JPWO2014097762A1 - Antibodies against highly pathogenic avian influenza - Google Patents

Abstract

The present invention relates to an antibody against the HA protein of influenza virus having a dissociation constant of at least 1 × 10 −10 M, a method for producing the antibody, a method for detecting an influenza virus by reacting an antibody with a biological sample, The present invention relates to a pharmaceutical composition for influenza virus.

Description

The present invention relates to a specific and anti-affinity antibody against highly pathogenic avian influenza, in particular a monoclonal antibody having a dissociation constant of at least 1 × 10 ⁻¹⁰ M.

  In general, humans are not infected with influenza infected by other animals, but are infected with seasonal A Hong Kong type (H3N1), A Soviet type (H1N1), type B influenza and the like. However, in recent years, it has been a problem that highly pathogenic influenza A (H5N1) derived from avian infectious type infects humans and develops severe pneumonia. In humans, since there is no previous infection, once a highly pathogenic influenza infection occurs, the infection spreads rapidly. Therefore, it is important to urgently develop therapeutic drugs in order to quickly diagnose the epidemic of highly pathogenic influenza (H5N1) and to carry out prevention, prevention, and vaccine administration. Recently, highly pathogenic influenza viruses have been successfully isolated in various regions, and antigens of various virus strains (clades) have been revealed. In the epidemic of highly pathogenic influenza, it is assumed that a variety of mutants are likely to appear if a pandemic epidemic occurs as in the case of seasonality. Therefore, the administration of anti-influenza drugs must be avoided as much as possible. Therefore, it is an urgent task to produce a simple test reagent capable of detecting a highly pathogenic influenza infection quickly and specifically and to focus on pinpoint treatment.

  To date, specific monoclonal antibodies have been made to detect H5N1 influenza and numerous attempts have been made. For example, monoclonal antibodies (Simmons CP et al., PLoS Med 4: e178, 2007 (Non-Patent Document 1)) prepared by immortalizing peripheral B cells after the H5N1 influenza infection, and library technology by phage display method Antibodies (Kashyap AK et al., PNAS 105: 5986, 2008 (Non-patent Document 2), Lim AP et al., Virol. J. 2008, 5: 130 (Non-patent Document 3)), mouse or rat Examples thereof include antibodies (US Pat. No. 7,879,326 (Patent Document 1)) produced by cell fusion by immunization with H5N1 antigen, and other monoclonal antibodies against highly pathogenic avian influenza (No. 4,625,485). Patent (Patent Document 2) There is.

However, the above antibodies still have problems regarding the reactivity to a wide range of virus strains and the detection sensitivity. For example, 11 types of antibodies from Simons et al. (Non-patent Document 1) have been reported from BF-1 to BF-19, but the virus neutralization activity is described in the report, but it is necessary for rapid diagnostic detection. The important antibody affinity for obtaining high sensitivity is a low level of KD = 9.25 × 10 ⁻⁷ M to 2.07 × 10 ⁻⁷ M. The antibody of Foun et al. (Patent Document 1) is an antibody using a peptide fragment of H5N1 as an immunogen, and the target epitope in this antibody is limited. The activity of the antibody is shown as a result of measurement by ELISA. For 6G6 antibody, OD = 0.604 at 1024-fold dilution, and OD = 0.139 for 7F6. It is difficult to say that the target measurement system has high affinity.
From such a situation, in order to recognize and quickly diagnose each clade (subtype) of H5N1 influenza virus equally and strongly, a high-affinity monoclonal antibody capable of binding to an antigen more strongly Desired.

US Pat. No. 7,879,326 Japanese Patent No. 4625485

Simons CP et al. , PLoS Med 4: e178, 2007 Kashap AK et al. , PNAS 105: 5986, 2008. Lim AP et al. , Virol. J. et al. 2008, 5: 130

  It is an object of the present invention to provide a specific and high affinity monoclonal antibody against highly pathogenic influenza.

  As a result of diligent research to solve the above-mentioned problems, the present inventor prepared an antigen using a recombinant vaccinia virus and immunized the GANP (registered trademark) transgenic mouse to obtain a highly pathogenic avian bird. The inventors have found that monoclonal antibodies against influenza virus can be produced and have completed the present invention.

That is, the present invention is as follows.
(1) An antibody against the HA protein of influenza virus having a dissociation constant of at least 1 × 10 ⁻¹⁰ M.
(2) The antibody according to (1), which has a dissociation constant of 1 × 10 ⁻¹⁰ M to 1 × 10 ⁻¹³ M.
(3) The antibody according to (1), wherein the influenza virus is an influenza H5 subtype virus.
(4) The antibody according to (3), wherein the influenza H5 subtype virus is influenza H5N1 virus.
(5) Influenza H5N1 virus is the Indogan Qinghai Lake strain (clade 2.2), Indonesia strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolian strain (A / whooper) (4) which is at least one selected from the group consisting of swan / Mongolia / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokaido / 1/2008: clade 2.3.2). The antibody described.
(6) The antibody according to (1), wherein the antibody is a monoclonal antibody.
(7) The antibody according to (6), which is produced by a hybridoma having a deposit number of NITE BP-01216 or NITE BP-01217, or a reception number of NITE ABP-01737 or NITE ABP-01738.
(8) The antibody according to (1), wherein the antibody is a chimeric antibody, a humanized antibody or a humanized antibody.
(9) An antibody that binds to a site to which the antibody according to (1) binds.
(10) The antibody fragment according to any one of (1) to (9).
(11) A hybridoma that produces the monoclonal antibody according to (6).
(12) A hybridoma whose accession number is NITE BP-01216 or NITE BP-01217, or whose reception number is NITE ABP-01737 or NITE ABP-01738.
(13) The method for producing the antibody according to (1), comprising the following steps:
(A) immunizing a non-human mammal with an influenza virus HA protein expressed using a recombinant vaccinia virus, and (b) collecting the antibody from the non-human mammal. .
(14) The method for producing a monoclonal antibody according to (6) or (7), comprising the following steps:
(A) immunizing a non-human mammal with an influenza virus HA protein expressed using a recombinant vaccinia virus;
(B) collecting antibody-producing cells from the immunized non-human mammal;
(C) The method comprising: fusing the antibody-producing cells obtained in step (b) and myeloma cells; and (d) collecting the antibody from the fused cells obtained in step (c).
(15) The method according to (13) or (14), wherein the non-human mammal is a GANP (registered trademark) transgenic non-human mammal.
(16) An influenza virus characterized by detecting influenza virus by contacting the antibody according to any one of (1) to (9) or the fragment according to (10) with a biological sample. Detection method.
(17) The method according to (16), wherein the influenza virus is an influenza H5 subtype virus.
(18) The method according to (17), wherein the influenza H5 subtype virus is influenza H5N1 virus.
(19) Influenza H5N1 virus has been identified as Indogan Qinghai Lake strain (clade 2.2), Indonesian strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolian strain (A / whooper) (18) which is at least one selected from the group consisting of swan / Mongolia / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokaido / 1/2008: clade 2.3.2). The method described.
(20) An influenza virus detection reagent comprising the antibody according to any one of (1) to (9) or the fragment according to (10).
(21) The reagent according to (20), wherein the influenza virus is an influenza H5 subtype virus.
(22) The reagent according to (21), wherein the influenza H5 subtype virus is influenza H5N1 virus.
(23) Influenza H5N1 virus can be found in Indogan Qinghai Lake strain (clade 2.2), Indonesia strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolia strain (A / whooper) (22) which is at least one selected from the group consisting of swan / Mongolia / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokaido / 1/2008: clade 2.3.2). The reagent described.
(24) A pharmaceutical composition for an anti-influenza virus comprising the antibody according to any one of (1) to (9) or the fragment according to (10).
(25) The pharmaceutical composition according to (24), wherein the influenza virus is an influenza H5 subtype virus.
(26) The pharmaceutical composition according to (25), wherein the influenza H5 subtype virus is influenza H5N1 virus.
(27) Influenza H5N1 virus has been identified as Indogan Qinghai Lake strain (clade 2.2), Indonesian strain (clade 2.1), Anhui strain (clade 2.3), Vietnamese strain (clade 1), Mongolian strain (A / whooper) (26) which is at least one selected from the group consisting of swan / Mongolia / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokaido / 1/2008: clade 2.3.2). The pharmaceutical composition as described.

  The present invention provides an antibody against a highly pathogenic avian influenza virus. The antibody of the present invention showed neutralizing activity against highly pathogenic avian influenza virus. Therefore, the antibody of the present invention is extremely useful as a reagent for detecting highly pathogenic avian influenza viruses or as a pharmaceutical composition for protecting against highly pathogenic avian influenza viruses.

It is a figure which shows the refinement | purification result of HA protein. It is a figure which shows the trimer formation and sugar chain modification of HA protein. It is a figure which shows the neutralization with respect to influenza virus H5N1 Vac-3 by the antibody of this invention, and a growth inhibition assay result. It is a figure which shows the neutralization and proliferation inhibition assay result with respect to highly pathogenic avian influenza virus H5N1 H5N1 A / whooper swan / Mongolia / 3/05 by the antibody of this invention. It is a figure which shows the neutralization and growth inhibition assay result with respect to the highly pathogenic avian influenza virus H5N1 A / whooper swan / Hokaido / 1/08 by the antibody of this invention. It is a figure which shows the therapeutic effect with respect to highly pathogenic avian influenza virus infection by the antibody of this invention. It is a figure which shows the therapeutic effect with respect to highly pathogenic avian influenza virus infection by the antibody of this invention.

Hereinafter, the present invention will be described in detail. This specification includes the contents of Japanese Patent Application No. 2012-278228 (application date: December 20, 2012), which is the basis for claiming priority of the present application.
The scope of the present invention is not limited to these descriptions, and modifications other than the following examples can be made as appropriate without departing from the spirit of the present invention.
All publications cited herein, for example, prior art documents, publications, patent publications and other patent documents, are hereby incorporated by reference in their entirety.

1. SUMMARY The present invention is a high affinity antibody against highly pathogenic avian influenza having a dissociation constant (KD) of at least 1 × 10 ⁻¹⁰ M, and a wide range of clade species, such as Indogan Qinghai Lake strain, Indonesian strain, Anhui strain , Which can be detected or protected with a single antibody against Vietnamese, Mongolian and Hokkaido strains.
Highly pathogenic avian influenza mediates chickens and is a highly lethal influenza that infects humans and has a different epidemic from seasonal influenza. At present, infection is being controlled by international containment of highly pathogenic avian influenza and limited vaccine administration. The key to this strategy is to quickly discover and diagnose highly infected flu patients and to take effective treatments and take measures to prevent the spread of infection. Accordingly, there is a need for a test reagent that can be easily and accurately diagnosed as quickly as possible with high sensitivity and accurately all of a wide range of highly pathogenic avian influenza.
In the present invention, hemagglutinin (HA) protein expressed using vaccinia virus is used as an antigen, and a highly pathogenic avian influenza is produced by using a high affinity monoclonal antibody production technique using GANP (registered trademark) transgenic mice. Viruses specifically and a wide range of clade species, namely, Indangan Qinghai Lake strain (A / Bar-Headed goose / Qinghai / 1A / 05: clade 2.2), Indonesia strain (A / Indonesia / 05/2005: clade 2) .1), Anhui strain (A / Anhui / 1194/2005: clade 2.3), Vietnam strain (A / Vietnam / 1194/2004: clade 1), Mongolian strain (A / whooper swan / Mongolia / 3 / 005: clade 2.2), Hokkaido strain (A / whooper swan / Hokkaido / 1/2008: clade 2.3.2) are prepared with high affinity antibodies capable of detecting all with a single type of antibody. Succeeded.

2. Antibody of the Invention The antibody of the present invention is a molecule that binds to the HA protein via the heavy and light chain variable regions VH and VL. The antibodies of the present invention represent either polyclonal or monoclonal immunoglobulin molecules such as IgG, IgA, IgM, IgE, IgD, or subclasses such as IgG1, IgG2, IgG3 or IgG4. The antibody of the present invention also includes functional fragments or subsequences of immunoglobulin molecules such as Fab, Fab ′, F (ab ′) ₂ , Fv, Fd, scFv and sdFv.
Hereinafter, the method for producing the antibody of the present invention will be described.

2-1. Antigen Preparation The antigen used in the present invention is the HA protein of influenza virus. This protein can be expressed using vaccinia virus.
<Production of recombinant vaccinia virus having HA protein gene of influenza virus>
The HA protein gene is registered with a predetermined accession number (Accession No.) in the GenBank database.
For example, H5 subtypes, for example, genes encoding HA proteins derived from influenza viruses belonging to each clade of H5N1, are registered in the GenBank accession number as follows.
Clade 1: _A / Vietnam / 1109 / 2004_EF541040
Clade 2.1: _A / Indonesia / 5 / 2005_EF541394
Clade 2.2: _A / bar-headed goose / Qinghai / 1A / 2005_DQ659327
Clade 2.3: _A / Anhui / 1 / 2005_DQ37171928

As a gene (cDNA) encoding the HA protein contained in the recombinant vaccinia virus of the present invention, in addition to the above genes, a part thereof or a mutant sequence thereof can be used. For example, DNA lacking part of the DNA encoding the HA protein of clade 1, DNA lacking part of the DNA encoding the HA protein of clade 2.1, and HA protein of clade 2.2 A DNA in which a part of the DNA is deleted or a DNA in which a part of the DNA encoding the HA protein of clade 2.3 is deleted can be used.
These HA protein genes have already been cloned and inserted into plasmids.

  Further, in the present invention, in addition to the above HA protein gene (in the present specification, the full-length base sequence or the base sequence of the protein coding region. Hereinafter, the same applies to other HA protein genes), as well as mutant forms thereof. Genes can also be used. For example, it hybridizes under stringent conditions with a sequence consisting of a base sequence complementary to the base sequence shown in EF541040 and is complementary to the base sequence shown in EF541394, which encodes the HA protein of clade 1. A string comprising a base sequence complementary to the base sequence shown in DQ659327, a DNA that encodes the HA protein of clade 2.1 and hybridizes with a sequence comprising a basic base sequence under stringent conditions Hybridizing under stringent conditions, and hybridizing under stringent conditions with a DNA sequence encoding the HA protein of clade 2.2, a sequence consisting of a base sequence complementary to the base sequence shown in DQ371728, And Clade 2.3 HA protein Etc. can be used a DNA over de.

  Examples of stringent conditions include 0.1 × SSC to 10 × SSC, 0.1% to 1.0% SDS, and 20 ° C. to 80 ° C., and more specifically, 37 ° C. to 56 ° C. An example is a condition in which after pre-hybridization at 30 ° C. for 30 minutes or more, washing is performed 1 to 3 times in 0.1 × SSC and 0.1% SDS at room temperature for 10 to 20 minutes. For the detailed procedure of the hybridization method, “Molecular Cloning: A Laboratory Manual (4th Edition)” (Cold Spring Harbor Laboratory Press (2012)) and the like can be referred to.

  Further, it has 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the nucleotide sequence shown in EF5410402, and Clade 1 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the DNA sequence encoding the HA protein of EF541394 And DNA encoding the HA protein of Clade 2.1, the base sequence shown in DQ659327 and 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or DNA having a homology of 99% or more and encoding the HA protein of clade 2.2, 50% or more, 60% or more, 70% or less with the nucleotide sequence shown in DQ371728 , 80%, 90%, 95%, have a 98% or 99% or more homology, and it is possible using DNA encoding the HA protein of clade 2.3.

The mutant gene is prepared using, for example, a site-directed displacement induction method described in “Molecular cloning: A Laboratory Manual (4th Edition)” or the like, or a mutation introduction kit using site-specific mutagenesis. can do. Examples of such mutagenesis kits include QuickChange ^™ Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTaylor ^™ Site-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRaectite-Site-Mit-Site-Mit-Site-Mit-Site-Mist Preferred examples include Mutan-Super Express Km and the like (manufactured by Takara Bio Inc.).

Therefore, the HA protein used as an antigen can be obtained by an ordinary genetic engineering technique based on the above gene information.
For example, in the present invention, the DNA encoding the HA protein is incorporated into a plasmid for homologous recombination and then introduced into an animal cell, followed by infecting the animal cell with the parent strain, vaccinia virus. After infection, the recombinant vaccinia virus may be cultured to express the HA protein. The HA protein can be expressed in large quantities in the form of complete sugar modification from the early stage to the late stage of vaccinia virus infection.

A homologous recombination plasmid can include a promoter, a gene to be expressed, and the like. Examples of animal cells include rabbit kidney cells, monkey kidney cells, and human fibroblasts. The expressed HA protein can be purified by a known technique using a His tag or the like. The purification method is not limited, and affinity chromatography, HPLC purification, and the like are employed. Confirmation that the purified HA protein is the target can be performed, for example, by Western blotting, SDS-polyacrylamide gel electrophoresis, or the like.
The majority of the HA protein expressed by the recombinant vaccinia virus forms a trimer similar to the HA protein on influenza virus particles and is used as an antigen.

2-2. Immunization The protein produced as described above is immunized by administration to a non-human mammal by itself or together with a carrier and a diluent, and the antibody titer is measured.
The type of non-human mammal to be immunized is not particularly limited, and examples include mice, rats, guinea pigs, rabbits, dogs, goats, etc., with mice being preferred.
In a preferred embodiment of the present invention, in order to produce the antibody of the present invention, a transgenic non-human mammal called “GANP (registered trademark)” having a high-specificity antibody-producing ability (WO 2004/040971) as an animal to be immunized. Can also be used.

GANP (registered trademark) transgenic non-human mammal is a non-human mammal into which a gene encoding a germinal center-associated nuclear protein has been introduced, and the animal is immunized with a predetermined antigen. Thus, it is an animal that can produce a high affinity or high specificity antibody against the antigen (WO 00/50611, Sakaguchi N. et al., J Immunol. 2005 Apr 15; 174 (8 ): 4485-94.).
For example, a GANP transgenic non-human mammal (for example, a mouse) can be produced by the method described in the above publication or Sakaguchi et al.

The total dose of the antigen per animal is 10 to 2000 μg. When immunizing an antigen, an adjuvant and an antigen solution are generally mixed. Examples of the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously, intraperitoneally, intramuscularly, subcutaneously into the footpad, or the like. Further, the immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 4 weeks.
When immunizing a GANP transgenic non-human mammal, it is not necessary to leave it until the absorbance level is lowered, and it is sufficient to perform the final immunization according to the immunization interval of a general monoclonal antibody production method.

The antibody titer can be examined using blood collected from the immunized animal. The obtained serum is serially diluted, and the antibody titer is measured by enzyme immunoassay (ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA (radioimmunoassay)) and the like.
As a result of the measurement, an animal having a high antibody titer against the HA protein is subjected to final immunization. However, the immunity of the antigen and the measurement of the antibody titer are not limited to the above measurement methods.
Thereafter, immunocompetent cells (such as spleen cells) or regional lymph nodes are removed several days after the final immunization day, preferably 2 to 5 days later. The interval between blood collections is 1 to 4 weeks, preferably 1 to 2 weeks after immunization.

2-3. Production of polyclonal antibody In the present invention, when obtaining a polyclonal antibody, blood is collected on the day when the desired antibody titer is shown to obtain antiserum. When antibody purification is required, purify by selecting a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or a combination thereof. Can do. Thereafter, the reactivity of the polyclonal antibody in the antiserum is measured by an ELISA method or the like.

2-4. Production of Monoclonal Antibody As a method for obtaining the monoclonal antibody of the present invention, first, the HANP transgenic non-human mammal is immunized with the HA protein, antibody-producing cells (for example, B cells) are collected from the immunized animal, and this antibody production is performed. Cells and myeloma cells are fused to produce a hybridoma (fusion cell line). And the target monoclonal antibody can be obtained by extract | collecting the antibody produced from this hybridoma.

(1) Preparation of antibody-producing cells Antibody-producing cells are prepared from spleen cells or lymph node cells or spleen or lymph nodes of animals such as immunized non-human mammals. Even when a GANP transgenic non-human mammal is used, antibody-producing cells can be collected from spleen cells, lymph node cells, spleen, lymph nodes, or the like in the same manner as described above.
Examples of lymph nodes include inguinal lymph nodes and mediastinal lymph nodes. Although it is not necessary to separate the antibody-producing cells from the collected cell population, it is desirable to separate only antibody-producing cells from the cell population. It is preferable to remove tissue debris and red blood cells as much as possible.

(2) Cell fusion As myeloma cells to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line to be used is preferably one that has the property that it cannot survive in a HAT selection medium (medium containing hypoxanthine, aminopterin, and thymidine) and can survive only in a state fused with antibody-producing cells. Examples of myeloma cells include P3X63-Ag. 8). U1 (P3U1), P3 / NS I / 1-Ag4-1 (NS1), etc. are mentioned.
Cell fusion is a commercially available medium such as DMEM or RPMI 1640 medium that does not contain fetal calf serum (FCS) or the like, and 1 × 10 ⁶ to 1 × 10 ⁷ cells / mL of spleen cells and / or lymph node cells. 1 × 10 ⁵ to 1 × 10 ⁶ myeloma cells are mixed (cell ratio of spleen cells and / or lymph node cells to myeloma cells is preferably 5: 1), and in the presence of a cell fusion promoter. Perform fusion. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 200 to 20,000 daltons can be used.

Moreover, it can also fuse | melt using the commercially available cell fusion apparatus using electrical stimulation (for example, electroporation). Furthermore, cells can be fused using Sendai virus. A person skilled in the art can fuse the antibody-producing cells and myeloma cells using a known cell fusion method.
After the fusion, the cells are diluted with, for example, a HAT medium prepared with RPMI 1640 medium containing 10 to 20% FCS, and then 0.5 to 3 × 10 ⁵ cells are seeded in each well of a 96-well culture plate, and a CO ₂ incubator Incubate at

(3) Establishment of hybridoma Next, a hybridoma producing the target antibody is selected from the cells after cell fusion treatment. 10-14 days after cell fusion, cells selected in HAT medium as described above form colonies. The culture supernatant of each well of the colony positive 96-well culture plate is collected and the antibody titer against HA is confirmed. As a confirmation method, an enzyme immunoassay (ELISA), a radioimmunoassay (RIA) or the like is performed. After confirming antibody production positive wells against HA, the cells are transferred to a 24-well or 12-well culture plate.

Here, the medium is preferably replaced with an HT medium (a medium containing hypoxanthine and thymidine) excluding aminopterin. HT medium is used as a recovery medium for hybridomas that continue to supply purine and pyrimidine precursors to the salvage pathway during the effect of residual aminopterin in the cells. After culturing for a while in HT medium, the antibody titer in the culture supernatant is confirmed again. Since the hybridoma is unstable because it is a fused cell, and it is highly likely that the antibody producing ability is lost immediately, it is preferable to confirm the antibody titer for the second time.
In the present invention, it is necessary to obtain a hybridoma having high specificity for HA without cross-reacting with SARS (Severe Accurate Respiratory Syndrome) spike (S) protein. It is preferable to confirm the cross-reactivity with the SARS S protein by ELISA, RIA or the like.

  The cells in the final selected well are cloned to make a single cell. For cloning, for example, the cell suspension is appropriately diluted with 10-20% FCS-containing RPMI 1640 medium, and then seeded with 0.3-2 cells in each well of a 96-well culture plate. The number of cells in each well of the 96-well culture plate is preferably seeded so that there is one cell in each well so that there is a high probability of one cell in each well. After cell seeding, the culture supernatant of the colony-positive well is collected 7 to 10 days later. At this time, it is preferable to confirm that it is a single colony after 3 to 5 days. The collected culture supernatant is checked for antibody titer. Again, clones with high specificity for HA and low cross-reactivity for SARS S protein are selected. Further, the number of cells in the selected well is increased to some extent to establish a hybridoma strain. Cloning may be performed several times as necessary.

(4) Preparation of monoclonal antibody An HA-specific monoclonal antibody is purified and collected from the established hybridoma strain by the following method. That is, a method of preparing an antibody from a culture supernatant cultured in a medium with reduced serum concentration, a method of preparing an antibody from a culture supernatant cultured in a commercially available serum-free medium, or injecting a hybridoma into the abdominal cavity of an animal. There is a method of collecting ascites and preparing antibodies from the ascites. The culture supernatant is collected from cells prepared at 0.1 to 4 × 10 ⁵ cells / mL and cultured for 1 to 2 weeks. In the case of ascites, 0.1 to 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and the same type of animal, and the hybridomas are proliferated in large quantities. And ascites is collected after 1-2 weeks.

Examples of the culture method include a method using a culture flask, a method using a spinner flask, a method using a shaker flask, and a method using a bioreactor.
Antibody purification methods include HA affinity column purification method, ammonium sulfate salting-out fraction purification method by gel filtration chromatography, ion exchange chromatography purification method, etc., selecting these known methods as appropriate, Or it can refine | purify by combining these.

(5) Deposit of microorganisms The cell lines (hybridomas) producing the monoclonal antibodies of the present invention are referred to as “3E10 (rVV cl2.2)” and “7G10 (rVV cl2.2)”, both of which are independent administrative corporation products. Deposited internationally based on the Budapest Treaty at the Japan Institute for Evaluation Technology Patent Microorganisms Deposit Center (2-5-8 KITESA Kamashita, Kisarazu City, Chiba Prefecture 292-0818) based on the Budapest Treaty (original deposit date: February 2012) 1 day). The accession numbers are NITE BP-01216 for “3E10 (rVV cl2.2)” and NITE BP-01217 for “7G10 (rVV cl2.2)”.
The cell lines (hybridomas) that produce the monoclonal antibodies of the present invention are also referred to as “8C1 (rVV cl2.2)” and “14A7 (rVV cl2.2)”, both of which are listed in the Budapest Microorganism Depositary Center. (The original deposit date: October 18, 2013). The receipt number is NITE ABP-01737 for “8C1 (rVV cl2.2)” and NITE ABP-01738 for “14A7 (rVV cl2.2)”.

2-5. Antibody Properties The antibody of the present invention specifically binds to HA and exhibits high affinity.
“Specific” means that it does not cross-react with subtypes other than the HA protein subtype used for immunization, and reacts only with the subtype protein used for immunization (binding / neutralization). For example, when H5N1 HA protein is used as an antigen as a subtype, the antibody of the present invention only reacts (binding / neutralizes) with H5 subtype protein or H5 subtype virus, and other H1N1, H1N2, etc. This means that there is no cross-reactivity with the subtype of. Furthermore, the antibody of the present invention specifically binds to different clades (eg, clade 1, 2.1, 2.2, 2.3) as long as they belong to the same subtype (eg, H5 subtype). can do.
Binding affinity can be determined by association constant (KA) and dissociation constant (KD). The affinity equilibrium constant (K) is expressed as a ratio of KA / KD. Its binding affinity can be detected as follows.

The binding affinity has a dissociation constant (KD) of at least 1 × 10 ⁻¹⁰ M, and is 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000 times, for example, with respect to this dissociation constant. Or 1000-10,000 times higher affinity. Specifically, antibodies of the present invention, the dissociation constant for the binding affinity of the HA protein (KD) ^{is, 1 × 10 -10 M, 5} × 10 -11 M, 1 × 10 -11 M, 5 × 10 - ¹² M, 1 × 10 ⁻¹² M, 5 × 10 ⁻¹³ M, 1 × 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 1 × 10 ⁻¹⁴ M, 5 × 10 ⁻¹⁵ M, or 1 × 10 ^{− 15} M, more preferably 1 × 10 ⁻¹⁰ M to 1 × 10 ⁻¹³ M. Alternatively, the value may be lower than these KDs and may have high affinity.

Here, when the dissociation constant (KD) of the antibody to be measured for affinity is within about 1 to 100 times the KD of the antibody of the present invention, the antibody is substantially the same as the antibody of the present invention. Is included in the present invention.
The association constant (KA) and dissociation constant (KD) can be measured using surface plasmon resonance (SPR), and it is possible to employ a known device and method for detecting the binding rate in real time and further monitoring it. (For example, Biacore (registered trademark) T200 (GE Healthcare), ProteON XPR36 (Bio-Rad), etc.). The KD value of a typical antibody as defined herein is sufficient to saturate half (50%) of the binding site of the HA antigen of the immobilized purified HA protein (A / Anhui / 1/2005). Defined as the concentration of antibody required.
In addition, the antibody of the present invention can specifically bind to multiple types of virus strains. Examples of the virus strains to be bound include A / whooper swan / Monglia / 03/2005, A / whooper swan / Hokaido / 1/2008, A / duck / Hokaido / Vac-3 / 2007.

2-6. Antibody Fragment and Recombinant Antibody In the present invention, a fragment of the above antibody is also provided. Here, the antibody fragment of the present invention is an antibody against HA and has the above KD value.
The antibody fragment means a partial region of the antibody of the present invention, and includes, for example, Fab, Fab ′, F (ab ′) ₂ , Fv, diabody (dibodies), dsFv (disulphide-stabilized Fv), scFv (single chain Fv) and the like. The antibody fragment can be obtained by cleaving the antibody of the present invention with various proteolytic enzymes according to the purpose.
For example, Fab can be obtained by treating antibody molecules with papain, and F (ab ′) ₂ can be obtained by treating antibody molecules with pepsin. Fab ′ can be obtained by cleaving the disulfide bond in the hinge region of F (ab ′) ₂ .

In the case of scFv, cDNA encoding the H chain V region and L chain V region of the antibody is obtained, and DNA encoding scFv is constructed. By inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression, scFv can be produced.
In the case of diabody, cDNA encoding the H chain V region and L chain V region of the antibody is obtained, and a DNA encoding scFv is constructed so that the length of the amino acid sequence of the peptide linker is 8 residues or less. A diabody can be produced by inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression.
In the case of dsFv, cDNA encoding the H chain V region and L chain V region of the antibody is obtained, and a DNA encoding dsFv is constructed. By inserting this DNA into an expression vector and introducing the expression vector into a host organism for expression, dsFv can be produced.

One preferred embodiment of the antibody of the present invention is a recombinant antibody. Examples of the recombinant antibody include, but are not limited to, chimeric antibodies, humanized antibodies, and humanized antibodies.
In the art, human antibodies or consensus residues thereof, or amino acid residues present in CDR regions are known (Kabat, Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human. Services.Public Health Service (1987)). Accordingly, human antibodies include antibodies in which one or more amino acid residues are replaced with amino acids present in any of the other human antibodies.

  When producing a humanized antibody, a so-called CDR grafting (CDR grafting) can be employed. CDR grafting refers to transplanting a complementarity determining region (CDR) from a variable region of a mouse antibody into a human variable region, wherein the framework region (FR) is derived from a human and the CDR is derived from a mouse. This is a method for producing a configured variable region. These humanized reshaped human variable regions are then linked to human constant regions. Methods for producing such humanized antibodies are well known in the art (see, for example, Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987)). .

  Humanized antibodies (fully human antibodies) generally have hypervariable regions that are antigen-binding sites in the V region, the other parts of the V region, and the structures of the constant regions that have the same structure as human antibodies. It is. However, the hypervariable site may be derived from another animal. Techniques for producing humanized antibodies are also known, and methods for producing gene sequences common to humans by genetic engineering techniques have been established. For example, a humanized antibody is a method using a human antibody-producing mouse having a human chromosomal fragment containing H and L chain genes of a human antibody (Tomizuka, K. et al., Nat. Genet., (1977) 16). , 133-143; Kuroiwa, Y. et al., Nucleic Acids Res., (1998) 26, 3447-3448; -73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers; Tomizuka, K. et al., Proc. Natl. Sci. USA, (2000) 97, 722-727, etc.), or a method for obtaining human antibodies derived from phage display selected from a human antibody library (Wormstone, IM et al., Investigative Ophthalmology & Visual Science., (2002) 43 (7), 2301-8; , Opthalmology, (2002) 109 (3), 427-431, etc.).

  In the present invention, the hybridoma of the present invention (for example, a hybridoma whose accession number is NITE BP-01216 or NITE BP-01217, or whose reception number is NITE ABP-01737 or NITE ABP-01738) or extracted from the hybridoma Chimeric antibodies, humanized antibodies, and humanized antibodies can be prepared using DNA or RNA as a raw material according to the well-known methods described above.

2-7. Further, the anti-HA antibody of the present invention includes, for example, an antibody that binds to a site (for example, an epitope) to which the antibody of the present invention binds (recognizes), preferably the accession number is NITE BP- Examples include an antibody that binds to a site (eg, an epitope) to which a monoclonal antibody produced by a hybridoma of 01216 or NITE BP-01217 or a receipt number of NITE ABP-01737 or NITE ABP-01738 binds.
The site to which the anti-HA antibody of the present invention binds is not limited as long as it is at least a part of the antigen HA.

3. Detection method Since HA can be used as a clinical marker for influenza, the antibody of the present invention is brought into contact with a biological sample, and HA in the biological sample is measured, thereby detecting or diagnosing influenza using the measurement result as an index. can do.
Therefore, the present invention provides a method for detecting influenza virus, which comprises detecting an influenza virus by bringing the antibody or fragment thereof of the present invention into contact with a biological sample.
First, a biological sample to be examined is collected from a subject such as an influenza patient, a patient suspected of influenza, or a health checkup examinee, and an HA measurement sample is prepared. The influenza virus to be detected is an influenza A virus or subtype, and examples include Indogan Qinghai Lake strain, Indonesia strain, Anhui strain, and Vietnam strain. Subtypes include, for example, H1N1, H1N2, H2N1, H2N2, H2N3, H3N2, H3N8, H4N6, H4N8, H5N1, H5N2, H5N3, H5N5, H5N8, H6N1, H6N2, H7N1, H7N9, H7N1, H7N9, H7N1, H7N9 H10N3, H11N1, H11N2, H11N9, H12N5, H13N6, H13N8, H15N8 or H16N3, and one or more of these are selected. In particular, an influenza H5 subtype influenza virus (for example, H5N1) is preferable.

Examples of the biological sample include blood, sputum, saliva, pharyngeal swab, nasal discharge, nasal discharge, and nasal aspirate.
In the detection method of the present invention, the antibody is contacted with a biological sample. “Contact” means that the antibody of the present invention or a fragment thereof (also referred to as an antibody or the like) and a biological sample to be tested are placed in the same environment so that they can be bound or reacted. For example, adding a biological sample to a container (well) containing an antibody or the like, adding an antibody or the like to a container (well) containing a biological sample, or culturing the antibody or the like and the biological sample in the same container It means to do. The measurement of HA can be performed by ELISA or EIA that is generally performed, and is not particularly limited.

  In particular, since HA derived from influenza patients contains the amino acid sequence of the HA protein shown in the above accession number, the antibody of the present invention that specifically binds to this amino acid sequence detects H5N1 influenza virus. Is particularly preferred. In the case of detection by ELISA, when the signal intensity is stronger than that of the negative control, it can be determined that influenza virus is contained in the test sample (ie, positive).

4). Kits and Reagents Containing Antibodies of the Present Invention In the present invention, antibodies against HA can be used as reagents or kits for detecting influenza viruses.
When the antibody of the present invention (for example, a monoclonal antibody) is used as an influenza detection or diagnostic agent, the monoclonal antibody can be combined with another solvent or solute to form a composition. For example, distilled water, pH buffer reagent, salt, protein, surfactant and the like can be combined. Monoclonal antibodies can also be enzyme-labeled. As a labeling enzyme, in addition to HRP (horseradish peroxidase), alkaline phosphatase, malate dehydrase, α-glucosidase, α-galactosidase, colloidal gold and the like can be used.

When the present invention is used in a kit, in addition to the antibody of the present invention, the above solvent, solute, enzyme labeling reagent, antigen-immobilized microplate, antibody dilution solution, OPD (orthophenylenediamine) tablet, substrate solution, A reaction stop solution, a concentrated washing solution, instructions for use, and the like can be included. In addition, a reaction medium such as a buffer solution that gives optimum conditions for the reaction, a buffer solution that is useful for stabilizing the reaction product, and a stabilizer for the reaction material may be included in the kit of the present invention.
In the present invention, a PCR method can also be employed to detect HA mRNA or DNA.
Primers used for PCR can also be included in the reagents or kits of the present invention. The region on which the primer is designed is not particularly limited as long as the HA protein can be detected.
The length of the primer is 18 to 24 bases, preferably 20 to 22 bases. The following can be illustrated as a base sequence of a primer.

5. Pharmaceutical Composition The pharmaceutical composition of the present invention contains the antibody of the present invention as an active ingredient, is used for anti-influenza virus, and is effective for the prevention or treatment of influenza.
In the present invention, the influenza virus that is the target of prevention or treatment of influenza is an influenza A virus or subtype, and examples include Indogan Qinghai Lake strain, Indonesia strain, Anhui strain, and Vietnam strain. Subtypes are, for example, H1N1, H1N2, H2N1, H2N2, H2N3, H3N2, H3N8, H4N6, H4N8, H5N1, H5N2, H5N3, H5N5, H5N8, H6N1, H6N2, H7N1, H7N1, H7N1, H7N2, H7N1, H7N2, H7N , H11N1, H11N2, H11N9, H12N5, H13N6, H13N8, H15N8 or H16N3 are selected from one or more. Among them, H5 subtype influenza virus (for example, H5N1) is preferable.

  The antibodies of the invention can prevent a subject from becoming infected with one or more influenza viruses (isolate or subtype) or reduce the subject's susceptibility to the infection. Also, one or more symptoms or complications associated with influenza infection, such as fatigue, chills, fever, body pain, headache, cough, sore throat, nasal congestion, sinus closure, pneumonia, bronchitis, ear infection, ear Reduce or eliminate pain, etc., or prevent hatred of these symptoms. Furthermore, it is possible to shorten the period of suffering from these symptoms.

  The antibody of the present invention can be administered alone or together with a pharmaceutically acceptable carrier or diluent as a pharmaceutical composition, and the administration can be performed once or divided into several times.

  As used herein, “pharmaceutically acceptable carrier” refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners. , Flavoring agents, solubilizers or other additives. By using one or more of such carriers, pharmaceuticals in the form of tablets, pills, powders, granules, injections, solutions, capsules, troches, elixirs, suspensions, emulsions or syrups, etc. A composition can be prepared. These pharmaceutical compositions can be administered orally or parenterally.

In the case of oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate and glycine can be used together with a disintegrant, a binder and the like. Examples of the disintegrant include starch, alginic acid, and certain types of silicate double salts. Examples of the binder include polyvinylpyrrolidone, sucrose, gelatin, and gum arabic. Further, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are very effective for tablet formation. When an aqueous suspension or elixir is used for oral administration, an emulsifier and a suspending agent may be used in combination, if necessary, and used with water, ethanol, propylene glycol, glycerin, etc., and diluents that combine them. .
Other forms for parenteral administration include injections that contain one or more active substances and are prescribed by conventional methods.

In the case of an injection, it is dissolved or suspended in a pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection to an antibody concentration of 1 μg / ml to 100 mg / ml. Can be manufactured. The injection thus prepared is a ratio of 1 μg / kg to 100 mg / kg, preferably 100 μg / kg to 15 mg / kg, in a single administration to a subject (for example, human influenza patient). And can be administered once to several times per day.
However, the dosage is not limited to this range, and may vary depending on the weight and symptoms of the patient and individual administration routes. The dose may vary depending on the sensitivity of the patient to be treated, the way the drug is prescribed, the period of administration and the interval between administrations. In some cases, a dose lower than the lower limit of the above range may be appropriate .

Examples of the administration form include intravenous injection, subcutaneous injection, intradermal injection, and the like, preferably intravenous injection. In addition, injections can be prepared as non-aqueous diluents (for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol), suspensions, or emulsions. Such sterilization of the injection can be performed by filtration sterilization through a bacteria-retaining filter, blending of a bactericide, or irradiation. The injection can be produced as a form prepared at the time of use. That is, it can be used as a sterile solid or powder composition by lyophilization, etc., and dissolved in sterile water for injection or other solvent before use.
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Preparation of high-affinity antibody against HA protein (1) Preparation of antigen Mass purification of histidine-tag (His-Tag) H5N1 antigen was performed by recombinant vaccinia virus (rVV). A recombinant plasmid in which the H5N1 influenza HA gene was introduced into the homologous recombination vector was introduced into rabbit kidney cells RK13 and infected with the parent strain vaccinia virus to obtain a recombinant vaccinia virus (rVV) expressing HA. After infecting mammalian cells, purification of His-Tag protein by a conventional method using TARON column chromatography was performed.

<Experimental method>
The HA protein gene of H5N1 HPAIV has already been cloned and inserted into a plasmid. The HA protein gene of H5N1 HPAIV HA gene (GenBank accession numbers: EF541040, EF541394, DQ65937, DQ371937) inserted into the plasmid was used as a PCR template. That is, H5N1 HPAIV HA protein gene region was prepared by performing PCR using each gene-specific primer using cDNA of H5N1 HPAIV HA protein gene as a template.
The resulting fragment was cloned into the plasmid pBMSF7C (Arch. Virol. Vol. 138, p. 315-330, 1994; see JP-A-6-237773).
Moreover, the expression promoter contained in the recombinant vaccinia virus of the present invention is inserted into the hemagglutinin (HA) gene region of vaccinia virus, and includes a poxvirus A type inclusion body (ATI) promoter and a plurality of repetitive vaccinia viruses. A hybrid promoter composed of a 7.5 kDa protein (p7.5) early expression promoter was used.

A recombinant vaccinia virus was produced by introducing the plasmid vector into a host vaccinia virus. The plasmid vector was introduced into the host by introducing plasmid vectors pBMSF7C-mCl1, pBMSF7C-mCl2.1, pBMSF7C-mCl2.2, and pBMSF7C-mCl2.3 into animal cells infected with the attenuated vaccinia virus LC16m8 strain, respectively. , Recombinant vaccinia viruses (RVV-Flu HA (H5-mCl1), RVV-Flu HA (H5-mCl2), which cause homologous recombination in the hemagglutinin (HA) gene region of vaccinia virus and express the HA protein gene of H5N1 HPAIV, respectively. .1), RVV-Flu HA (H5-mCl2.2), RVV-Flu HA (H5-mCl2.3) were prepared.
The HA protein was expressed by infecting the produced recombinant vaccinia virus into RK-13 cells, which are a rabbit kidney cell line. Subsequently, purification of HA protein to which His-Tag was added by a conventional method using TARON column chromatography was performed.

<Purification result>
The purification results of HA protein (Western blotting and CBB staining) are shown in FIG.
The obtained purified HA protein was designated as “H5mcl2.2 antigen”.
As a result of Western blotting and native gel electrophoresis analysis, it was confirmed that this protein was an antigen that formed a trimer and was subjected to sugar chain modification in the same manner as virus particle hemagglutin (HA) (FIG. 2). ). In FIG. 2, the left panel is a diagram showing the HA protein constituting the influenza virus particle, the middle panel is a diagram showing the purified HA protein expressed by rVV, and the right panel is the diagram showing the purified HA protein. It is a figure which shows having received sugar chain modification. The purified protein yield was 0.5-2 mg.

(2) Immunization of antigen Purified HA protein (H5mcl2.2 antigen) obtained as described above was immunized subcutaneously in a GANP (registered trademark) -transgenic mouse (Tg mouse) of Balb / c background. The immunization method was mixed with complete Freund's adjuvant, and after 4 weeks, mixed with incomplete Freund's adjuvant for booster immunization. After further boosting three times, the spleen was removed 3 days after the final immunization, and lymphocytes were fused with myeloma P3U1 cells using polyethylene glycol. After the fusion, the cells were seeded in a 96-well plate at an equivalent of 1 × 10 ⁵ cells / well of myeloma. Mouse thymocytes of 2 × 10 ⁵ cells / well were used as feeder cells, and the feeder cells and recombinant IL-6 (0.25 unit / ml) were added to the wells and cultured. At the time of culture, HAT (hypoxanthine, aminopterin, thymidine) was selected.

(3) Screening for antigen-specific high-affinity monoclonal antibody Specific antibody activity in the hybridoma supernatant was measured by ELISA using recombinant antigen.
Based on the OD ₄₈₅ measurement value of peroxidase reaction (substrate is OPD) in the absence of normal negative control and supernatant, 0.1 or less was determined as negative, and 1.0 or more was measured as a positive clone. The positive clone was re-examined, the clone was expanded and cultured in a 48-well plate for 3 days, the supernatant was collected, and after confirming that the activity was again OD ₄₈₅ > 0.5, the limiting dilution method was applied. 5 / well and 2 / well were cloned using 96-well plates.
Cloning was performed according to a conventional method, and cell culture was performed in the presence of recombinant IL-6 0.25 unit / ml in 96 wells in the presence of ⁴ × 10 ⁵ mouse thymocytes feeder cells. A subclone having the same activity as that of the original cell was established and stored in the proliferating clone, and used for the subsequent analysis. The above operation was performed using RPMI-1640, 10% -heat-immobilized fetal bovine serum, and HT (hypoxanthine, thymidine) medium.
The obtained clones are shown in Table 1.

In Table 1, clones indicated as “3E10”, “7G10”, “8C1”, and “14A7” represent deposit clones having the following deposit numbers or receipt numbers, respectively.
“3E10”: NITE BP-01216
“7G10”: NITE BP-01217
“8C1”: NITE ABP-01737
“14A7”: NITE ABP-01738

GANP H5 monoclonal antibody specificity evaluation Evaluation of monoclonal antibodies for development of immunochromatographic diagnosis of highly pathogenic avian influenza virus H5 subtype <Method>
In this example, the reactivity of the monoclonal antibody prepared in Example 1 was evaluated.
The virus was evaluated by fluorescent immunostaining of infected cells of highly pathogenic and low pathogenic H5N1 viruses, and other strains were examined by ELISA using HA recombinant proteins.
・ Fluorescent immunostaining method: Infect cells with virus, fix PFA, react with antibody culture supernatant and observe stained image with fluorescence microscope.
ELISA method: H5HA recombinant protein was immobilized on a plate, and the antibody culture supernatant was reacted. An HRP-labeled anti-mouse IgG antibody (SouthernBiotech) was used as a secondary antibody, and the absorbance was measured after chemical color development.

<Evaluation results>
Of the 35 clones shown in Table 1, 10 clones showed a positive reaction to H5 virus Mongolia, and the others showed different reactivity depending on the clone.
Only 5H7-11-11-1 showed weak reactivity in H3N2 virus-infected cells (Table 2).
In Table 2, a circle (◯) means positive, and a cross (×) means negative.

Moreover, the dissociation constant of each antibody was measured using ProteON XPR36 (Bio-Rad). The results are shown in Table 3.

2. Evaluation of antibody combination on immunochromatography <Method>
Antibody combinations on immunochromatographic strips using 9 out of 10 clones listed in Table 2 and 11 clones of 7A5-1 and 29.18 clones (Balb / c-derived control clones) listed in Table 1 Evaluation was performed.
As antigens, Mongolia and Hokaido, which are highly pathogenic H5N1 viruses, and Vac3, which is a low pathogenic H5N1 virus, are used, and virus extracts made by Adtech Co. at concentrations of 1 × 10 ⁵ to 1 × 10 ⁷ (pfu / ml), respectively. Dissolved in and added. The positive line was visually evaluated after 15 minutes.

<Evaluation results>
As a result of examining combinations (90 ways) of each antibody clone and each virus strain, 7 kits showing strong positive reaction to Mongolia and showing weak reactivity to Hokaido, but solid-phased antibody and colloidal gold Selected as candidate antibody pairs with labeled antibodies (Table 4).
In Table 4, double circles (◎) represent a strong reaction, circles (○) represent equivalent reactions, triangles (△) represent weak reactions, and crosses (×) It means that it did not react.

2-1 Specificity Evaluation Using Recombinant HA Protein <Method>
Of the selected 7 kits, 4 highly reactive candidate kits were used to evaluate the specificity for the H5 subtype and other subtype recombinant HA proteins (Sino Biological Inc.). The evaluation method was made from 0 to 8 levels with no reactivity, and the positive intensity increased as the number increased. In addition, compared with the color sample, a strong reaction is indicated by a double circle (◎), a weak reaction is indicated by a triangle (Δ), and no reaction is indicated by a cross (×).

<Evaluation results>
It was No. 17 (solid phase 8C1-gold colloid labeled 5H7) that showed the widest reactivity with the H5 subtype.
Table 5 shows the H5 subtype specificity evaluation results using the candidate immunochromatography kit.
In Table 5, symbols such as circles and triangles are the same as described above. The number indicates the signal level when compared with the color sample.

In addition, Table 6 shows the evaluation results of the crossability using the candidate immunochromatography kit.

2-2 Recognition method of H5 subtype by immunochromatography using fluorescent labeling Fluorescent latex beads (Fujikura Kasei 3602-613) are labeled instead of colloidal gold for two highly reactive combinations. The signal was determined by a fluorescent immunochromatographic reader (Konica Minolta). A plurality of types of viruses of the H5 subtype were used as measurement targets.

Labeling method 0.3 mg of antibody was mixed with 1.3 ml of 0.4% fluorescent bead suspension and allowed to react at room temperature for 1 hour. After discarding the supernatant by centrifugation, 1% BSA solution was added and blocked for 1 hour. After discarding the supernatant by centrifugation, it was washed with 0.1% Trion X-100 solution at pH 7.7, suspended in 1% BSA solution at pH 7.7, and incubated at 37 ° C. for 48 hours. The fluorescent bead labeled antibody thus produced was used in place of the colloidal gold labeled antibody.

Immunochromatography method 100 μl of H5 subtype virus suspension (105 pfu / reaction) was dropped onto the strip and set in an immunochromatography reader. Positive reactions are measured at 15 and 20 minutes by immunochromatography reader. A signal / background ratio (S / B value) exceeding 1 was measured as a positive reaction.

Result No. 17 reacted to cover the Eurasian type H5 subtype, but the North American type H5 subtype could not be recognized (Table 7). On the other hand, no. 45 could not recognize part of the Eurasian type H5 subtype, but it could recognize the North American type (Table 7).
+ Indicates positive and-indicates negative.

2-3 H5 subtype minimum detection sensitivity test method using fluorescent immunochromatography The minimum detectable amount of virus was measured by the fluorescent immunochromatography method described above.
Immunochromatography Multi-diluted 100 μl of H5 subtype virus suspension was dropped onto the strip and set in an immunochromatography reader. Positive reactions are measured at 15 and 20 minutes by immunochromatography reader. A signal / background ratio (S / B value) exceeding 1 was measured as a positive reaction.
The S / B value was plotted against the amount of virus in the measurement limit multi-stage diluted state, and an approximate line was drawn. The intersection point with the S / B value = 1, which is the cutoff value, was calculated and used as the measurement limit.

Results The detection limit of each kit was as shown in Table 8. This sensitivity was several tens of times higher than that of the colloidal gold method.
The numbers in the table represent the minimum infectivity titer (pfu) required for human reaction.

2-4 Cross-reactivity test method for non-H5 subtype virus using fluorescent immunochromatography The cross-reactivity for non-H5 subtype virus was examined by the fluorescent immunochromatography method described above.
Immunochromatography Multi-diluted 100 μl of non-H5 subtype virus suspension (107 pfu / reaction) was dropped onto the strip and set in an immunochromatography reader. Positive reactions are measured at 15 and 20 minutes by immunochromatography reader. A signal / background ratio (S / B value) exceeding 1 was measured as a positive reaction.

Results Non-H5 subtype viruses were examined from H1 to H16 subtypes, but no crossing was observed. The high specificity of the method was shown (Table 9).
+ Indicates positive and-indicates negative.

2-5 Cross-reactivity test method for non-H5 subtype HA protein using fluorescent immunochromatography The cross-reactivity for non-H5 subtype virus-purified HA protein was examined by the fluorescent immunochromatography method described above.
Immunochromatography Multi-diluted 100 μl of non-H5 subtype virus suspension (107 pfu / reaction) was dropped onto the strip and set in an immunochromatography reader. Positive reactions are measured at 15 and 20 minutes by immunochromatography reader. A signal / background ratio (S / B value) exceeding 1 was measured as a positive reaction.

Results A number of non-H5 subtype HA proteins were examined, but no cross-reactivity was found. The high specificity of the method was shown (Table 10).
From the above, it was shown that the antibody of the present invention can diagnose all H5 subtypes depending on the combination.

Evaluation of neutralizing / inhibitory activity of GANP-Tg mouse-derived hybridoma clones against H5N1 influenza virus HA protein (1)
In this example, the neutralizing / inhibitory activity of the GANP-Tg mouse-derived hybridoma culture supernatant against the low pathogenic influenza virus H5N1-Vac3 strain was evaluated.
<Method>
The inhibition test for the influenza virus H5N1-Vac3 strain was performed according to the following procedure.
1) Three days before the assay, MDCK cells were seeded on a 6-well plate at 2 × 10 ⁵ cells / well.
2) 0.1 ml of influenza virus H5N1 (1 × 10 ³ PFU / ml) virus solution diluted with MEM medium containing 1% BSA and 0.1 ml of hybridoma culture supernatant diluted 100 times, 1000 times, and 10000 times Were mixed and neutralized at 37 ° C. for 1 hour.
3) 0.1 ml of a mixture of influenza virus solution and hybridoma culture supernatant was added to each well and spread throughout.
4) Adsorbed at 37 ° C. for 1 hour. The plate was shaken every 15 minutes.
5) The virus solution was removed and washed once with BSA (−) / MEM.
6) Hybridoma culture supernatant 1/100, 1/1000, 1/10000 amount added, 10 μg / ml acetyltrypsin, 0.8% agarose added, 1 ml BSA / MEM 2 ml was added to each well.
7) After agarose coagulation, the plate was turned upside down and placed in a CO ₂ incubator.
8) Cultured at 37 ° C. for 2 days.
9) 10% formalin was added. Fixed at room temperature for 1 hour.
10) Formalin and agarose medium were removed and washed with water.
11) Stained with 1% crystal violet for 1 hour.
12) The number of plaques was determined after washing and drying.

<Result>
FIG. 3 shows a photograph of the plate after culture. Compared to the negative control (no addition), 3E10, 10B9, 14A7 and 3B9 showed a clear difference in plaque size and number. The amount of virus used was confirmed by performing back titration (no addition), but it was a predetermined amount of virus.

Evaluation of neutralizing / inhibiting activity of GANP-Tg mouse-derived hybridoma clones against H5N1 influenza virus HA protein (2)
In this example, neutralization / inhibitory activity was evaluated using the same H5N1 clade (clade 2.2) highly pathogenic avian influenza virus as the antigen used for antibody production. Nine types (9 types in Table 2, excluding 12B1-26-31-13) that showed neutralizing activity in Example 2 or 3 or strong binding properties by immunofluorescent staining (IF) and ELISA In vitro evaluation of the neutralizing / inhibitory activity of the Tg mouse-derived hybridoma culture supernatant against the highly pathogenic avian influenza virus (H5N1A / whooper swan / Mongolia / 3/05) strain was performed.

<Method>
The experiment was conducted in the P3 facility. Viral infectivity was determined by plaque-forming ability.
An inhibition test against the influenza virus H5N1 A / whooper swan / Mongolia / 3/05 strain was performed according to the following procedure.
1) Three days before the assay, MDCK cells were seeded on a 6-well plate at 2 × 10 ⁵ cells / well.
2) 0.1 ml of influenza virus H5N1 (1 × 10 ³ PFU / ml) virus solution diluted with MEM medium containing 1% BSA and 0.1 ml of hybridoma culture supernatant diluted 100 times, 1000 times or 10000 times Were mixed and neutralized at 37 ° C. for 1 hour.
3) 0.1 ml of a mixture of influenza virus solution and hybridoma culture supernatant was added to each well and spread throughout.
4) Adsorbed at 37 ° C. for 1 hour. The plate was shaken every 15 minutes.
5) The virus solution was removed and washed once with BSA (−) / MEM.
6) Hybridoma culture supernatant 1/100, 1/1000, 1/10000 amount added, 10 μg / ml acetyltrypsin, 0.8% agarose added, 1 ml BSA / MEM 2 ml was added to each well.
7) After agarose coagulation, the plate was turned upside down and placed in a CO ₂ incubator.
8) Cultured at 37 ° C. for 3 days.
9) 10% formalin was added. Fixed at room temperature for 1 hour.
10) Formalin and agarose medium were removed and washed with water.
11) Stained with 1% crystal violet for 1 hour.
12) The number of plaques was determined after washing and drying.
After measuring the plaque forming value of H5N1 and determining the dilution conditions for forming 50-60 plaques per plate, 100 μl of the hybridoma culture supernatant was added to the plate under these conditions to neutralize the activity to prevent virus infection As measured.

<Result>
The results are shown in FIG.
In FIG. 4, the numbers described in the upper left of each plate indicate the number of plaques when the hybridoma culture supernatant (No. 1 to 4) is added at 10 ⁻² , 10 ⁻³ , and 10 ⁻⁴ dilution. The control group was Back titration (Back), and the virus solution used for neutralization value measurement was added in stock solution (1 time) and diluted 10 times.
In addition, as a control of the antiviral effect, Relenza (GlaxoSmithKline) was added at concentrations of 1 μM, 0.1 μM, and 0.01 μM, and it was confirmed that plaques could appear.
As shown in FIG. 4, four types of clones 3B9-11-6-9, 3E10-8-1-11, 10B9-6-3-31, 14A7-3-21-1 are highly pathogenic avian influenza. The neutralizing / growth inhibitory activity against the virus (H5N1 A / whooper swan / Mongolia / 3/05) strain was shown.

Evaluation of neutralizing / inhibiting activity of GANP-Tg mouse-derived hybridoma clones against H5N1 influenza virus HA protein (3)
In the present Example, it carried out using the highly pathogenic avian influenza virus of H5N1 clade (clade 2.3.2) different from the antigen used at the time of antibody preparation. Derived from 4 types of GANP-Tg mice, 3B9-11-6-9, 3E10-8-1-11, 10B9-6-3-31, 14A7-3-21-1, which showed neutralizing activity in Example 4 The neutralizing / inhibitory activity against the highly pathogenic avian influenza virus (H5N1 A / whooper swan / Hokkaido / 1/08) strain of hybridoma ascites was evaluated.

<Method>
An inhibition test against the influenza virus H5N1 A / whooper swan / Hokkaido / 1/08 strain was performed according to the following procedure.
1) Three days before the assay, MDCK cells were seeded on a 6-well plate at 2 × 10 ⁵ cells / well.
2) 0.1 ml of influenza virus H5N1 (1 × 10 ³ PFU / ml) virus solution diluted with MEM medium containing 1% BSA and 0.1 ml of hybridoma culture supernatant diluted 1000 times, 10000 times, and 100,000 times Were mixed and neutralized at 37 ° C. for 1 hour.
3) 0.1 ml of a mixture of influenza virus solution and hybridoma culture supernatant was added to each well and spread throughout.
4) Adsorbed at 37 ° C. for 1 hour. The plate was shaken every 15 minutes.
5) The virus solution was removed and washed once with BSA (−) / MEM.
6) Hybridoma culture supernatant 1/1000, 1/10000, 1/100000 was added, 10 μg / ml acetyltrypsin, 0.8% agarose and 1 ml BSA / MEM 2 ml were added to each well.
7) After agarose coagulation, the plate was turned upside down and placed in a CO ₂ incubator.
8) Cultured at 37 ° C. for 3 days.
9) 10% formalin was added. Fixed at room temperature for 1 hour.
10) Formalin and agarose medium were removed and washed with water.
11) Stained with 1% crystal violet for 1 hour.
12) The number of plaques was determined after washing and drying.

<Result>
As shown in FIG. 5, the 3B9-11-6-9 clone showed neutralization / growth inhibitory activity against the highly pathogenic avian influenza virus (H5N1 A / whooper swan / Hokaido / 1/08) strain.

Protective effect of HA protein / GANP-Tg mouse-derived hybridoma IgG against highly pathogenic avian influenza virus infection in mice In vitro test (Example 5), clone 3B9-11-6-9 was cloned into H5N1 A / whooper swan / Mongolia / 3/05 (clade2.2) strain and H5N1 A / whooper swan / Hokkaido / 1/08 strain (clade 2.3.2) neutralization and growth inhibitory activity against highly pathogenic avian influenza viruses were observed In this example, the therapeutic effect (in vivo test) on highly pathogenic avian influenza virus-infected mice was examined.

<Method>
Seven days before administration of the hybridoma, Crlj: CD1-Fxn1 nu / nu (7 weeks old) mice were treated with pristane, and after 7 days, 5 × 10 ⁶ hybridoma cells (3B9-11-6-9) were added to each of four mice. Intraperitoneal injection. Two weeks later, 35 ml to 50 ml of ascites was obtained and IgG antibody purification was performed. 12-30 mg IgG antibody was purified from ascites using HiTrap Protein G HP column.
H5N1 A / whooper swan / Hokaido / 1/08 (200 PFU / mouse; 3.3 MLD50) was used as the attacking virus, and Balb / c mice (8-10 weeks old, pupa) were used as animals. As a control group, a control vehicle treatment group uses the reagents used for antibody purification (93.5 mM Glycine-HCl, 65.4 mM Tris-HCl: PBS (−) = 0.835: 1.164), and a test antibody Used 8 animals in each group. In addition, 4 animals / group were necropsied 3 days after the infection, and 4 animals / group were necropsied at the end of the experiment.
Non-infected (diluted solution) inoculated group (4 mice per group) was used for monitoring body weight change. The antibody amount was 15 mg / kg / mouse, and were administered to Day 0 (immediately before virus infection) and Day 2 from the orbital vein nest. The method is shown in FIG.

<Result>
The results are shown in FIG.
FIG. 6 Panel B shows the change in body weight after challenge infection with influenza virus. In the infected mouse group administered with 14A7-3-21-1 clone (●) and the infected mouse group administered with Vehicle (（), significant weight loss was observed after 6 days of infection. In contrast, no decrease in body weight was observed in the infected mouse group (■) and the non-infected mouse group (♦) administered with the 3B9-11-6-9 clone.
FIG. 6 panel C shows the change in survival rate after challenge infection. On the 11th day of infection, the infected mouse group administered with 14A7-3-21-1 clone (●) had a survival rate of 25%, and the infected mouse group administered with Vehicle (▲) had a survival rate of 50%. In contrast, in the infected mouse group (■) and the non-infected mouse group (♦) to which the 3B9-11-6-9 clone was administered, no decrease in the survival rate was observed.
From the above results, the 3B9-11-6-9 clone having a wide binding ability to H5N1 highly pathogenic avian influenza virus showed a remarkable therapeutic effect even against highly pathogenic avian influenza virus infection.

Protective effect of HA protein / GANP-Tg mouse-derived hybridoma IgG against highly pathogenic avian influenza virus infection in mice In an in vitro test (Example 6), H3N1 A / whooper swan / Hokaido in 3B9-11-6-9 clone / 1/08 strain (clade 2.3.2) showed a therapeutic effect on highly pathogenic avian influenza virus. In this example, after infection with highly pathogenic avian influenza virus, 4, 6, Eight days later, the antibody was administered to examine the therapeutic effect on mice (in vivo test).

<Method>
Seven days before the administration of the hybridoma, Crlj: CD1-Fxn1 nu / nu (7 weeks old) mice were treated with pristane, and seven days later, 5 × 10 ⁶ hybridoma cells (3B9-11-6-9) were added to each of four mice. Intraperitoneal injection. Two weeks later, 35 ml to 50 ml of ascites was obtained and IgG antibody purification was performed. 12-30 mg IgG antibody was purified from ascites using HiTrap Protein G HP column.
H5N1 A / whooper swan / Hokaido / 1/08 (200 PFU / mouse; 3.3MLD50) was used as the attacking virus, and Balb / c mice (9 weeks old, pupa) were used as animals. As a control group, a control vehicle treatment group uses the reagents used for antibody purification (93.5 mM Glycine-HCl, 65.4 mM Tris-HCl: PBS (−) = 0.835: 1.164), and a test antibody Used 5 animals in each group. Surviving animals were necropsied at the end of the experiment.
Non-infected (diluted solution) inoculated group (4 mice per group) was used for monitoring body weight change. The amount of antibody was 15 mg / kg / mouse, and it was administered to Day 0, Day 4, Day 6, and Day 8 from the orbital vein nest.

<Result>
The results are shown in FIG.
FIG. 7 is a diagram showing the results of studying the therapeutic effect of anti-HA monoclonal antibody administration (BALB / crice (Female, 9-week-old), H5N1 HPAIV 300 PFU, 15 mg / kg 3B9 administration group).
FIG. 7 panel A shows the change in body weight after challenge infection with influenza virus. In the infected mouse group (●) in which the 3B9-11-6-9 clone was administered to Day 8 and the non-administered mouse group (□), significant weight loss was observed after 6 days of infection. In contrast, no decrease in body weight was observed in the infected mouse group (Δ) and the non-infected mouse group (♦) in which the 3B9-11-6-9 clone was administered to Day 0. In the group of infected mice in which the 3B9-11-6-9 clone was administered to Day 4 (）) and Day 6 (）), a temporary decrease in body weight was not observed.
FIG. 7 panel B shows the change in survival rate after challenge infection. On the 14th day of infection, an infected mouse group (●) administered to the infected mouse group Day0 (Δ), Day4 (（), Day6 (◯), and Day8 administered with the 3B9-11-6-9 clone is shown. The survival rates were Day 4 (（) 75%, Day 6 (（) 50%, and Day 8 (●) 0%. In contrast, in the infected mouse group (■) and the non-infected mouse group (♦) in which the 3B9-11-6-9 clone was administered to Day 0 (Δ), no decrease in the survival rate was observed.
From the above results, the 3B9-11-6-9 clone having a wide binding ability to H5N1 highly pathogenic avian influenza virus shows a remarkable therapeutic effect even after administration after infection with highly pathogenic avian influenza virus. It was.

According to the present invention, a high-affinity monoclonal antibody (KD = 1 × ^{10 to 10} M level) capable of recognizing all of Indonesian strains, Anhui strains, and Vietnamese strains that are highly toxic influenza is provided. The antibody of the present invention makes it possible to produce an ultrasensitive diagnostic kit. Further, since it is effective not only for detection of viruses of various clades but also for inhibition of virus growth, it is extremely useful for medical and clinical examinations.

The hybridomas “3E10 (rVV cl2.2)” and “7G10 (rVV cl2.2)” are both registered by the National Institute of Technology and Evaluation for Microorganisms (Kazusa Kamashizu, Kisarazu City, Chiba Prefecture 292-0818). -5-8 Deposited internationally at NITE Biotechnology Headquarters (Patent Microorganism Deposit Center) based on the Budapest Treaty (original deposit date: February 1, 2012).
The accession number is NITE BP-01216 for “3E10 (rVV cl2.2)” and NITE BP-01217 for “7G10 (rVV cl2.2)”.
Hybridomas “8C1 (rVV cl2.2)” and “14A7 (rVV cl2.2)” were both internationally deposited at the above-mentioned patent microorganism deposit center based on the Budapest Treaty (original deposit date: October 18, 2013) .
The receipt number is NITE ABP-01737 for “8C1 (rVV cl2.2)” and NITE ABP-01738 for “14A7 (rVV cl2.2)”.

SEQ ID NO: 1 synthetic DNA
SEQ ID NO: 2: Synthetic DNA
[Sequence Listing]

Claims (27)

An antibody against the HA protein of influenza virus having a dissociation constant of at least 1 × 10 ⁻¹⁰ M. The antibody according to claim 1, wherein the dissociation constant is 1 × 10 ⁻¹⁰ M to 1 × 10 ⁻¹³ M.   The antibody according to claim 1, wherein the influenza virus is an influenza H5 subtype virus.   The antibody according to claim 3, wherein the influenza H5 subtype virus is an influenza H5N1 virus.   Influenza H5N1 virus is found in Indogan Qinghai Lake strain (clade 2.2), Indonesian strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolian strain (A / whooper swan / Mongolia) The antibody according to claim 4, which is at least one selected from the group consisting of / 3/2005: clade 2.2) and Hokkaido strain (A / whose swan / Hokaido / 1/2008: clade 2.3.2). .   The antibody according to claim 1, wherein the antibody is a monoclonal antibody.   7. The antibody of claim 6, wherein the antibody is produced according to a deposit number of NITE BP-01216 or NITE BP-01217, or a receipt number of NITE ABP-01737 or NITE ABP-01738.   The antibody according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody or a humanized antibody.   An antibody that binds to a site to which the antibody of claim 1 binds.   The antibody fragment according to any one of claims 1 to 9.   A hybridoma producing the monoclonal antibody according to claim 6.   A hybridoma whose accession number is NITE BP-01216 or NITE BP-01217, or whose reception number is NITE ABP-01737 or NITE ABP-01738. The method for producing an antibody according to claim 1, comprising the following steps:
(A) immunizing a non-human mammal with an influenza virus HA protein expressed using a recombinant vaccinia virus, and (b) collecting the antibody from the non-human mammal. . It is a manufacturing method of the monoclonal antibody of Claim 6 or 7, Comprising: The following processes:
(A) immunizing a non-human mammal with an influenza virus HA protein expressed using a recombinant vaccinia virus;
(B) collecting antibody-producing cells from the immunized non-human mammal;
(C) The method comprising the steps of fusing the antibody-producing cells obtained in step (b) and myeloma cells, and (d) collecting the antibody from the fused cells obtained in step (c).   The method according to claim 13 or 14, wherein the non-human mammal is a GANP® transgenic non-human mammal.   A method for detecting influenza virus, comprising detecting the influenza virus by bringing the antibody according to any one of claims 1 to 9 or the fragment according to claim 10 into contact with a biological sample.   The method according to claim 16, wherein the influenza virus is an influenza H5 subtype virus.   The method according to claim 17, wherein the influenza H5 subtype virus is an influenza H5N1 virus.   Influenza H5N1 virus is found in Indogan Qinghai Lake strain (clade 2.2), Indonesian strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolian strain (A / whooper swan / Mongolia) The method according to claim 18, which is at least one selected from the group consisting of: / 3/2005: clade 2.2) and Hokkaido strain (A / whose swan / Hokaido / 1/2008: clade 2.3.2). .   An influenza virus detection reagent comprising the antibody according to any one of claims 1 to 9 or the fragment according to claim 10.   The reagent according to claim 20, wherein the influenza virus is an influenza H5 subtype virus.   The reagent according to claim 21, wherein the influenza H5 subtype virus is an influenza H5N1 virus.   Influenza H5N1 virus is found in Indogan Qinghai Lake strain (clade 2.2), Indonesian strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolian strain (A / whooper swan / Mongolia) The reagent according to claim 22, wherein the reagent is at least one selected from the group consisting of / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokkaido / 1/2008: clade 2.3.2). .   The pharmaceutical composition for anti-influenza viruses containing the antibody of any one of Claims 1-9, or the fragment | piece of Claim 10.   The pharmaceutical composition according to claim 24, wherein the influenza virus is an influenza H5 subtype virus.   26. The pharmaceutical composition according to claim 25, wherein the influenza H5 subtype virus is an influenza H5N1 virus.   Influenza H5N1 virus is found in Indogan Qinghai Lake strain (clade 2.2), Indonesian strain (clade 2.1), Anhui strain (clade 2.3), Vietnam strain (clade 1), Mongolian strain (A / whooper swan / Mongolia) The pharmaceutical according to claim 26, which is at least one selected from the group consisting of / 3/2005: clade 2.2) and Hokkaido strain (A / whooper swan / Hokaido / 1/2008: clade 2.3.2). Composition.