JPH06100594A - Anti-human influenzal virus antibody - Google Patents

Abstract

PURPOSE:To obtain anti-human influenzal virus antibody and an immunogenic artificial polypeptide. CONSTITUTION:The objective anti-human influenzal virus antibody is capable of recognizing the truncal region in a hemagglutinin molecule of human influenzal A type viruses H1N1 and H2N2 and having the neutralizing activity but unrecognizing the truncal region of H3N2 and having no neutralizing activity. Furthermore, the objective immunogenic artificial polypeptide has substantially the same antigenicity as that of the truncal regions of the H1N1 and H2N2. This antibody is useful for diagnosing, treating, etc., the A type virus and the polypeptide is useful as a vaccine.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody against hemagglutinin of human influenza A virus, and a polypeptide containing an antigenic site recognized by the antibody.

[0002]

BACKGROUND OF THE INVENTION There are three types of influenza virus (A, B and C), which cause a worldwide pandemic of influenza, and many deaths are caused by human influenza A virus. Influenza A virus is further classified into many subtypes according to the antigenicity of viral surface proteins haemagglutinin (hereinafter abbreviated as HA) and neuraminidase (hereinafter abbreviated as NA), and H1N1 is a human influenza A virus. Three types are currently known: subclass, H2N2 subclass, and H3N2 subclass. HA of this influenza A virus is a globular region (
It is composed of two different regions, a head region) and a stem region, and the globular region contains a receptor binding site for the virus to bind to target cells.
A is involved in the hemagglutination activity of A, while the stem region contains a fusion peptide required for membrane fusion between the viral envelope and the endosomal membrane of cells and is involved in the fusion activity [Wiley et al., Annual. Review of Biochemistry (Ann. Rev. Biochem.), Volume 56,
365-394 (1987)]. The anti-HA antibodies conventionally obtained as anti-HA antibodies that recognize the H1N1 subtype and H2N2 subtype all recognize the globular region of HA. However, this region is the site where antigenic variation is most likely to occur, and therefore these antibodies do not commonly react with subtypes of human influenza A virus, and with the antigenic change of HA of the virus, It loses recognizability. On the other hand, Green et al. Synthesized a polypeptide from the amino acid sequence of the stem region of one HA of the H3N2 subtype, and obtained an antibody against this polypeptide. Japanese activity is weak (Table 59-50
1714), and the polypeptide itself used as an antigen did not react with rabbit antiviral sera obtained by immunization with H3N2 subtype, and there was also a problem in terms of antigenicity [Cell, No. 28, pp. 477-487 (1982)].

[0003]

[Problems to be Solved by the Invention] Human influenza A
Type virus periodically changes the type of HA and NA, causing a pandemic, and even if vaccinated before the winter season, when the influenza is outbreak, another type of virus causes an influenza pandemic, so the effect of the vaccine Can often not be expected. However, in the HA molecule and NA molecule, an antigenic site that is common to the virus subtypes and is unlikely to cause antigenic mutation,
In particular, if an antibody that recognizes a three-dimensional structure and has virus-neutralizing activity can be obtained, this antibody can be used for diagnosis, prevention, and treatment of the onset of infection by type A virus, and the antigen site itself can also be used. , Will be useful as a vaccine. An object of the present invention is to provide an antibody having cross-recognizing ability for influenza A virus subtypes and having virus neutralizing activity, and an antigenic site polypeptide that can be used as a vaccine.

[0004]

Means for Solving the Problems To outline the present invention, the first invention of the present invention relates to an anti-human influenza virus antibody, which is characterized by having the following characteristics (a) and (b). (A) Recognizing the stem regions of the H1N1 and H2N2 subtype HA molecules of the human influenza A virus, but not the H3N2 subtype HA molecule stem regions. (B) H3N3 has a neutralizing activity on H1N1 and H2N2 subtypes of human influenza A virus.
N2 subtype has no neutralizing activity. A second invention of the present invention relates to an immunogenic artificial polypeptide, which is characterized by having substantially the same antigenicity as the stem region in the HA molecule of H1N1 subtype and H2N2 subtype of human influenza A virus. To do.

As a result of earnest studies, the present inventors have found that the H1N1 subtype and H2N of human influenza A virus are
Antibodies against the commonly conserved antigenic sites in the stem regions of the two subtypes were identified as H1N1 subtype and H2
It was found that the antibody has a strong neutralizing activity against N2 subtype virus, and that the antibody is extremely useful for treatment and prevention of influenza, and that the polypeptide containing the antigenic site is extremely useful as a vaccine. Completed the invention.

Human influenza A virus H1N
The antibody of the present invention that recognizes the common site in the HA molecular stem region of the 1 subtype and the H2N2 subtype and neutralizes the virus can be prepared as a monoclonal antibody as follows. For example, a virus particle selected from the following antigens, H1N1 subtype or H2N2 subtype, H1
As the N1 subtype, for example, A / Bangkok / 10/8
3, A / Yamagata / 120/86, A / Osaka / 930
/ 88, A / Suita / 1/89 (above, preserved strain of the Institute for Microbial Diseases, Osaka University), A / PR / 8/34 [Influenza (H1N1), ATCC VR-95], A1 / FM
/ 1/47 [Influenza A (H1N1), ATCC
VR-97], A / New Jersey / 8/76 [Influenza A (H1N1), ATCCVR-897], A /
NWS / 33 [Influenza A (H1N1), ATC
CVR-219], A / Weiss / 43 [influenza A (H1N1), ATCCVR-96], A / WS / 3
3 [Influenza A (H1N1), ATCC VR-
825], and as the H2N2 subtype, for example, A / Okud
a / 57, A / Adachi / 2/57, A / Kumamoto / 1 /
65, A / Kaizuka / 2/65, A / Izumi / 5/65
(Above, Osaka University Research Institute for Microbial Diseases), A2 / Japa
n / 305/57 [Influenza A (H2N2), A
TCC VR-100], or an HA molecule obtained from these viruses, or an HA polypeptide prepared by using a gene recombination technique, or a recognition site of the antibody of the present invention, that is, an antigen site of the stem region of the HA molecule. Immunization with a recombinant polypeptide contained in the molecule, or a synthetic polypeptide containing the site in the molecule, the resulting spleen cells are fused with, for example, mouse myeloma cells, and from the resulting hybridoma, It has binding and neutralizing activity to H1N1 and H2N2 subtype viruses, and has H3N2 subtype viruses such as A / Fukuoka / C29 / 85, A / Sichuan / 2 /
87, A / Ibaraki / 1/90, A / Suita / 1/90
(Above, preserved strain of the Institute for Microbial Diseases, Osaka University), A / Port C
halmers / 1/73 [Influenza A (H3N2),
ATCC VR-810], A2 / Aichi / 2/68
[Influenza A, ATCC VR-547], and B-type viruses such as B / Nagasaki / 1/87 (Osaka University Research Institute for Microbial Diseases), B / Allen / 45 [influenza B, ATCC VR-102] Has no binding and neutralizing activity, and H1N1 subtype and H2
Select antibody-producing cells that recognize the N2 subtype HA molecule, but do not inhibit the hemagglutination activity involving the globular region in the HA molecule, but inhibit the membrane fusion activity involving the stem region in the HA molecule. , Can be prepared by culturing the cells.

The preparation of this hybridoma is carried out based on Nature, 256, 495-497 (1975). Balb for immunization
/ C mouse, Balb / c mouse and other mouse F1
A mouse or the like is used. Immunization against one mouse
For example, virus particles (100 to 1000 HA units) are used as an antigen, and the treatment is performed, for example, three times in 2 to 5 months. The breeding of mice and the collection of splenocytes follow conventional methods.

As a myeloma cell, SP2 / 0-Ag
14 (ATCC CRL1581), p3 x 63Ag8
U. 1 (ATCC CRL1597), p3 x 63Ag
8 (ATCC TIB9), p3 × 63-Ag8.65.
3 (ATCC CRL1580) and the like are preferably used. Spleen cells and myeloma cells were mixed at a ratio of 1: 1 to 10: 1, and fusion was performed using NaCl (about 0.85%), dimethyl sulfoxide [10 to 20% (v / v)] and polyethylene having a molecular weight of 1000 to 6000. This is done by incubating the mixture of both cells in a phosphate buffer (pH 7.2-7.4) containing glycol at 35-37 ° C for 1-5 minutes. The fused cells are selected using HAT medium as the growing cells. Cloning of the fused cells is repeated at least 3 times by the limiting dilution method.

When the hybridoma is cultured in the same manner as ordinary animal cells, the antibody of the present invention can be obtained in the medium as a result. In addition, the antibody of the present invention can be accumulated in ascites by transplanting the hybridoma into the abdominal cavity of a pristane-treated nude mouse or Balb / c mouse and proliferating it. That is, 0.5 to 1 mg of pristane was inoculated into the abdominal cavity of these mice, and 5 to 10 ⁶ to 1 × 10 ⁷ hybridomas were transplanted to the abdominal cavity of the mice 2 to 3 weeks thereafter. Ascites usually accumulates after 7 to 10 days and is collected. Monoclonal antibodies in culture and ascites fluid are purified by conventional means.

The obtained monoclonal antibody neutralizes the virus by recognizing the stem regions of HA molecules of H1N1 subtype and H2N2 subtype and inhibiting the membrane fusion activity of the virus. It has properties.

(A) By a staining test, MDCK infected with H1N1 subtype and H2N2 subtype respectively
It recognizes cells (ATCC CCL34) and not MDCK cells infected with the H3N2 subtype. In the staining test, four kinds of antibodies (monoclonal antibody of the present invention, rabbit anti-mouse immunoglobulin G serum, goat anti-rabbit immunoglobulin G serum, peroxidase-rabbit antiperoxidase complex) were used, and Journal of Clinical Microbiology (J. Clin. Microbiol),
Vol. 28, pp. 1308 to 1313 (1990).

(B) By immunoprecipitation method, HA molecules of H1N1 subtype and H2N2 subtype are recognized, and H3
It does not recognize HA molecules of the N2 subtype.

(C) In the hemagglutination test, the hemagglutination activity of each of the H1N1 subtype, H2N2 subtype and H3N2 subtype is not inhibited.

(D) Recognizing the common conserved region unique to the stem region in the H1N1 subtype and H2N2 subtype HA molecules, which is identified by genetic analysis encoding the HA molecule, It does not recognize the common storage area specific to the trunk area.

The gene encoding the HA molecule is analyzed as follows. MDCK cells are infected with virus particles, the infected cells are collected the next day, and viral RNA is extracted from the cells using guanidine isothiocyanate. Next, H1
N-, H2N2, H3N2 subtype (-) chain RN
An oligonucleotide primer containing a sequence complementary to the 3'end of A, for example, primer 5 represented by SEQ ID NO: 5 in the sequence listing was prepared, and cDNA was prepared using the primer.
To synthesize. To amplify the cDNA, H1N1,
An oligonucleotide primer containing a sequence complementary to the 3'end of the (+) strand RNA of each of H2N2 and H3N2 subtypes, for example, primer 6 represented by SEQ ID NO: 6 in the sequence listing was prepared, and the primer and primer 5 PCR using cDNA (Polymerase chain reaction)
The method enables efficient amplification. Amplified DNA
The HA gene of about 1.7 kbp therein was separated by agarose gel electrophoresis, and then, for example, using primers 5 and 6, a second P
CR is performed, and the amplified DNA is centrifuged using 20% (w / v) polyethylene glycol 6000 / 2.5M NaCl to obtain a purified precipitate fraction. Next, a sequence primer selected from the HA gene sequences of each subclass of virus was prepared, and the primer was [γ- ³² P] ATP.
After labeling with the labeled primer, the labeled fraction was annealed with the above-mentioned purified fraction, and the dideoxy method [BioTechniques, 9th method] using a thermal cycler was used.
Vol. 66-72 (1990)].

For example, the primers 7 to 14 represented by SEQ ID NOS: 7 to 14 of the sequence listing are sequence primers for the H1N1 subtype, and SEQ ID NO: 1 of the sequence listing
Primers 15-23 represented by 5-23 are H
A sequence primer for 2N2 subtype,
Primers 24 to 26 represented by SEQ ID NOs: 24 to 26 in the sequence listing are sequence primers for H3N2 subtype. In addition, by using the primers 9 and 13 as PCR primers and the primers 11 and 12 as sequence primers, H1
A part of the gene encoding the stem region of the HA molecule of N1 subtype is efficiently amplified and analyzed. Also, PCR
By using the primers 17 and 21 as the primers for use and the primers 19 and 20 as the sequence primers, a part of the gene encoding the stem region of the HA molecule of the H2N2 subtype is efficiently amplified,
Parsed. Further, by using the primers 24 and 26 as PCR primers and the primers 25 and 26 as sequence primers, H3N
A part of the gene encoding the stem region of the HA molecule of two subtypes is efficiently amplified and analyzed.

The common conserved region in HA molecules among virus subtypes is represented by SEQ ID NO: 1 in the sequence listing of the stem region in HA molecules of H1N1 subtype and H2N2 subtype found by the present inventors. And the GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the sequence listing. FIG. 1 is a schematic diagram of the tertiary structure of the HA molecule [Wiley et al., Nature, No. 289].
Vol., Pp. 373-378 (1981)]. A in the figure
Both polypeptide sequences shown in the region and the B region are located close to each other in the center of the stem region of the HA molecule as shown in FIG. 1, and the monoclonal antibody C179 which is an example of the antibody of the present invention.
Is the TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing of the stem region in the HA molecule and SEQ ID NO: 2 in the sequence listing.
The GITNKVNSVIEK polypeptide sequence represented by

(E) In the neutralizing activity test, it inhibits the plaque forming ability or focus forming ability of the H1N1 subtype and H2N2 subtype, but does not inhibit the plaque forming ability or focus forming ability of the H3N2 subtype. The neutralizing activity test is performed by the pull reduction neutralization test or the influenza virus rapid focus reduction neutralization test described in the above-mentioned Journal of Clinical Microbiology. That is, mixing the antibody and virus,
After incubating for a certain period of time, MDCK cells were infected, and the neutralizing ability was judged by the decrease of appearance of plaques or foci.

(F) In the fusion activity test, it inhibits the membrane fusion activity of H1N1 subtype and H2N2 subtype, but does not inhibit the membrane fusion activity of H3N2 subtype. Fusion activity test is Nature, Volume 300, 658-65
Follow the method described on page 9 (1982). That is, CV
-1 cells (ATCC CCL70) were infected with the virus, then treated with an antibody, and the ability to inhibit fusion activity was determined by the presence or absence of polykaryon formation.

The antibody according to the present invention binds to the stem region in the HA molecule, inhibits the membrane fusion activity of H1N1 subtype and H2N2 subtype, and strongly neutralizes the infectivity of the virus strain. Therefore, the antibody according to the present invention can be used for the prevention and treatment of influenza by H1N1 subtype and H2N2 subtype. At this time, the antibody may be usually administered to an adult in an amount of about 0.5 to 5000 mg, preferably 5 to 500 mg. The antibody of the present invention is formulated by mixing with a commonly used excipient, physiological saline, glucose solution, mannitol, methylcellulose, zetillan, and the like. This agent can be a lyophilized product, which can be redissolved immediately before use with an isotonic solution such as physiological saline, 5% glucose solution, Ringer's solution or the like. Further, for humans, the antibody of the present invention is preferably chimerized and used as a chimeric antibody which is difficult to be recognized as a foreign substance in the human body, and is used as an artificial antibody in which only an antigen recognition site is transplanted to a human type antibody. It is suitable.

Further, the antibody-inducing polypeptide of the present invention can be used as a vaccine for influenza. That is, when a polypeptide having substantially the same antigenicity as the stem region in HA molecules of H1N1 subtype and H2N2 subtype is used as an immunogen, H1N1
It is possible to prevent or treat subtype and H2N2 subtype influenza outbreaks. The immunogenic polypeptides include H1N1 subtypes, HA molecules prepared from H2N2 subtypes, HA polypeptides prepared by gene recombination techniques, and stem regions prepared by enzymatic degradation of HA molecules or HA polypeptides. A polypeptide or the like can be used. In addition, TGLR represented by SEQ ID NO: 1 in the sequence listing is formed in the molecule by gene recombination technology or chemical synthesis method.
N polypeptide sequence and represented by SEQ ID NO: 2 in the sequence listing
Preparing a polypeptide having a GITNKVNSVIEK polypeptide sequence, the configuration of which is a configuration in a natural HA molecule having substantially the same antigenicity as these sequences,
You may use it. For example, the HA gene is prepared from viral RNA, the gene is cleaved with a restriction enzyme, the vector is integrated into a vector, and the gene is expressed in animal cells such as CV-1 cells, whereby the desired polypeptide can be obtained.

When these polypeptides are used as vaccines, their antigenicity can be increased by selecting an appropriate carrier. As the carrier, for example, albumin, polyamino acid, etc. may be used. The vaccine of the present invention may be administered by a conventional active immunization method, that is, a prophylactically or therapeutically effective and immunogenic amount is administered once or multiple times in a manner compatible with the dosage formulation. In addition, the vaccine may be formulated as usual, and may contain an adjuvant for improving the immune response.

[0023]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of virus A / PR / 8/34 and A / Ba as H1N1 subtypes
ngkok / 10/83, A / Yamagata / 120/86, A
/ Osaka / 930/88, A / Suita / 1/89, Al
/ FM / 1/47, A / Okud as H2N2 subtype
a / 57, A / Adachi / 2/57, A / Kumamoto / 1 /
65, A / Kaizuka / 2/65, A / Izumi / 5/6
5, A2 / Aichi / 2/6 as H3N2 subtype
8, A / Fukuoka / C29 / 85, A / Sichuan / 2 /
87, A / Ibaraki / 1/90, A / Suita / 1/9
B / Nagasaki / 1/87 was used as the 0 type and B type viruses, and each of them was inoculated into the allantoic cavity of 11-day-old embryonated chicken eggs and cultured at 34 ° C. for 4 days, and then each virus was collected.

Example 2 Preparation of Monoclonal Antibody (1) A / Okuda / 57 virus (320 HA units) prepared in Example 1 was suspended in Freund's complete adjuvant before use, and at 1-month intervals, Balb / c. The mice were immunized twice by intraperitoneal injection, and one month later, the same antigen (32
(0 HA unit) in PBS was used for boosting by intraperitoneal injection. Three days later, the spleen was removed from the mouse to prepare splenocytes. P3 x 63Ag for mouse myeloma
8 was cultured in DME medium supplemented with 10% fetal bovine serum for 2 days after passage, and washed with physiological saline before cell fusion to prepare. Next, splenocytes and myeloma cells were mixed at a cell ratio of 1: 5, centrifuged to remove the supernatant, and the precipitated cell mass was thoroughly loosened, and then stirred to mix 1 ml of a mixed solution [polyethylene glycol- 4000 (2g), MEM
(2 ml), dimethyl sulfoxide] and added for 5 minutes 37
After keeping the temperature at 0 ° C, MEM was slowly added so that the total amount of the solution became 10 ml. Next, after centrifugation, the supernatant was removed and the cells were loosely loosened. Normal medium [PRMI-
1640 to which 10% fetal bovine serum was added] 30 ml was added, and the cells were gently suspended using a measuring pipette.

Dispense the suspension into a 96-well culture plate,
The cells were cultured at 37 ° C. for 24 hours in an incubator containing 5% CO ₂ . Next, HAT medium was added and the cells were cultured for 10 to 14 days. Subsequently, a part of the culture supernatant was collected and a hybridoma was screened.

(2) In order to obtain a monoclonal antibody that cross-reacts among influenza A virus subtypes, the above-mentioned culture supernatant that has not been diluted is used as a primary antibody, and three subtypes (H1N1, H2N2, H3N
A staining test of MDCK cells infected with each of 2) was performed. The dyeing test was performed according to the method described in the above-mentioned Journal of Clinical Microbiology. That is, 96-well microtiter plate (Falcon 30
72: Becton Dickinson), each subtype strain of human influenza A virus (H1N1: A)
/ Yamagata / 120/86, H2N2: A / Okuda / 5
7, H3N2: A / Fukuoka / C29 / 85) -infected MDCK cells were washed with PBS (pH 7.4),
Immobilize with anhydrous ethanol at room temperature for 10 minutes, and then fix these with 4 kinds of antibodies [the above-mentioned culture supernatant containing a monoclonal antibody, 1000-fold diluted solution of rabbit anti-mouse immunoglobulin G serum (manufactured by Organotechnica), goat 5 of anti-rabbit immunoglobulin G serum (manufactured by Organotechnica)
A 00-fold diluted solution and a 1000-fold diluted solution of peroxidase-rabbit anti-peroxidase complex (manufactured by Organotechnica) were continuously reacted for 40 minutes each, and washed with PBS. Finally, the peroxidase reaction was performed by the method of Graham, Karnovsky, using 0.01% H ₂ O ₂ and 0.3 mg / ml 3,3'-diaminobenzidine tetrahydrochloride in PBS. of
Histo Chemistry and Sight Chemistry (J. Histochem. Cytochem.), Volume 14, 291-
302 (1966)]. The stained cells were observed under a normal light microscope to show that the H1N1 subtype-infected M
Antibodies that respectively recognize DCK cells and MDCK cells infected with H2N2 subtype were selected. Next, the cells in the hole in which the cells confirmed to produce the antibody are taken out, and the limiting dilution method is performed 3 times to clone the target cells, and the cloned hybridoma strain is hybridoma C17.
9. The monoclonal antibody produced by the hybridoma was designated as monoclonal antibody C179.

(3) Balb / c mice treated with pristane were intraperitoneally administered with the above hybridoma strain at 5 × 10 ⁶ mice / mouse. After 10 to 21 days, ascites was collected from the mouse in which ascites cancer was induced, and solid components were removed by centrifugation at 3000 rpm for 5 minutes to prepare an ascites fluid. About 1 mg of the monoclonal antibody C179 (hereinafter simply abbreviated as C179) was contained in 1 ml of ascites fluid. C179
Was an IgG2a type antibody purified with Protein A-Sepharose 4B (Pharmacia).

Example 3 Properties of Monoclonal Antibody (1) A 100-fold dilution of the ascites fluid described in Example 2- (3) was serially diluted, and the staining test described in Example 2- (2) was performed. The antigen recognition was examined. A / PR / 8/34, A / Bangkok / 10 as H1N1 subtype
/ 83, A / Yamagata / 120/86, A / Osaka / 9
30/88, A / Suita / 1/89, H2N2 subtype as A / Okuda / 57, A / Adachi / 2/57,
A / Kumamoto / 1/65, A / Kaizuka / 2/65, A
/ Izumi / 5/65, A / Ai as H3N2 subtype
chi / 2/68, A / Fukuoka / C29 / 85, A / Si
chuan / 2/87, A / Ibaraki / 1/90, A / Suita
/ 1/90, and B / Nagasaki / 1/87 was used as the influenza B virus. C179 recognizes all H1N1 and H2N2 subtypes,
The H3N2 subtype and B virus were not recognized.

(2) The neutralizing activity of the antibody was measured by the rapid focus reduction neutralization test of influenza virus as described above in the archives of virology (Arch. Virol.
), 86, 129-135 (1985), Microbiology and Immunology (Microbiol. I).
mmunol.), 29, 327-335 (198).
It carried out according to 5). As the ascites fluid of Example 2- (3) was used as the antibody, and the antibody was added with a 3-fold volume of a solution of a receptor-degrading enzyme (RDE: Takeda Pharmaceutical Co., Ltd.) before use.
After reacting at ℃ for 18 hours, heat treatment at 56 ℃ for 45 minutes
DE was inactivated and finally prepared as a 16-fold dilution of ascites fluid, which was used as a test fluid, and the measurement was carried out as follows.

That is, the 96-well microplate has MDC
10 ⁴ K cells / well were dispensed, and the next day, the antibody (16
4-fold diluted solution (double-diluted solution) and each virus solution described in Example 3- (1) prepared in 30 focus forming units / hole were mixed in equal amounts and incubated at 37 ° C. for 1 hour. Next, 25 μl of this mixed solution was dispensed into each well of the microtiter plate containing the MDCK cells, and the mixture was kept at 37 ° C. for 30 minutes.
I kept it warm. Next, remove the solution in each well, wash each well with PBS, and then add 0.5% tragacanth gum (Wako Pure Chemical Industries).
And MEM containing 5 μg / ml trypsin was added. After incubating at 37 ° C. for 20 to 24 hours, the additive solution was removed, each well was washed with PBS, and the cells were treated with absolute ethanol at room temperature for 10 minutes to fix the cells. Next, it was dried and cells were stained according to the staining test described in Example 2- (2). After staining, wash the cells with tap water, dry,
The number of stained foci was counted under an optical microscope.

C179 inhibited the focus formation of all H1N1 and H2N2 subtypes and showed strong virus neutralizing activity, while it did not affect the focus formation of H3N2 subtype and B virus. In addition, similar results were shown in the pull reduction neutralization test.

(3) The hemagglutination inhibitory activity (HI) of the antibody was measured as follows. An RDE-treated antibody (32-fold dilution) was used in the same manner as in Example 3- (2) to prepare a serial dilution, and then each virus (16HA) described in Example 3- (1) was prepared.
Unit) and allowed to react for 30 minutes at room temperature. afterwards,
The effect of the antibody on the hemagglutination activity of each virus was examined by adding chicken erythrocytes and mixing them well. C179 did not affect the hemagglutinating activity of all subtype viruses.

(4) The fusion inhibitory activity of an antibody is measured by the above-mentioned Nature, Vol. 300, pp. 658-659 (198).
The method of 2) was modified and performed. That is, a monolayer culture of CV-1 cells was infected with each virus strain described in Example 3- (1). 24 hours later,
Wash twice with MEM, then add trypsin (10 μg /
(ml) in DMEM for 15 minutes at 37 ° C. Next, the cells were washed twice with DMEM, then a DMEM dilution of the ascites fluid of Example 2- (3) was added, and the mixture was incubated at 37 ° C for 30 minutes. Thereafter, cells, fusion medium was adjusted to pH 5.0 (Na ₂ CO ₃ with no additive PRMI, 0.2% bovine serum albumin, 10 mM MES, 10 mM HEPES) at, 37 ° C., and treated for 2 minutes, followed by After washing twice with DMEM and removing the fusion medium, the mixture was incubated with DMEM containing 2% fetal bovine serum at 37 ° C. for 3 hours. Next, the cells were fixed with anhydrous methanol and stained with Giemsa, and then the presence or absence of polykaryon formation was determined by an optical microscope. C179 blocked polykaryon formation of all H1N1 and H2N2 subtypes, but not H3N2 and B virus polykaryon formation.

As mentioned above, C179 is H1N1 subtype, H
The antibody specifically recognized the 2N2 subtype, inhibited viral membrane fusion, and exhibited neutralizing activity. The results are shown in Table 1.

[0036]

[Table 1]

The numbers in the table are the dilution factors of the ascites fluid of Example 2- (3), and the staining titer was the maximum dilution factor of the ascites fluid capable of staining cells in the staining test, and the neutralization activity was a control. The maximum dilution factor of the ascites fluid that can suppress the appearance of foci is shown up to half of the number of foci in the antibody-free section of. Further, + means that polykaryon formation was completely inhibited by the antibody solution of the ascites fluid diluted 1000 times, and − means that no inhibition was observed even when the 10 times diluted ascites fluid was used. means. In addition, no HI activity was observed using the 32-fold diluted ascites fluid.

Example 4 Determination of Epitope (1) It was determined by immunoprecipitation reaction that the recognition protein of C179 was an HA molecule. That is, H2N2
After subtype A / Okuda / 57 was adsorbed and infected with MDCK cells for 30 minutes, 10 μCi of methionine in the medium was added.
The infected cells were labeled by culturing for 24 hours in MEM substituted with " ³⁵ S] methionine.
PA buffer [50 mM Tris (pH 7.4), 150 mM
NaCl, 1 mM EDTA, 1% Nonidet P-40,
1% deoxycholic acid, 0.1% SDS]. Subsequently, the insoluble matter was removed by centrifugation to obtain a supernatant. Next, the supernatant was mixed with C179 and incubated at 4 ° C. for 1 hour, Protein A-Sepharose CL4B beads were added, and the mixture was kept at room temperature for 2 hours to adsorb the immunoprecipitate to the beads. The beads were then collected and washed 5 times with RIPA buffer, then boiled to release the C179 binding protein. Next, the protein was subjected to SDS-12.5% polyacrylamide gel electrophoresis, the gel was fixed, immersed in 1 M sodium salicylate and dried, and autoradiography was performed. The labeled protein to which C179 binds shows that the A / Okuda / 57 H
It was identified as the A molecule. Similar tests were performed using the H1N1 subtype, other H2N2 subtypes, and H3N2 subtype, respectively. C179 was specifically immunoprecipitated with HA molecules of all H1N1 and H2N2 subtypes, but showed no binding with HA molecules of H3N2 subtype.

(2) MDCK cells infected with the H1N1 subtype or the H2N2 subtype in the presence of C179 were cultured to obtain an antigen mutant strain which was not sensitive to C179. That is, A / Su of H1N1 subtype as parent strain
ita / 1/89, H2N2 subtype A / Izumi / 5
MDC infected with the virus by using E./65
K cells were cultivated in the presence of C179, and mutant strains capable of growing even in the presence of C179 were purely separated from plaques of MDCK cells, and A / Suita / 1/89 mutant strains were A / Su.
The mutant strains of ita / 1/89 (R) and A / Izumi / 5/65 were designated as A / Izumi / 5/65 (R), respectively. The 2 mutants showed no reactivity to C179 in the staining test and the neutralization test. In addition, both mutant strains are MD
The plaque-forming ability in CK cells was also weak, it did not show pathogenicity in experimental animal mice, and it was a weakly infected strain that could grow only in cultured cells.

(3) In order to identify the antigen recognition site of the antibody, the gene encoding the HA molecule (hereinafter abbreviated as HA gene) was analyzed. (A) Synthesis of primer: primer 5 to primer 2
6 was synthesized using a DNA synthesizer, and after deprotection, ion exchange HPLC (TSK gel, DEAE-2SW column)
And purified by Sep-pak C18 to obtain about 50 μg of each DNA.

(B) MDCK cells infected with A / Suita / 1/89 were collected, guanidine isothiocyanate was added, and the cells were lysed by repeating injection with a syringe 5 times. Layer the cell extract after lysis on cesium chloride solution,
Ultracentrifugation was performed. The precipitate at the bottom of the centrifuge tube was dissolved in a buffer solution, treated with phenol and chloroform, and then precipitated with ethanol, and the recovered RNA was used as a sample of viral genomic RNA. Then using primer 5 c
DNA synthesis, followed by primer 5, primer 6
Was used to amplify the synthetic cDNA by the PCR method. Next, the amplified cDNA was separated by agarose gel electrophoresis, and a 1.7 kbp cDNA band corresponding to the HA gene was eluted. This cDNA was further amplified by PCR using primers 5 and 6, and the amplified fragment was
0% (w / v) polyethylene glycol 6000, 2.
A 5 M NaCl solution was added at 60% (v / v) and centrifuged to obtain a purified precipitate fraction.

Next, H labeled with [γ- ³² P] ATP
The nucleotide sequence of the purified gene was analyzed by the dideoxy method using the thermal cycler described in the above Biotechnics using the 1N1 subtype sequencing primers, primers 7 to 14, as follows. Ie 2
95 pmol of primer and 1 pmol of purified fragment
Annealing was carried out by heating at ℃ for 3 minutes and quenching. Tack polymerase is then added to the buffer in a buffer containing deoxynucleotides and dideoxynucleotides.
The temperature was kept at 10 ° C for 10 minutes to carry out a polymerase extension reaction.
Then, in order to completely carry out the extension reaction, the reaction was transferred to a thermal cycler reaction. Thermal cycler conditions are 90 ° C
1 minute, 55 ℃ 2 minutes, 72 ℃ 3 minutes cycle 10
I went there. Next, the cycle-finished product was heated in the presence of formamide at 95 ° C. for 3 minutes and then rapidly cooled in ice, and electrophoresed on an 8% denaturing polyacrylamide gel. After the electrophoresis, the gel was dried and exposed with an X-ray film, and then the base sequence was read to determine the base sequence of the entire HA gene shown in SEQ ID NO: 27 in the sequence listing.

(C) HA of A / Suita / 1/89 (R)
The analysis of the nucleotide sequence of the gene was performed using Example 4- (3)-(b)
Was carried out according to the method of 1. above, the base sequence of the entire HA gene was determined, and the HA gene of the parent strain was compared. The HA gene of the mutant strain had three nucleotide substitutions. That is, G of base number 627 of the HA gene of the parent strain shown in SEQ ID NO: 27 of the sequence listing was mutated to A, G of base number 736 was mutated to A, and C of base number 1018 was mutated to A. . The HA molecule is cleaved at one site by a protease to activate the infectivity of the virus. The larger polypeptide after cleavage of the protease is called HA1 and the smaller one is called HA2. And both of these polypeptides are linked by an SS bond,
This mutation involved amino acid substitutions at positions 189, 225 and 318 of HA1. Amino acid residues 189 and 225 are in a highly variable region and
The 18th substitution, Thr → Lys (ACA → AAA at the nucleotide level), was responsible for the non-responsiveness of the mutant strain to C179. In addition, the amino acid numbers of the HA molecules in this specification are virus (Volume), Vol. 11, Vol. 257.
~ H266 numbering system described on page 266 (1961).

(D) A / Izumi / 5/65 and A / Izum
The analysis of the base sequence of each HA gene of i / 5/65 (R) was carried out according to the method of Example 4- (3)-(b) except that the H2N2 subtype sequencing primer and primer 15- 23 was used. A / Izumi
The base sequence of the HA gene of / 5/65 is shown in SEQ ID NO: 28 in the sequence listing. The HA gene of the mutant strain had one nucleotide substitution. The T at the base number 1197 of the HA gene of the parent strain shown in SEQ ID NO: 28 is mutated to A, and this mutation is accompanied by amino acid substitution at the 52nd position of HA2. This 52nd substitution, Val → Glu. (GTA → GAA at nucleotide level) is a mutant C1
He was responsible for 79 non-responsiveness.

(E) H1N1 subtype A / PR / 8
/ 34, A / Bangkok / 10/83, A / Yamagata / 1
20/86, A / Osaka / 930/88, H2N2 subtype A / Okuda / 57, A / Adachi / 2/57, A
/ Kumamoto / 1/65, A / Kaizuka / 2/65, H3
N2 / Subtype A2 / Aichi / 2/68, A / Fukuok
a / C29 / 85, A / Sichuan / 2/87, A / Ibar
H of HA molecule of aki / 1/90, A / Suita / 1/90
In order to identify the amino acid sequence near the 318th position of A1 and the amino acid sequence near the 52nd position of HA2, a part of the base sequence of each HA gene was analyzed.

Example 4 for the H1N1 subtype
CDNA of RNA genome of each virus was synthesized according to the method of (3)-(b), and PCR amplification was performed on this cDNA using primers 9 and 13. DN obtained
The nucleotide sequence was determined by the dideoxy method using a thermal cycler using the A fragment as a template and the primers 11 and 12.

Regarding the H2N2 strain, Example 4- (3)-
According to the method of (b), cDNA of RNA genome of each virus
A was synthesized, and primers 17, 2 were prepared for this cDNA.
PCR amplification was performed using 1. Using the obtained DNA fragment as a template and primers 19 and 20, the nucleotide sequence was similarly determined by the dideoxy method.

Regarding the H3N2 strain, Example 4- (3)-
CD for viral genomic RNA according to the method of (b)
NA was synthesized and a primer 24 was added to this cDNA,
PCR amplification was performed using No. 26. Using the obtained DNA fragment as a template and primers 25 and 26, the nucleotide sequence was similarly determined by the dideoxy method.

For the H1N1 subtype and H2N2 subtype, 31 of the HA1 region represented by SEQ ID NO: 1 in the sequence listing is used.
The TGLRN polypeptide sequence of 8th to 322nd (A region) and the GITNKVNSVIEK polypeptide sequence of 47th to 58th of HA2 region (B region) represented by SEQ ID NO: 2 in the sequence listing are conserved, while H3N2 is conserved. The subtype includes 318 of the HA1 region represented by SEQ ID NO: 3 in the sequence listing.
-322 TGMRN polypeptide sequence (A 'region)
And 47-of the HA2 region represented by SEQ ID NO: 4 in the sequence listing.
The 58 th QINGKLNR (L / V) IEK polypeptide sequence (B 'region) is conserved. One amino acid differs between the A region and the A'region, and a difference of 5 to 6 amino acid residues is observed between the B region and the B'region. The difference between these two regions is the difference in antigen recognizability of the antibody, and is the reason why the antibody could not react with the H3N2 subtype in the serological and fusion inhibition tests.

As shown in FIG. 1, the TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing of the A region and the GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the sequence listing of the B region are the HA molecule. They are located close to each other in the center of the stem region, C179 recognizes both sequences, and the site is an epitope of C179. C179 is this H
It binds to the stem region of the A molecule, inhibits the membrane fusion function of the HA molecule, and neutralizes the virus.

H1N1 subtype: SEQ ID NO: 2 in Sequence Listing
Base numbers 1017 to 1031 of the HA gene of A / Suita / 1/89 shown in 7 encode the A region, and base number 1
191-1226 code the B region. SEQ ID NO: 29 in the sequence listing shows a part of HA gene of A / PR / 8/34, and its nucleotide numbers 76 to 90 encode A region,
The base numbers 250 to 285 encode the B region. SEQ ID NO: 30 in the sequence listing is A / Bangkok / 10/83
Of the HA gene, base numbers 76 to 90 thereof encode the A region, and base numbers 250 to 285 thereof encode the B region. Sequence number 31 in the sequence listing is A / Yama
A part of the HA gene of gata / 120/86 is shown, and its base numbers 76 to 90 encode the A region, and its base number 2
50 to 285 encode the B region. SEQ ID NO: 32 in the sequence listing shows a part of HA gene of A / Osaka / 930/88, its base numbers 76 to 90 encode A region, and its base numbers 250 to 285 encode B region. .

H2N2 subtype: SEQ ID NO: 2 in Sequence Listing
Base numbers 1007 to 1021 of the HA gene of A / Izumi / 5/65 shown in 8 encode the A region, and base number 1
181-1216 code the B region. SEQ ID NO: 33 of the sequence listing shows a part of HA gene of A / Okuda / 57, and its nucleotide numbers 94 to 108 encode A region,
The base numbers 268 to 303 encode the B region. Sequence number 34 in the sequence listing is H of A / Adachi / 2/57
A part of the A gene is shown, and its base numbers 103 to 117 encode the A region and its base numbers 277 to 312 encode the B region. Sequence number 35 in the sequence listing is A / Kuma
A part of the HA gene of moto / 1/65 is shown, and its base numbers 104 to 118 encode the A region, and its base number 2
78 to 313 encode the B region. SEQ ID NO: 36 in the sequence listing shows a part of HA gene of A / Kaizuka / 2/65, the base numbers 88 to 102 encode the A region, and the base numbers 262 to 297 encode the B region. .

H3N2 subtype: SEQ ID NO: 3 in Sequence Listing
7 is A2 / Aichi / 2/68, SEQ ID NO: 38 is A / Fuku
oka / C29 / 85, SEQ ID NO: 39 is A / Sichuan / 2
/ 87, SEQ ID NO: 40 shows a part of the HA gene of A / Ibaraki / 1/90, SEQ ID NO: 41 shows A / Suita / 1/90, and each base number 84 to 98 is A '.
It encodes a region, and each base number 258 to 293 encodes a B'region.

Example 5 Preventive Effect Against Influenza Virus A mouse infection experiment of influenza virus was carried out to obtain C1.
The preventive effect of 79 was investigated. C on 10 Balb / c mice
179 PBS solution (1 mg / ml) was intraperitoneally administered at 1 ml per mouse. A1 / FM / 1/47 (4000 HA units) of H1N1 subtype diluted 1000 times after 1 day
Was inoculated intranasally with 25 μl each. As a control, 12 mice were inoculated with the virus only.

As shown in FIG. 2, 8 mice in the control group died (2 mice after 5 days, 5 mice after 6 days, 1 mouse after 8 days). Also, other surviving mice were markedly debilitated. On the other hand, the C179-administered mice were normal, and all were well after 14 days. FIG. 2 is a graph showing the survival rate of the C179-administered group and the non-administered group, wherein the vertical axis represents the survival rate and the horizontal axis represents the number of days after virus infection.

