CA2121559C - Immunogenic artificial polypeptide - Google Patents

Abstract

[Object] To provide an anti-human influenza virus antibody and an immunogenic artificial polypeptide.
[Constitution] An anti-human influenza virus antibody which recognizes the stem regions of haemagglutinin molecules of the H1N1 and H2N2 subtypes and has a neutralization activity but does not recognize the stem region of the H3N2 subtype and has no neutralization activity. An immunogenic artificial polypeptide having an antigenicity substantially same as the stem regions of haemagglutinin molecules. An immunogenic artificial polypeptide having an antigenicity substantially same as the stem regions of haemagglutinin molecule and lacking the globular head region of haemagglutinin molecules.
[Effect] This antibody is useful in the diagnosis and treatment of influenza A virus, while these polypeptides are useful as a vaccine.

Description

[Designation of Document] SPECIFICATION
itle of the Invention] ~ I~UNOGENIC ARTIFICIAL POLYPEPTIDE
[DE~tailed Description of the Invention]
[Field of Industrial. Application]
This invention relates to a polypeptide containing an antigen site which is recognized by an antibody against the stem region in haemagglutinin molecule of human influenza A virus and a gene coding for this polypeptide.
[Prior Art]
There are three types (A, B and C) of influenza viruses and the worldwide prevalence of influenza costing a large number of deaths is caused by human .influenza A virus.
Influenza A virus is further classified into various subtypes depending on the antigenicities of haemagglutinin (hereinafter referred to s:~mply as HA) and neuraminidase (hereinafter referred to s=imply as NA) which are viral surface prc>teins. There have been known so far three subtypes of human influenza A viruses, namely, the H1N1, H2N2 and H3N2 subtypes.
The HA of influenza A virus comprises two structurally distinct regions, namely, a globular head region and a stem region. The globular head region contains a receptor binding site which is responsible for virus attachment to a target cell and participates in the haemagglutination activity of HA. On the other hand, the stem region contains a fusion peptide which is necessary for membrane fusion between the viral envelope and an endosomal membrane of the cell and thus relates to fusion activity [Whey et al., Ann. Rev. Hiochem., 56, 365 - 394 ( 1~~87 ) ] .
All of anti-HA antibodies, which have been obtained hitherto as an antibody capable of recognizing the H1N1 and H2N2 subtypes; recognize the globwlar head region of HA. However, this region mast frequently undergoes antigen mutation.
Therefore; these antibodies ate not common to the subtypes of ./ ' human infl~unza A virus and, further, lose the recognizing ability with antigenic-changes in the HA of the virus.
On the other hand, Green et al. have synthesized a polypeptide based on an amino acid sequence in the stem region of HA of the H3N2 subtype and obtained antibodies against this polypeptide. However, these ~antibodies have,a_low neutralization activity ,(International Publication No. WO 84f00687 of ' March 1, 1984) '~' ~ , Furthermore, the polypeptide per se employed as an antigen does not react with rabbit antiviral serum obtained by immunizing with the H3N2 subtype, which suggests that there is a problem from the viewpoint of antigenicity too [Cell, 28, 477 - 487 (1982y). , The infectivity of the HA of influenza A virus is activated when the HA is cleaved at one site with a protease. The larger polypeptide thus obtained is called HA1 while the smaller one HA2. It is believed that between these polypeptide HA2 will undergo less antigen mutation due to the subtype.
In East German Patent Laid-Open No. 228737, H. Glathe et. al. describe that HA2 is taken out by treating viral particles successively with an acid and trypsin or with a reducing agent alone.
By these treatments, however, HA molecules are destroyed in the stereostructure and irreversibly denatured. As a result, the HA2 thus obtained does not have its inherent stereostructure. In addition, the above-mentioned patent is silent whether the _____.~.~
....~-.._......_.~_...,~.~. _ .

~1~1~~9 efficacy of the obtained HA2 as a vaccine has been specifically confirmed c>r not.
(Problems to be Solved by the Invention]
Human influenza A virus periodically changes types of HA and NA and thus causes wide prevalence. It is often observed that vaccinization before winter, i.e, the season of prevalence, produces no effect, since the prevalence is caused by a virus of a different type.
However, if an antigen site which is common to virus subtypes in HA and NA molecules and hardly undergoes antigen mutation can be acquired, this antigen site is usable as a vaccine.
It is an object of the present invention to provide an antigen site polypeptide, which is w~ecognized by an antibody capable of recognizing the subtypes of human influenza A virus and usable as a ~~accine, and a gene coding for this polypeptide.
[Means for Solving the Problems]
To sum up, the first invention relates to an immunogenic artificial polypeptide characterized by having an antigenicity substantially same as that of the stem region in HA molecule of human influnza A virus.
The second invention relates to an immunogenic artificial polypeptide characterized_by having an antigenicity substantially same as that of the stem region in HA molecule of human influenza A virus and lacking a globular head region of HA
molecule.
The third invention relates to a gene coding for the immunogenic artificial polypeptide of the first invention.
The forth invention relates to a gene coding for the immunogenic artificial polypeptide of the second invention.

As the results of intensive studies, the present inventors have found out that a poTypeptide containing an antigen site, which has been conserved in common in the stem region in HA molecule of the H1N1 and H2N2 subtypes of human influenza A virus, and another polypeptide containing an antigen site, which has been conserved in common in the~stem region in HA molecule of the H3N2 subtype, are highly useful as a vaccine.
Then they have constructed a gene coding for the above-mentioned polypeptide which is useful in the production of this polypeptide through genetic engineering techniques, thus completing the present invention.
Examples of the immunogenic artificial polypeptide of the present invention, which has an antigenicity substantially the same as the stem region of HA molecule of~the influenza A
viruses and lacks a globUlal' head region of HA molecules , _ includes polypeptide which Lacks a globler head regions of HA
molecule by artificial proteolysis, and which is expressed by the HA gene lacking specif icaly a globular head regions of AA
molecules_ These polypeptides should only have the configuration which the antibody recognizing an antigen site common to the stem regions of HA molecule specificaly can recognize, may lack some part of the molecule or also may have the additional amino acid sequence.
Furthermore, these polypeptides may be partially digested with a protease in the process for producing the same by the protein engineering or genetic engineering technique. ' Namely, the expression."having an antigenicity substantially the same as that of the stem region in aA molecule" as used herein means that the polypeptide has an antigenicity of both of the HA1 and HA2 in the stem region of HA molecule which is efficiently usable as a vaccine. Therefore such a polypeptide comprising HA2 alone, the inherent stereostructure of which has been destroyed due to denaturation, as the one reported by H. Glathe et, al. as cited above is f=_xcluded from the scope of the present invention.
As examples of the immunogenic artificial polypeptide of the present invention which is the most effective as a vaccine, t he following ones may be cited.
(1) An immunogenic artificial polypeptide which <~ontains at least a TGLRN polypeptide sequence represented by the SEQ ID No. 1 in the sequence .Listing and a GITNKVNSVIEK polypeptide sequence represented by the SEQ ID No. 2 in the sequence .Listing in the molecule and has an antigenicity wherein the configuration of these sequences is substantially the same as that of the stem region of hemagglutinin molecule of the H1N1 and H2N2 subtypes.
(2) An immunogenic artificial polypeptide which contains at least a TGMRN polypeptide sequence repre-aented by the SEQ ID No. 3 in the sequence listing and a QINGKLNR (L/v) IEK polypeptide sequence represented by the SEQ ID No. 4 in the sequence listing in the molecule and has an antigenicity wherein the c~onf iguration of these sequences is substantially the same as that of the stem region of hemagglutinin molecule of the H3N2 subtype.

2121~5~

(3) An immunogenic artificial polypeptide of the ~econd~, invention of the present invention separated from hemagglutinin molecule of human influenza A virus which has been treated with a protease.
The antibody, which recognizes a site common to the stem regions in HA molecules of the H1N1 and H2N2 subtypes of human inf luenza A virus and has a neutralization activity for the H1N1 and H2N2 subtypes of human influenza A virus, can be prepared as a monoclonal antibody in the following manner. A
mammal such as mouse, guinea pig or rabbit is immunized with an antigen. As the antigen, viral particles selected from among those of the H1N1 and H2N2 subtypes may be used. Examples of virus strains of the H1N1 subtype include A/Bangkok/10/83, A/Yamagata/120/86, A/Osaka/930/88, A/Suita/1/89 (each being a stock of the Research 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), ATCC: VR-897], A/NWS/33 [influenza A (H1N1), ATCC VR-219], A/Weiss/43 [influenza A (H1N1), ATCC VR-96] and A/wS/33 [influenza A (H1N1), ATCC VR-825]. Examples of strains of the H2N2 subtype include A/Okuda/57, A/Adachi/2/57, A/Kumamoto/ 1/65, A/Kaizuka/2/65, A/Izumi/5/65 (each being a stock of the Research Institute for Microbial Diseases, Osaka Llniversity) and A2/Japan/305/57 [influenza A (H2N2), ATCC VR-7.00]. Alternately, the mammal can be immunized with an HA
molecule obtained from these viruses, an HA polypeptide prepared by using the genetic recombination.technology, a recombinant polypeptide containing the recognition site of the antibody of the present invention, namely, the antigen site of the stem region of an HA molecule therein or a synthetic polypeptide containing the antigen site of the stem region of an HA molecule 212~~~~
therein. Next, spleen cells obtained from the animal thus ~nmunized are fused with myeloma cells. From the hybridomas thus obtained, cells which produce an antibody having the characteristics (A) to (C) as will be specified below are selected and incubated to thereby give the target antibody according to the present invention.
(A) It has an avidity and a neutralization activity for viruses of the above-mentioned H1N1 and H2N2 subtypes.
(B) It has neither any avidity nor any neutralization activity for viruses of t:he H3N2 subtype such as A/Fukuoka/C29/85, A/Sichuan/2/87, A/Ibaraki/1/90, A/Suita/1/90, A/Kitakyushu/159/93 (each being a stock of the Research Institute for Microbial Diseases, Osaka University), A/Port C:halmers/1/73 (influenza A (H3N2), ATCC VR-810] and A2/Aichi/2/68 [influenza A, ATCC VR547] and influenza B viruse strains such as B/Nagasaki/1/87 (a stock of the Research Institute for Microbial Diseases, Osaka University) and B/Allen/45 [influenza B, ATCC VR-102].
(c) It recognizes HA molecules of the H1N1 and H2N2 subtypes, does not inhibit the haemagglutination activity for which the globular head region of the HA molecule is responsible, but inhibits the membrane fusion activity for which t:he stem region of the HA molecule is responsible.
These hybridomas are prepared in accordance with the description of Nature, 256, 495 - 497 (1975). As a mouse to be immunized; a Balb/c mouse and an F1 mouse obtained by mating a Balb/c mouse with another mouse of a different series may be used. The immunization is effected, for example, thrice within 2 too 5 months by using 100 to 1000:HAU/animal of viral particles as an antigen. The feeding of the mouse and the collection of .its spleen cells are carried out in a conventional manner.

As the myeloma cells, SP2/0-Agl4 (ATCC CRL1581), ~~3x63Ag8U.1 (ATCC CRL1597), p3x63Ag8 (ATCC TIB9) or p3x63-Ag8.
653 (ATCC CRL1580,) may be suitably employed. The spleen cells and the myeloma cells are mixed together at a ratio of from 1 .
1 to 10 ..1. The fusion is effected by maintaining the mixture of these cells at 35 to 37°C in a phosphate buffer solution {pH
7.2 - 7.4) containing NaCl (about 0.850 , dimethyl sulfoxide [10 - 20~ (v/v)] and polyethylene glycol of a molecular weight of 1000 to 6000 for 1 to 5 minutes. By using an HAT medium, cells growing thereon are selected as fused cells. The fused cells are cloned by repeating the limiting dilution procedure at least thrice.
The hybridomas are incubated by a method commonly used for incubating animal cells. Thus the antibody of the present invention can be obtained in the medium. Alternately, the hybridomas may be transplanted into the peritoneal cavity of a nude mouse or a Balb/c mouse treated with pristane and grown therein. As a result, the antibody of the present invention can be accumulated in the asc:ites. Namely, 0.5 to 1 mg of pristans ' i.s inoculated into the peritoneal cavity of the mouse. Two to 3 weeks thereafter, 5 x 106 to 1 x 10' hybridomas are transplanted into the peritoneal cavity of the animal. Then the ascites, which is usually accumulated after 7 to 10 days, is taken out.
The monoclonal antibody contained in the culture and the ascites may be purified by a conventional method.
The monoclonal antibody thus obtained recognizes the stem regions of HA molecules of the H1N1 and H2N2 subtypes and inhibits the membrane fusion activity of these viruses to thereby neutralize these viruses. Now the properties of this antibody will be described in greater detail.
(a) The results of the staining test indicate that the g antibody of the present invention recognizes MDCK cells (ATCC
~CL34) infected with the H1N1 and H2N2 subtypes but does not recognize MDCK cells infected with the H3N2 subtype. The staining test is effected in accordance with the method described in J. Clin. Microbiol., 28, 1308 - 1313 (1990) by using four antibodies, namely, the monoclonal antibody of the present invention, rabbit anti-mouse immunoglobulin G serum, coat anti-rabbit immunoglobulin G serum, and peroxidase-rabbit anti-peroxidase complex.
(b) The results of the immunoprecipitation test indicate that the antibody of the present invention recognizes HA
molecules of the H1N1 and H2N2 subtypes but does not recognize an HA molecule of the H3N2 subtype.
(c) In the haemagglutination test, the antibody of the present invention does not inhibit the hemagglutination .activities of the H1N1, H2N2 and H3N2 subtypes.
(d) The antibody of the present invention recognizes a common conserved region characteristic of the stem regions of HA
molecules of the H1N1 and H2N2 subtypes, which is specified by .
analyzing genes coding for the HA molecules, but does not recognize a common conserved region characteristic of the stem region of an HA molecule of the H3N2 subtype.
A gene coding for the HA molecule (hereinafter referred to simply as HA gene) is analyzed by the following method.
MDCK cells are infected with viral particles and the infected cells are harvested on the following day. Viral RNAs in the cells are extracted by using guanidine isothiocyanate. Next, an oligonucleotide primer complementary to the 3' terminus of the negative strand RNA of each of the H1N1, H2N2 and H3N2 subtypes (for example, the primer 5 represented by the SEQ ID
No. 5 in the sequence listing) is prepared and cDNAs are 21215~~
synthesized by using this primer. To amplify these cDNAs, another oligonucleotide primer complementary to the 3' terminus of the positive strand RNA of each of the H1N1, H2N2 and H3N2 aubtypes (for example, the primer 6 represented by the SEQ ID
i~o. 6 in the sequence listing) is prepared. Then the cDNAs can be efficiently amplified by the polymerase chain reaction (PCR) method with the use of the primers 5 and 6. An HA gene of about 1.7 kbp contained in an amplified DNA is separated by agarose gel electrophoresis and then the second PCR is effected by using, for example, the primers 5 and 6. The DNA thus amplified is centrifuged by using 20~ (w/v) polyethylene glycol 6000/2.5 M
NaCl to thereby give a purified precipitate fraction.
Subsequently, sequence primers selected from among HA gene sequences of the subclasses of viruses are prepared. After labeling these primers with [~-'Zp]ATP, the labeled primers are annealed with the above-mentioned purified fraction, followed by sequencing by the dideoxy method with the use of a thermal cycler [Bio-Techniques, 9, 66 - 72 (1990)].
For example, the primers 7 to 14 represented respectively by the SEQ ID Nos. 7 to 14 in the sequence listing are sequence primers for the H1N1 subtype, the primers 15 to 23 represented respectively by the SEQ ID Nos. 15 to 23 in the sequence listing are sequence primers for the H2N2 subtype, and the primers 24 to 26 represented respectively by the SEQ ID Nos. 24 to 26 in the sequence listing are sequence primers for the H3N2 subtype. A
part of the gene coding for the stem region of the HA molecule of the H1N1 subtype can be amplified and analyzed at a high efficiency by using the primers 9 and 13 as PCR primers and the primers 11 and 12 as sequence primers. A part of the gene coding for the stem region of the HA molecule of the H2N2 subtype can be amplified and analyzed at a high efficiency by using the 2121~5~
primers 17 and 21 as PCR primers and the primers 19 and 20 as sequence primers. Further, a part of the gene coding for the stem region of the HA molecule of the H3N2 subtype can be amplified and analyzed at a high efficiency by using the primers 24 and 26 as PCR primers and the primers 25 and 26 as sequence primers.
As common conserved regions in HA molecules of H1N1 and H2N2 subtypes, the TGLRN polypeptide sequence represented by the SEQ ID No. 1 in the sequence listing and the GITNKVNSVIEK
polypeptide sequence represented by the SEQ ID No. 2 in the sequence listing in the stem regions in the HA molecules of the H1N1 and H2N2 subtypes, which have been found out by the present :inventors, can be cited. Fig. 1 is a schematic view of the tertiary structure of an HA molecule [Wiley et al., Nature, 289, 373 - 378 (1981)] and shows the position of the common conserved .regions in HA molecules of H1N1 and H2N2 subtypes. As Fig. 1 shows, these polypeptide sequences, represented by the A region and the B region in the figure, are close to each other at the center of the stem region of the HA molecule. A monoclonal antibody C179, which is an example of the antibody of the present invention and produced by Hybridoma C179 (FERM BP-4517), recognizes A region (the TGLRN polypeptide sequence represented by the SEQ ID No. 1 in the sequence listing) and B region (the GITNRVNSVIEK polypeptide sequence represented by the SEQ ID No.
2 in the sequence listing) in the stem region of this HA
molecule.
(e) In the neutralization activity test, the antibody of the present invention inhibits the plaque- or focus-forming abilities of the H1N1 and H2N2 subtypes but does not inhibit the plaque- or focus-forming ability of the H3N2 subtype. The neutralization activity test is carried out by the plaque reduction neutralization test or the influenza virus rapid focus seduction neutralization test described in the above-mentioned Journal of Clinical Microbiology. More specifically, the antibody is mixed with an virus and kept warm for a given period of time. Then MDCK cells are infected therewith and the neutralization activity is judged based on the reduction in the plaques or foci.
(f) In the fusion activity test, the antibody of the present invention inhibits the membrane fusion activities of the H1N1 and H2N2 subtypes but does not inhibit that of the H3N2 subtype. The fusion activity test is effected in accordance with a method described in Nature, 300, 658 - 659 (1982).
Specifically, CV-1 cells (ATCC CCL70) are infected with a virus and treated with an antibody. Then the ability to inhibit the fusion activity is determined by examining the formation of polykaryons.
The monoclonal antibody C179 can bind to the stem regions of HA molecules, inhibit the membrane fusion.activity of the EilN1 and H2N2 subtypes and markedly nentralizes the infections powers.of these virus strains. Accordingly, the polypeptide capable of inducing the antibody which binds to the stem regions of HA molecules of H1N1 and H2N2 subtypes, inhibits the membrane fusion activities of the H1N1 and H2N2 subtypes and markedly neutralizes the infections powers of these viruses (hereinafter this type antibody is referred to simply as C179 type antibody) is usable as a vaccine for influenza. Namely, the prevalence of influenza caused by the H1N1 and H2N2 subtypes can be prevented and treated by using a polypeptide, which has an antigenicity substantially the same as the stem regions of HA molecules of.
the H1N1 and H2N2 subtypes, as an immunogen. Examples of the immunogenic polypeptide include HA'molecules prepared from the 2121~~9 H1N1 and H2N2 subtypes and an HA polypeptide constructed by the genetic recombination technology. However, the globular head region of HA molecule is easy to become antigenic epitope and most frequently undergoes antigen mutation. So, a polypeptide having a stem region of HA molecule and lacking the globular head region of HA molecule is more effective as an antigen polypeptide which can induce C179 type antibody.
The polypeptide having an antigenicity which is substantially same as that of the stem region of HA molecule and lacking the globular head region of HA modecule (hereinafter this polypeptide is referred to simply as stem region polypeptide) is obtained by enzymatic digestion and deletion of a globular head region of. HA molecule or an HA polypeptide.
For example, the stem region polypeptide can be prepared by limitedly digesting HA molecules purified from viral particles of the H1N1 or H2N2 subtype with a protease. Alternately, the stem region polypeptide prepared by treating each of viral particles, a split vaccine obtained by inacaivating viral particles, or an extract obtained by treating viral particles with a surfactant with a protease may be used. As the protease to be used herein,,proteinase which can digest the globular head region in HA molecules without causing the loss of the antigenicity of the stem region are desirab7_e. As an example of the proteinase usable in the present invention, Proteinase K (EC 3.4.21.14; manufactured by Boehringer), which is an alkaline proteinase produced by Tritirachium album, may be cited. By using a proteinase which is comparable to this Proteinase K in the achievement of the digestion results, the stem region polypeptide of .he present invention can be prepared. It is also possible to combine a proteinase with a peptidase and conduct the treatment with the peptidase after the completion of the treatment with the proteinase.
Since HA molecules exist in the form of rigid trimers i_n a solution, they are hardly digested with a protease. Accordingly HA molecules can be efficiently treated with the protease in the presence of a modifier such as guanidine hydrochloride or urea. The modifier may be used at such a concentration as to allow the digestion by the protease without causing irreversible denaturation of the target stem region polypeptide. When urea is used as the modifier, the digestion with the protease may be effected in the presence of from 0.1 to B M, preferably from 1 to 3 M of urea. This protease-treatment can be performed by using a resin such as Sepharose on which the protease has been immobi:Lized. After the completion of the reaction, the protease-immobilized resin can be easily eliminated by centrifugation. The modifier and low molecular weight matters in the reaction mixture can be eliminated by dialysis. Thus protease-treated HA molecules can be prepared. The molecular weight of the protease-treated HA molecules can be measured by gel electrophoresis. Further, the target stem region polypeptide can be confirmed by measuring the avidity of the protease-treatment product for. C179 type antibody and its haemagglutination activity.
The stem region polypeptide obtained by the protease-treatment is a polypeptide having an »-antigenicity substantially the same as that of the stem region in HA molecule .(an avidity-for C179 type antibody) and lacking the biological activity of the globular head region thereof (a hemagglutination activity). It consists of a polypeptide part originating in the HA1 stem region in HA molecule and another polypeptide part originating in HA2 therein.
In this point, this polypeptide essentially differs from the above-mentioned vaccine of H. Glathe et. al.
which consists of a polypeptide originating in HA2 alone.
The polypeptide having an antigenicity which is substantially same as that of the stem region of HA molecule and lacking the globular head region of HA molecule is obtained by genetic recombination or by chemical synthesis. For example it is possible to get the polypeptide as follows. HA gene is prepared from a viral RNA, and a gene encoding a globular head region is deleted from HA gene by using some restriction enzyme or using PCR method. