virus titers in the lungs and the protective ability of IYv + 141 + 167 was higher than that of the first vaccination mice The results were better than those of the mice.

In the case of administration together with the immunostimulant, viruses were not detected in the lungs after 3 to 5 days of infection in the mice that had been subjected to the second inoculation (FIG. 4B). However, mice immunized with NIBRG-268 did not show effective protection (Figs. 4A and B).

These results suggest that HA NLG variants as described above may have an excellent prophylactic effect in connection with the development of an effective H7N9 vaccine.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. For example, if the techniques described are performed in a different order than the described methods, and / or if the described components are combined or combined in other ways than the described methods, or are replaced or substituted by other components or equivalents Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

<110> KOREA UNIVERSITY <120> RECOMBINANT AVIAN INFLUENZA VIRUS HAVING IMPROVED IMMUNOGENICITY          AND VACCINE COMPOSITION COMPRISING THE SAME <130> APC-2016-0326 <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 560 <212> PRT <213> influenza virus <400> 1 Met Asn Thr Gln Ile Leu Val Phe Ala Leu Ile Ala Ile Ile Pro Thr   1 5 10 15 Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val Ser Asn Gly Thr              20 25 30 Lys Val Asn Thr Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr          35 40 45 Glu Thr Val Glu Arg Thr Asn Ile Pro Arg Ile Cys Ser Lys Gly Lys      50 55 60 Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly  65 70 75 80 Pro Pro Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile                  85 90 95 Glu Arg Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn             100 105 110 Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys         115 120 125 Glu Ala Met Gly Phe Thr Tyr Ser Gly Ile Arg Thr Asn Gly Thr Thr     130 135 140 Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp 145 150 155 160 Leu Leu Ser Asn Thr Asp Asn Ala Ser Phe Pro Gln Met Thr Lys Ser                 165 170 175 Tyr Lys Asn Thr Arg Lys Ser Pro Ala Leu Ile Val Trp Gly Ile His             180 185 190 His Ser Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn         195 200 205 Lys Leu Val Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro     210 215 220 Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Leu Ser Gly Arg Ile Asp 225 230 235 240 Phe His Trp Leu Met Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe                 245 250 255 Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys             260 265 270 Ser Met Gly Ile Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu Gly         275 280 285 Asp Cys Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln     290 295 300 Asn Ile Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Lys Gln 305 310 315 320 Arg Ser Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Ile Pro                 325 330 335 Lys Gly Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly             340 345 350 Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala         355 360 365 Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile     370 375 380 Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Gln 385 390 395 400 Gln Phe Glu Leu Ile Asp Asn Glu Phe Asn Glu Val Glu Lys Gln Ile                 405 410 415 Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile Thr Glu Val Trp Ser             420 425 430 Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp         435 440 445 Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg Val Lys Arg Gln     450 455 460 Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe 465 470 475 480 His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr                 485 490 495 Asp His Ser Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile             500 505 510 Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val Ile Leu Trp Phe         515 520 525 Ser Phe Gly Ala Ser Cys Phe Ile Leu Leu Ala Ile Val Met Gly Leu     530 535 540 Val Phe Ile Cys Val Lys Asn Gly Asn Met Arg Cys Thr Ile Cys Ile 545 550 555 560 <210> 2 <211> 465 <212> PRT <213> influenza virus <400> 2 Met Asn Pro Asn Gln Lys Ile Leu Cys Thr Ser Ala Thr Ala Ile Thr   1 5 10 15 Ile Gly Ala Ile Ale Val Leu Ile Gly Ile Ale Asn              20 25 30 Ile Gly Leu His Leu Lys Pro Gly Cys Asn Cys Ser His Ser Gln Pro          35 40 45 Glu Thr Asn Thr Ser Gln Thr Ile Ile Asn Asn Tyr Tyr Asn Glu      50 55 60 Thr Asn Ile Thr Asn Ile Gln Met Glu Glu Arg Thr Ser Arg Asn Phe  65 70 75 80 Asn Asn Leu Thr Lys Gly Leu Cys Thr Ile Asn Ser Trp His Ile Tyr                  85 90 95 Gly Lys Asp Asn Ala Val Arg Ile Gly Glu Ser Ser Asp Val Leu Val             100 105 110 Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Glu Cys Arg Phe Tyr         115 120 125 Ala Leu Ser Gln Gly Thr Thr Ile Arg Gly Lys His Ser Asn Gly Thr     130 135 140 Ile His Asp Arg Ser Gln Tyr Arg Ala Leu Ile Ser Trp Pro Leu Ser 145 150 155 160 Ser Pro Pro Thr Val Tyr Asn Ser Arg Val Glu Cys Ile Gly Trp Ser                 165 170 175 Ser Thr Ser Cys His Asp Gly Lys Ser Arg Met Ser Ile Cys Ile Ser             180 185 190 Gly Pro Asn Asn Asn Ala Ser Ala Val Val Trp Tyr Asn Arg Arg Pro         195 200 205 Val Ala Glu Ile Asn Thr Trp Ala Arg Asn Ile Leu Arg Thr Gln Glu     210 215 220 Ser Glu Cys Val Cys His Asn Gly Val Cys Pro Val Val Phe Thr Asp 225 230 235 240 Gly Ser Ala Thr Gly Pro Ala Asp Thr Arg Ile Tyr Tyr Phe Lys Glu                 245 250 255 Gly Lys Ile Leu Lys Trp Glu Ser Leu Thr Gly Thr Ala Lys His Ile             260 265 270 Glu Glu Cys Ser Cys Tyr Gly Glu Arg Thr Gly Ile Thr Cys Thr Cys         275 280 285 Arg Asp Asn Trp Gln Gly Ser Asn Arg Pro Val Ile Gln Ile Asp Pro     290 295 300 Val Ala Met Thr His Thr Ser Gln Tyr Ile Cys Ser Pro Val Leu Thr 305 310 315 320 Asp Asn Pro Arg Pro Asn Asp Pro Asn Ile Gly Lys Cys Asn Asp Pro                 325 330 335 Tyr Pro Gly Asn Asn Asn Asn Gly Val Lys Gly Phe Ser Tyr Leu Asp             340 345 350 Gly Ala Asn Thr Trp Leu Gly Arg Thr Ile Ser Thr Ala Ser Arg Ser         355 360 365 Gly Tyr Glu Met Leu Lys Val Pro Asn Ala Leu Thr Asp Asp Arg Ser     370 375 380 Lys Pro Ile Gln Gly Gln Thr Ile Val Leu Asn Ala Asp Trp Ser Gly 385 390 395 400 Tyr Ser Gly Ser Phe Met Asp Tyr Trp Ala Glu Gly Asp Cys Tyr Arg                 405 410 415 Ala Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Asp Lys             420 425 430 Val Trp Trp Thr Ser Asn Ser Ser Val Ser Met Cys Ser Ser Thr Glu         435 440 445 Phe Leu Gly Gln Trp Asn Trp Pro Asp Gly Ala Lys Ile Glu Tyr Phe     450 455 460 Leu 465 <210> 3 <211> 498 <212> PRT <213> influenza virus <400> 3 Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp   1 5 10 15 Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met              20 25 30 Ile Gly Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys          35 40 45 Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu      50 55 60 Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu  65 70 75 80 Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile                  85 90 95 Tyr Arg Arg Val Asn Gly Lys Trp Met Arg Glu Leu Ile Leu Tyr Asp             100 105 110 Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Asp Asp         115 120 125 Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn     130 135 140 Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160 Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser                 165 170 175 Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu             180 185 190 Leu Val Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg         195 200 205 Gly Glu Asn Gly Arg Lys Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn     210 215 220 Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Lys Ala Met Met Asp 225 230 235 240 Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Phe Glu Asp Leu                 245 250 255 Thr Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His             260 265 270 Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Pro Ala Val Ala Ser Gly         275 280 285 Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe     290 295 300 Arg Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu 305 310 315 320 Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala                 325 330 335 Ala Phe Glu Asp Leu Arg Val Leu Ser Phe Ile Lys Gly Thr Lys Val             340 345 350 Val Pro Arg Gly Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn         355 360 365 Glu Asn Met Glu Thr Met Glu Ser Ser Thr Leu Glu Leu Arg Ser Arg     370 375 380 Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg 385 390 395 400 Ala Ser Ala Gly Gln Ile Ser Ile Gln Pro Thr Phe Ser Val Gln Arg                 405 410 415 Asn Leu Pro Phe Asp Arg Thr Thr Val Met Ala Ala Phe Thr Gly Asn             420 425 430 Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met         435 440 445 Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe     450 455 460 Glu Leu Ser Asp Glu Lys Ala Ala Ser Pro Ile Val Pro Ser Phe Asp 465 470 475 480 Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr                 485 490 495 Asp Asn         <210> 4 <211> 252 <212> PRT <213> influenza virus <400> 4 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro   1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe              20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys Thr          35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe      50 55 60 Thr Leu Thr Val Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val  65 70 75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala                  85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala             100 105 110 Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met         115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe     130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu                 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met             180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln         195 200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Ser Ser     210 215 220 Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys                 245 250 <210> 5 <211> 97 <212> PRT <213> influenza virus <400> 5 Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly   1 5 10 15 Cys Arg Cys Asn Gly Ser Ser Asp Pro His Ala Ala Ala Asn Ile              20 25 30 Ile Gly Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu Phe Phe          35 40 45 Lys Cys Ile Tyr Arg Arg Phe Lys Tyr Gly Leu Lys Gly Gly Pro Ser      50 55 60 Thr Glu Gly Val Pro Lys