[0056]

INDUSTRIAL APPLICABILITY According to the present invention, human influenza A
Antibodies useful for diagnosis of type I virus, prevention of infection, and treatment are provided. The antigenic site recognized by this antibody is widely conserved among viral subtypes and is capable of inducing neutralizing antibodies, and polypeptides containing this site are important as vaccines.

[Sequence list]

SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment SEQ ID NO: 2 Sequence length: 12 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment SEQ ID NO: 3 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment SEQ ID NO: 4 Sequence length: 12 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence characteristics: 9th Xaa is Val or Leu Is. SEQ ID NO: 5 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence AGCAAAAGCA GGGGATAAT 19 SEQ ID NO: 6 Sequence length : 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence AGTAGAAACA AGGGTGTTTT T 21 SEQ ID NO: 7 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence TCTTTTCGAG TACTGTGTCA ACA 23 SEQ ID NO: 8 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence GCCCCACTAC AATTGGGGAA ATG 23 SEQ ID NO: 9 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence Type: Other nucleic acid (synthetic DNA) sequence TTTTACAGAA ATTTGCTATG GCTG 24 sequence Number: 10 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence ACTCCCCTAT TGTGACTGGG TGTA 24 SEQ ID NO: 11 Sequence Length : 22 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GGTTATCATC ATCAGAATGA AC 22 SEQ ID NO: 12 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence AGTTCACCTT GTTTGTAATC CCGT 24 SEQ ID NO: 13 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence CCATTTTTTA CTCTTTCCAT GCAT 24 SEQ ID NO: 14 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other nucleic acid (synthetic DNA) sequence ATCTACTCAA CTGTCGCCAG TTCA 24 sequence Issue: 15 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence TTGTGTCGAC CTTCTCTGTG GAA 23 SEQ ID NO: 16 Sequence length : 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence TGTAGCATTG CCGGATGGCT 20 SEQ ID NO: 17 Sequence length: 23 Sequence type: Nucleic acid Strand Number of: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence ATTATCCGGT TGCCAAAGGA TCG 23 SEQ ID NO: 18 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand topology : Linear sequence type: Other nucleic acid (synthetic DNA) sequence GAGAGCACTG GTAATCTGTT GCA 23 SEQ ID NO: 19 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence type : Other nucleic acid (synthetic DNA) sequence CCATCAAATG CCTTTTGAGT GGA 23 SEQ ID NO: 20 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single-strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence ACTAGAAGCT CAGCATTGTA TGT 23 SEQ ID NO: 21 Sequence length: 24 Sequence Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence CATGCATTCA TCATCACATT TGTG 24 SEQ ID NO: 22 Sequence length: 23 Sequence type: Nucleic acid Number of strands : Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence CATACTTGGG ATAATCATAC GTC 23 SEQ ID NO: 23 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Direct Chain type of sequence: Other nucleic acid (synthetic DNA) sequence GCCATTTATG CTACAGTAGC AGG 23 SEQ ID NO: 24 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other Nucleic acid (synthetic DNA) sequence of GATCAGATTG AAGTGACTAA TGCT 24 SEQ ID NO: 25 Column length: 24 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GAATGCATCA CTCCAAATGG AAGC 24 SEQ ID NO: 26 Sequence length: 23 Sequence Type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence AGGTCCTGAA TTCTCCCTTC TAC 23 SEQ ID NO: 27 Sequence length: 1754 Sequence type: Nucleic acid Number of strands : duplex topology: linear sequence type: cDNA-to genomic RNA origin: organism: a / Suita / 1/89 SEQ GGATAATAAA TACAACCAAA ATGAAAGCAA AACTACTAGT CCTGTTATGT GCATTTACAG 60 CTACAGATGC AGACACAATA TGTATAGGCT ACCATGCGAA CAACTCAACC GACACTGTTG 120 ACACAGTACT TGAGAAGAAC GTGACAGTGA CACACTCTGT CAACCTACTT GAGGACAGTC 180 ACAACGGAAA ACTATGTCGA CTAAAAGGAA TAGCCCCACT ACAATTGGGT AATTGCAGCA 240 TTGCCGGATG GATCTTAGGA AACCCAGAAT GCGAATCACT GTTTTCTAAG GAATCATGGT 300 CCTACATTGC AGAAACACCA AACTCCGAGA ATGGAACATG TTACCCAGGG TATTTCGCCG 360 ACTATGA A ACTGAGGGAG CAATTGAGTT CAGTATCATC ATTCGAGAGA TTCGAAATAT 420 TCCCCAAAGA AAGCTCATGG CCCAACCACA CCGTAACCAA AGGAGTAACG GCATCATGCT 480 CCCATAATGG GAAAAGCAGT TTTTACAGAA ATTTGCTATG GCTGACGGGG AAGAATGGCT 540 TGTACCCAAA TCTGAGCAAG TCCTATGTGA ACAACAAAGA GAAAGAAGTC CTTGTACTAT 600 GGGGTGTTCA TCACCCGTCT AACATAGGGG ACCAAAGGGC CATCTATCAT ACAGAAAATG 660 CTTATGTCTC TGTAGTGTCT TCACATTATA GCAGGAGATT CACCCCAGAA ATAGCAAAAA 720 GACCCAAAGT AAGAGGTCAA GAAGGAAGAA TTAACTACTA CTGGACTCTG CTGGAACCCG 780 GGGACACAAT AATATTTGAG GCAAATGGAA ATCTAATAGC GCCATGGTAT GCTTTCGCAC 840 TGAGTAGAGG CTTTGGGTCA GGAATCATCA CCTCAAACGC ATCAATGGAT GAATGTGACG 900 CGAAGTGTCA AACACCCCAG GGAGCTATAA ACAGTAGTCT TCCTTTCCAG AATGTACACC 960 CAGTCACAAT AGGAGAGTGT CCAAAGTATG TCAGGAGTAC AAAATTAAGG ATGGTTACAG 1020 GACTAAGGAA CATCCCATCC ATTCAATCCA GAGGTTTGTT TGGAGCCATT GCCGGTTTCA 1080 TTGAAGGGGG GTGGACTGGA ATGATAGATG GATGGTATGG TTATCATCAT CAGAATGAAC 1140 AAGGATCTGG CTATGCTGCG GATCAAAAAA GCACACAAAA TGCCATTAAC GGAATTACAA 1200 ACAAGGTGAA TTCTGTAATC GAG AAAATGA ACACTCAATT CACAGCTGTG GGCAAAGAAT 1260 TCAACAAATT AGAAAGAAGG ATGGAATACT TAAATAAAAA AGTTGATGAT GGATTTCTGG 1320 ACATTTGGAC ATATAATGCA GAATTGTTGG TTCTACTGGA AAATGAAAGG ACTTTGGATT 1380 TTCATGACTC AAATGTGAAG AATCTGTATG AGAAAGTAAA AAGCCAATTA AAGAATAATG 1440 CCAAAGAAAT AGGATACGGG TGTTTTGAAT TCTACCACAA GTGTAACAAT GAATGCATGG 1500 AAAGTGTGAA AAATGGAACT TATGACTATC CAAAATATTC CGAGGAATCA AAGTTAAACA 1560 GGGAAAAAAT TGATGGAGTG AAATTGGAAT CAATGGGAGT CTATCAGATT CTGGCGATCT 1620 ACTCAACTGT CGCCAGTTCA CTGGTGCTTT TGGTCTCCCT GGGGGCAATC AGCTTCTGGA 1680 TGTGTTCTAA TGGGTCTTTG CAGTGTAGAA TATGCATCTG AGACCAGAAT TTCAGAAATA 1740 TAAGAAAAAA CACC 1754 SEQ ID NO: 28 Sequence length: 1728 Sequence type: Nucleic acid Number of strands: Double-stranded RNA to linear genomic sequence : Organism name: A / Izumi / 5/65 Sequence ATAGACAACC AAAAGCATAA CAATGGCCAT CATCTATCTC ATACTCCTGT TCACAGCAGT 60 GAGGGGGGAC CAGATATGCA TTGGATACCA TGCCAATAAT TCCACAGAAA AGGTCGACAC 120 AATTCTAGAG CGGAATGTCA CTGTGACTCA TGCCAAGGAC ATCCTTGAGA AGACCCACAA 180 CGGAAAGCTA TGCAAACTAA ACGGAATCCC TCCACTTGAA CTAGGGGACT GTAGCATTGC 240 CGGATGGCTC CTTGGAAATC CAGAATGTGA TAGGCTTCTA AGGGTGCCAG AATGGTCCTA 300 TATAATGGAG AAAGAAAACC CGAGATACAG TTTATGTTAC CCAGGCAACT TCAATGACTA 360 TGAAGAATTG AAACATCTCC TCAGCAGCGT AAAACATTTC GAGAAAGTAA AGATTCTGCC 420 CAAAGATAGA TGGACACAGC ATACAACAAC TGGAGGTTCA AAGGCCTGCG CAGTGTCAGG 480 TAAACCATCA TTCTTCAGGA ACATGGTCTG GCTGACAAAG AAAGGACCAA ATTATCCGGT 540 TGCCAAAGGA TCGTACAACA ATACGAGCGG AGAGCAAATG CTAATAATTT GGGGAGTGCA 600 CCATCCTAAT GATGAGGCAG AACAAAGAGC ATTGTACCAG GAAGTGGGAA CCTATGTTTC 660 CGCAAGCACA TCAACATTGA ACAAAAGGTC AATCCCTGAA ATAGCAGCAA GGCCTAAAGT 720 GAATGGACTA GGAAGTAGAA TGGAATTCTC TTGGACCCTC TTGGATGTGT GGGACACCAT 780 AAATTTTGAG AGCACTGGTA ATCTAGTTGC ACCAGAGTAT GGATTCAAAA TATCGAAAAG 840 AGGTAGTTCA GGGATCATGA AGACAGAAGG AACACTTGGG AACTGTGAGA CCAAATGCCA 900 AACTCCTTTG GGAGCAATAA ATACAACACT ACCTTTTCAC AATGTCCACC CACTGACAAT 960 AGGTGAATGC CCCAAATATG TAAAATCGGA GAAATTGGTC TTAGCAACA G GACTAAGGAA 1020 TGTTCCCCAG ATTGAATCAA GAGGATTGTT TGGGGCAATA GCTGGCTTTA TAGAAGGAGG 1080 ATGGCAAGGA ATGGTTGATG GTTGGTATGG ATACCATCAC AGCAATGACC AGGGATCAGG 1140 GTATGCAGCA GACAAAGAAT CCACTCAAAA GGCATTTGAT GGAATCACCA ACAAGGTAAA 1200 TTCTGTGATT GAAAAGATGA ACACCCAATT TGAAGCTGTT GGGAAAGAAT TCAATAATTT 1260 AGAGAAAAGA CTGGAGAACT TGAACAAAAA GATGGAAGAC GGGTTTCTAG ATGTGTGGAC 1320 ATACAATGCT GAGCTTCTAG TTCTGATGGA AAATGAGAGG ACACTTGACT TCCATGATTC 1380 TAATGTCAAG AACCTGTATG ATAAAGTCAG AATGCAGCTG AGAGACAACG TCAAAGAACT 1440 AGGAAATGGA TGTTTTGAAT TTTATCACAA ATGTGACGAT GAATGCATGA ATAGTGTGAA 1500 AAACGGGACG TATGATTATC CCAAGTATGA AGAAGAATCT AAACTAAATA GAAATGAAAT 1560 CAAAGGGGTA AAATTGAGCA GCATGGGGGT TTACCAAATT CTTGCCATTT ATGCTACAGT 1620 TGCAGGTTCT CTGTCACTGG CAATCATGAT GGCTGGGATC TCTTTCTGGA TGTGCTCCAA 1680 CGGGTCTCTG CAGTGCAGAA TCTGCATATG ATTGTAATTT ATTTTATA 1728 SEQ ID NO: 29 length of sequence: 442 SEQ type: Nucleic acid Number of strands: Duplex Topology: Linear Sequence type: cDNA to genom ics RNA Origin: Organism Name: A / PR / 8/34 SEQ CCTTTCCAGA ATATACACCC AGTCACAATA GGAGAGTGCC CAAAATACGT CAGGAGTGCC 60 AAATTGAGGA TGGTTACAGG ACTAAGGAAC ATCCCGTCCA TTCAATCCAG AGGTCTATTT 120 GGAGCCATTG CCGGTTTTAT TGAAGGGGGA TGGACTGGAA TGATAGATGG ATGGTATGGT 180 TATCATCATC AGAATGAACA GGGATCAGGC TATGCAGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GGATTACAAA CAAGGTGAAC TCTGTTATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GTAAAGAATT CAACAAATTA GAAAAAAGGA TGGAAAATTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTAGT TCTACTGGAA 420 AATGAAAGGA CTCTGGATTT CC 442 SEQ ID NO: 30 Sequence length: 424 Sequence type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleotide type: Nucleic acid type to genomic RNA Origin: Organism: A / Bangkok / 10/83 Sequence CCTTTCCAGA ATGTACACCC AGTCACAATA GGAGAGTGCC CAAAGTACGT CAGGAGTACA 60 AAATTAAGGA TGGTTACAGG ACTAAGGAAC ATCCCATGAACGATTGAATGACGATGGA 180AGAGACATGACGAGAGACGA TATGCTGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GGATTACAAA CAAGGTGAAC TCTGTAATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GTAAAGAATT CAACAAATTA GAAAAAAGGA TGGAAAACTT AAATAGTAGAAATT ATA: AGTAAAGCT ATT: AAAAAAAGT : linear sequence type: cDNA-to genomic RNA origin: organism: a / Yamagata / 120/86 SEQ CCTTTCCAGA ATGTACACCC AGTCACAATA GGAGAGTGCC CAAAGTATGT CAGGAGTACA 60 AAATTAAGGA TGGTTACAGG ACTAAGGAAC ATCCCATCCA TTCAATCCAG AGGTTTGTTT 120 GGAGCCATTG CCGGTTTCAT TGAAGGGGGG TGGACTGGAA TGATAGATGG ATGGTATGGT 180 TATCATCATC AGAATGAACA AGGATCTGGC TATGCTGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GGATTACAAA CAAGGTGAAT TCTGTAATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GCAAAGAATT CAACAAATTA GAAAGAAGGA TGGAAAACTT AAATAAAAAA 360 GTTGATGATG AGA: ATGA4GAAGAATGA4GAAGAAG Type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Osaka / 930/88 Sequence CCTTTCCAGA ATGTACACCC AGTCACAATA GGAGAGTGCC CAAAGTATGT CAGGAGTACA 60 AAATTAAGGA TGGTTACAGG ACTAAGGAAC ATCCCATCCA CATCATCATCCA GGAGCCATTG CCGGTTTCAT AGAAGGGGGG TGGACTGGAA TGATAGATGG ATGGTATGGT 180 TATCATCATC AGAATGAACA AGGATCTGGC TATGCTGCGG ATCAAAAAAG CACACAAAAT 240 GCCATTAACG GAATTACAAA CAAGGTGAAT TCTGTAATCG AGAAAATGAA CACTCAATTC 300 ACAGCTGTGG GCAAAGAATT CAACAAATTA GAAAGAAGGA TGGAAAACTT AAATAAAAAA 360 GTTGATGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTGGT TCTACTGGAA 420 AATGAAAGG 429 SEQ ID NO: 33 SEQ length: 400 type of sequence : Number of nucleic acid strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Okuda / 57 Sequence GCAATAAATA CAACATTACC TTTTCACAAT GTCCACCCAC TGACAATAGG TGAGTGCCCC 60 AAATATGTAA AATCGGAGAA GTTGGTCTAGGAATGAGTCCCCGAATCAGTACCCTAGAAAGTAGCCTAGAAA 120G GGCAATAGCT GGTTTTATAG AAGGAGGATG GCAAGGAATG 180 GTTGACGGTT GGTATGGATA CCATCACAGC AATGACCAGG GATCAGGGTA TGCAGCAGAC 240 AAAGAATCCA CTCAAAAGGC ATTTGATGGA ATCACCAACA AGGTAAATTC TGTGATTGAA 300 AAGATAAACA CCCAATTTGA AGCTGTTGGG AAAGAATTCG GTAACTTAGA GAAAAGACTG 360 GAGAACTTGA ACAAAAAGAT GGAAGACGGG TTTCTAGATG 400 SEQ ID NO: 34 SEQ Length: 409 SEQ types: the number of nucleic acid strands: Double-Strand Topology: Linear Sequence Type: cDNA to genomic RNA Origin: Organism Name: A / Adachi / 2/57 Sequence CGCCTTGGAG CAATAAATAC AACATTGCCT TTTCACAATG TCCACCCACT GACAATAGGT 60 GAGTGCCCCAATATGTAAA ATCGGAGAAT ATCAGAGAATAG 120 CCAGAGTT ATCAGAAGACATACTACAGAAGGACT CAAGGAATGG TTGATGGTTG GTATGGATAC CATCACAGCA ATGACCAGGG ATCAGGGTAT 240 GCAGCAGACA AAGAATCCAC TCAAAAGGCA TTTGATGGAA TCACCAACAA GGTAAATTCT 300 GTGATTGAAA AGATGAACGA CCAATTTGAAAAGACAACTTGAAAAGAGAACTAGAACGATAACTTAGAA AGA Length: 410 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Kumamoto / 1/65 sequence CTCCTTTGGA GCAATAAATA CAACATTACC TTTTCACAAT GTCCACCCAC TGACAATAGG 60 TGAATGCCCC AAATATGTAA AATCGGAGAA ACTGGTCTTA GCAACAGGAC TAAGGAATGT 120 TCCCCAGATT GAATCAAGAG GATTGTTTGG GGCAATAGCT GGCTTTGTAG AAGGAGGATG 180 GCAAGGAATG ATTGATGGTT GGTATGGATA CCATCACAGC AATGATCAGG GATCAGGGTT 240 TGCAGCAGAC AAAGAATCCA CTCAAAAGGC ATTTGATGGA ATCACCAACA AGGTAAATTC 300 TGTGATTGAA AAGATGAACA CCCAATTTGA AGCTGTTGGG AAAGAATTCA ATAATTTAGA 360 GAAAAGACTG GAGAACTTGA ACAAAAGGAT GGAAGACGGG TTTCTAGATG 410 SEQ ID NO: 36 SEQ length: 394 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Kaizuka / 2/65 Sequence AATACAACAC TACCTTTTCA CAATGTCCAC CCACTGACAA TAGGTGAATG CCCCAAATAT 60 GTAAAATCGG AGAAATTGGT CTTAGCAACA GG ATGTTCCCCA GATTGAATCA 120 AGAGGATTGT TTGGGGCA AT AGCTGGCTTT ATAGAAGGAG GATGGCAAGG AATGGTTGAT 180 GGTTGGTATG GATACCATCA CAGCAATGAC CAGGGATCAG GGTATGCAGC AGACAAAGAA 240 TCCACTCAAA AGGCATTTGA TGGAATCACC AACAAGGTAA ATTCTGTGAT TGAAAAGATG 300 AACACCCAAT TTGAAGCTGT TGGGAAAGAA TTCAATAATT TAGAGAAAAG ACTGGAGAAC 360 TTGAACAAAA AGATGGAAGA CGGGTTTCTA GATG 394 SEQ ID NO: 37 Length of sequence: 329 SEQ types: the number of nucleic acid strands : Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A2 / Aichi / 2/68 Sequence ATGACAAGCC CTTTCAAAAC GTAAACAAGA TCACATATGG AGCATGCCCC AAGTATGTTA 60 AGCAAATGGGCATGTGAACATGGACAGCAGCTAGAGCTCGACTAGAGCTGCACAGACCAGTACCAGAGAGCCAACGAG 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC CATCGACCAA ATCAATGGGA AATTGAACAG GGTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAATCGAA AAGGAATTC 329 SEQ ID NO: 38 Sequence length: Nucleotide sequence: Nucleotide sequence: Nucleotide sequence: Nucleotide sequence: 334 sequences Nucleic acid sequence: Nucleotide sequence: 334 sequences Type: cDNA-to genomic RNA origin: Organism: A / Fukuoka / C29 / 85 SEQ ATGACAAACC CTTTCAAAAT GTAAACAAGA TCACATATGG GGCATGTCCC AGGTATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCATATTCGG CGCAATAGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGTT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAATCGAA AAGGAATTCT CAGA 334 SEQ ID NO: 39 Sequence length: 329 Sequence type: Nucleic acid Strand number: Double stranded Topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Sichuan / 2/87 SEQ ATGACAAACC CTTTCAAAAT GTAAACAAGA TCACATATGG GGCATGTCCC AGATATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCATATTCGG CGCAATAGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGCT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGA CGAACG 300 AGAAATTCCA TCAAACCGAA AAGGAATTC 329 SEQ ID NO: 40 Sequence length: 334 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism: A / Ibaraki / 1/90 sequence ATGACAAACC CTTTCAAAAT ATAAACAGGA TCACATATGG GGCATGTCCC AGATATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAGAAA CAAACTAGAG 120 GCATATTCGG CGCAATCGCA GGTTTCATAG AAAATGGTTG GGAGGGAATG GTAGACGGTT 180 GGTACGGTTT CAGGCATCAA AATTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAATCGAA AAGGAATTCT CAGA 334 SEQ ID NO: 41 sequence of Length: 329 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to genomic RNA Origin: Organism name: A / Suita / 1/90 Sequence ATGACAAACC CTTTCAAAAT GTAAACAGGA TCACATATGG GGCATGTCCC AGATATGTTA 60 AGCAAAACAC TCTGAAATTG GCAACAGGGA TGCGGAATGT ACCAGAAAAA CAAACTAGGG 120 GCATATTCGG CGCAATCGCA GGTTTCATAG AAAA TGGTTG GGAGGGAATG GTAGACGGTT 180 GGTACGGTTT CAGGCATCAA AACTCTGAGG GCACAGGACA AGCAGCAGAT CTTAAAAGCA 240 CTCAAGCAGC AATCGACCAA ATCAACGGGA AACTGAATAG GTTAATCGAG AAGACGAACG 300 AGAAATTCCA TCAAACCGAA AAGGAATTC 329

[Brief description of drawings]

FIG. 1 is a schematic diagram of the tertiary structure of a hemagglutinin molecule.

FIG. 2 is a diagram showing the survival rate of influenza virus-infected groups.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI Technical indication location A61K 39/395 D 9284-4C S 9284-4C C12N 15/44 ZNA C12P 21/02 C 8214-4B 21/08 8214-4B G01N 33/53 D 8310-2J 33/569 L 9015-2J // (C12P 21/02 C12R 1:91) (C12P 21/08 C12R 1:91)

Claims (4)

[Claims] 1. An anti-human influenza virus antibody having the following characteristics (a) and (b): (A) Recognizes the stem region of hemagglutinin molecule of H1N1 and H2N2 subtypes of human influenza A virus, but does not recognize the stem region of hemagglutinin molecule of H3N2 subtype. (B) H3N3 has a neutralizing activity on H1N1 and H2N2 subtypes of human influenza A virus.
N2 subtype has no neutralizing activity. 2. The anti-human influenza virus antibody according to claim 1, which has the following characteristics (a) and (b). (A) The TGLRN polypeptide sequence represented by SEQ ID NO: 1 in the sequence listing of the stem region in the hemagglutinin molecule of the H1N1 subtype and H2N2 subtype of human influenza A virus, and represented by SEQ ID NO: 2 in the sequence listing GITNKV
Recognize NSVIEK polypeptide sequence. (B) TGMRN polypeptide sequence represented by SEQ ID NO: 3 in the sequence listing of the stem region in the hemagglutinin molecule of the H3N2 subtype of human influenza A virus and QINGKLNR (L / V represented by SEQ ID NO: 4 in the sequence listing). ) I
It does not recognize the EK polypeptide sequence. 3. H1 of human influenza A virus
An immunogenic artificial polypeptide having substantially the same antigenicity as the stem region in hemagglutinin molecules of N1 and H2N2 subtypes. 4. At least SEQ ID NO: 1 in the sequence listing in the molecule
And a GITNKVNSVIEK polypeptide sequence represented by SEQ ID NO: 2 in the sequence listing, wherein the configurations of both sequences are H1N1 subtype and H2
The immunogenic artificial polypeptide according to claim 3, which has substantially the same antigenicity as the stem region in the N2 subtype hemagglutinin molecule.