Ser Met Arg Glu Glu Tyr Arg Lys Glu Gln  65 70 75 80 Gln Ser Ala Val Asp Ala Asp Asp Gly His Phe Val Ser Ile Glu Leu                  85 90 95 Glu     <210> 6 <211> 230 <212> PRT <213> influenza virus <400> 6 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp   1 5 10 15 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe              20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser          35 40 45 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile      50 55 60 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr  65 70 75 80 Met Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Glu                  85 90 95 Glu Met Ser Arg Glu Trp Ser Met Leu Ile Pro Lys Gln Lys Val Ala             100 105 110 Gly Pro Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile         115 120 125 Ile Leu Lys Ala Asn Phe Ser Ile Phe Asp Arg Leu Glu Thr Leu     130 135 140 Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Ile Val Gly Glu Ile 145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Ala Glu Asp Val Lys Asn                 165 170 175 Ala Val Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Asp Asn Thr Val             180 185 190 Arg Val Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser Asn Glu         195 200 205 Asn Gly Arg Pro Pro Leu Thr Pro Lys Gln Lys Arg Glu Met Ala Gly     210 215 220 Thr Ile Arg Ser Glu Val 225 230 <210> 7 <211> 121 <212> PRT <213> influenza virus <400> 7 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Asp Ile Leu Leu Arg Met   1 5 10 15 Ser Lys Met Gln Leu Glu Ser Ser Ser Glu Asp Leu Asn Gly Met Ile              20 25 30 Thr Gln Phe Glu Ser Leu Lys Leu Tyr Arg Asp Ser Leu Gly Glu Ala          35 40 45 Val Met Arg Met Gly Asp Leu His Ser Leu Gln Asn Arg Asn Glu Lys      50 55 60 Trp Arg Glu Gln Leu Gly Gln Lys Phe Glu Glu Ile Arg Trp Leu Ile  65 70 75 80 Glu Glu Val Arg His Lys Leu Lys Val Thr Glu Asn Ser Phe Glu Gln                  85 90 95 Ile Thr Phe Met Gln Ala Leu His Leu Leu Leu Glu Val Glu Gln Glu             100 105 110 Ile Arg Thr Phe Ser Phe Gln Leu Ile         115 120 <210> 8 <211> 716 <212> PRT <213> influenza virus <400> 8 Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu   1 5 10 15 Ala Glu Lys Thr Met Lys Glu Tyr Gly Glu Asp Leu Lys Ile Glu Thr              20 25 30 Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr          35 40 45 Ser Asp Phe His Phe Ile Asn Glu Gln Gly Glu Ser Ile Ile Val Glu      50 55 60 Leu Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu  65 70 75 80 Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn                  85 90 95 Thr Thr Gly Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr             100 105 110 Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg Glu Val His         115 120 125 Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His     130 135 140 Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Arg Ala Asp 145 150 155 160 Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe                 165 170 175 Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg             180 185 190 Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr         195 200 205 Gly Thr Met Arg Lys Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser     210 215 220 Ser Leu Glu Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly 225 230 235 240 Tyr Ile Glu Gly Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg                 245 250 255 Ile Glu Pro Phe Leu Lys Thr Thr Pro Arg Pro Leu Arg Leu Pro Asn             260 265 270 Gly Pro Pro Cys Ser Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu         275 280 285 Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu     290 295 300 Tyr Asp Ala Ile Lys Cys Met Arg Thr Phe Phe Gly Trp Lys Glu Pro 305 310 315 320 Asn Val Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Leu                 325 330 335 Ser Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Ile Glu Asn Glu Glu             340 345 350 Lys Ile Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp         355 360 365 Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Asp Asp Cys     370 375 380 Lys Asp Val Gly Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Leu 385 390 395 400 Arg Ser Leu Ala Ser Trp Ile Gln Asn Glu Phe Asn Lys Ala Cys Glu                 405 410 415 Leu Thr Asp Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val             420 425 430 Ala Pro Ile Glu His Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ser         435 440 445 Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr     450 455 460 Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe 465 470 475 480 Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg                 485 490 495 Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg             500 505 510 Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr         515 520 525 Asp Pro Arg Leu Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu     530 535 540 Ile Gly Asp Met Leu Leu Arg Ser Ala Ile Gly Gln Val Ser Arg Pro 545 550 555 560 Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys                 565 570 575 Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile             580 585 590 Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr         595 600 605 Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser     610 615 620 Pro Lys Gly Val Glu Glu Ser Ser Ile Gly Lys Val Cys Arg Thr Leu 625 630 635 640 Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu                 645 650 655 Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu             660 665 670 Arg Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu         675 680 685 Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala     690 695 700 Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Ser 705 710 715 <210> 9 <211> 757 <212> PRT <213> influenza virus <400> 9 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn   1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His              20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln          35 40 45 Tyr Ser Glu Lys Gly Arg Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro      50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser  65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu                  85 90 95 Glu Ser His Gly Ile Phe Glu Asn Ser Cys Ile Glu Thr Met Glu             100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr         115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala     130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser 145 150 155 160 Gly Arg Leu Ile Asp Phe Leu Lys Asp Val Met Glu Ser Met Lys Lys                 165 170 175 Glu Glu Met Gly Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg             180 185 190 Asp Asn Met Thr Lys Lys Met Ile Thr Gln Arg Thr Ile Gly Lys Arg         195 200 205 Lys Gln Arg Leu Asn Lys Arg Gly Tyr Leu Ile Arg Ala Leu Thr Leu     210 215 220 Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu                 245 250 255 Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro             260 265 270 Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys         275 280 285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly     290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala 305 310 315 320 Met Ile Thr Tyr Met Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val                 325 330 335 Leu Ser Ile Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly             340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile         355 360 365 Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser     370 375 380 Thr Arg Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Glu Gly Thr 385 390 395 400 Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser                 405 410 415 Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Arg Tyr Thr             420 425 430 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala         435 440 445 Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp     450 455 460 Arg Phe Tyr Arg Thr Cys Lys Leu Leu Gly Ile Asn Met Ser Lys Lys 465 470 475 480 Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe                 485 490 495 Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe             500 505 510 Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr         515 520 525 Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala     530 535 540 Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560 Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Ile                 565 570 575 Lys Lys Leu Trp Glu Gln Thr Arg Ser Ser Ays Gly Leu Leu Val Ser             580 585 590 Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu         595 600 605 Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu     610 615 620 Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Met 625 630 635 640 Asn Asn Ala Val Met Met Pro Ala His Gly Pro Ala Lys Asn Met Glu                 645 650 655 Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg             660 665 670 Ser Ile Leu Asn Thr Ser Gln Arg Gly Val Leu Glu Asp Glu Gln Met         675 680 685 Tyr Gln Arg Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser     690 695 700 Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys                 725 730 735 Lys Glu Glu Phe Thr Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu             740 745 750 Leu Arg Arg Gln Lys         755 <210> 10 <211> 759 <212> PRT <213> influenza virus <400> 10 Met Glu Arg Ile Lys Glu Leu Arg Asn Leu Met Ser Gln Ser Arg Thr   1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys              20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys          35 40 45 Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Thr      50 55 60 Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys  65 70 75 80 Met Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val                  85 90 95 Thr Trp Trp Asn Arg Asn Gly Pro Met Thr Asn Thr Val His Tyr Pro             100 105 110 Lys Ile Tyr Lys Thr Tyr Phe Glu Arg Val Glu Arg Leu Lys His Gly         115 120 125 Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg     130 135 140 Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155 160 Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile                 165 170 175 Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu             180 185 190 Leu Gln Asp Cys Lys Ile Ser Pro Leu Met Val Ala Tyr Met Leu Glu         195 200 205 Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr     210 215 220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225 230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Lys Asn Asp Asp Val Asp                 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Ala Val             260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln         275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu     290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser                 325 330 335 Ser Val Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu             340 345 350 Lys Ile Arg Val His Glu Gly Ser Glu Glu Phe Thr Met Val Gly Arg         355 360 365 Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu     370 375 380 Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly                 405 410 415 Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His             420 425 430 Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn         435 440 445 Trp Gly Val Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu     450 455 460 Pro Asp Met Thr Pro Ser Ile Glu Met Ser Met Arg Gly Val Arg Ile 465 470 475 480 Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val                 485 490 495 Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Val Leu             500 505 510 Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr         515 520 525 Ile Thr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser     530 535 540 Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val 545 550 555 560 Lys Ile Gln Trp Ser Gln Asn Pro Thr Met Leu Tyr Asn Lys Met Glu                 565 570 575 Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ile Arg Gly Gln Tyr             580 585 590 Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly         595 600 605 Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala     610 615 620 Pro Pro Lys Gln Ser Arg Met Gln Phe Ser Ser Phe Thr Val Asn Val 625 630 635 640 Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe                 645 650 655 Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala             660 665 670 Gly Thr Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser         675 680 685 Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Arg Arg Tyr     690 695 700 Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu 705 710 715 720 Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys                 725 730 735 Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys             740 745 750 Arg Ile Arg Met Ala Ile Asn         755

Claims (21)

The recombinant influenza virus comprising H7 hemagglutinin (HA) wherein the amino acids at positions 141 and 167 are glycosylated.
delete The method according to claim 1,
Wherein said amino acid is asparagine.
The method of claim 3,
Wherein said glycosylation is N-linked glycosylation (NLG).
The method according to claim 1,
The H7 hemagglutinin is derived from avian influenza virus, recombinant influenza virus.
6. The method of claim 5,
Wherein said avian influenza virus is H7N1, H7N2, H7N3, H7N7 or H7N9 subtype avian influenza virus.
The method according to claim 1,
Neuraminidase, NP, Nucleoprotein, Matrix protein, NS, Nonstructural protein, PA (Polymerase acidic protein), PB1 (Polymerase basic protein 1), and PB2 (Polymerase basic protein 2). 2. The recombinant influenza virus according to claim 1,
8. The method of claim 7,
The neuraminidase is a recombinant influenza virus derived from H7N9 subtype avian influenza virus.
8. The method of claim 7,
(NP), a matrix protein (M), a nonstructural protein (NS), a polymerase acidic protein (PA), a polymerase basic protein (PB1), and a polymerase basic protein 2) is derived from the H1N1 subtype influenza virus, recombinant influenza virus.
9. A vaccine composition comprising the recombinant influenza virus according to any one of claims 1 to 9 as an active ingredient.
11. The method of claim 10,
A vaccine composition, further comprising an adjuvant.
12. The method of claim 11,
The immune enhancer may be selected from the group consisting of complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), Squalene, Squalane, aluminum hydroxide, aluminum phosphate Aluminum phosphate, and saponin.
12. The method of claim 11,
A vaccine composition, further comprising a pharmaceutically acceptable carrier.
11. The method of claim 10,
Lt; RTI ID = 0.0 &gt; influenza &lt; / RTI &gt; virus.
11. The method of claim 10,
Wherein the vaccine is at least one selected from the group consisting of a subunit vaccine, a synthetic vaccine, and a genetic engineering vaccine.
9. A pharmaceutical composition for the prevention or treatment of influenza infectious diseases, which comprises the recombinant influenza virus according to any one of claims 1 to 9 as an active ingredient.
17. The method of claim 16,
Wherein the influenza infectious disease is at least one selected from the group consisting of sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea.
9. A composition for the diagnosis of an influenza virus infection comprising the recombinant influenza virus according to any one of claims 1 to 9 or an antigen thereof.
An influenza virus infection diagnosis kit comprising the diagnostic composition of claim 18.
18. A method for providing information about the diagnosis of influenza virus infection, comprising the step of treating the diagnostic composition of claim 18 to a sample.
21. The method of claim 20,
Wherein the sample is at least one selected from the group consisting of cells expected to be infected with influenza virus or infected cells, blood, urine, saliva, and tissue.

Images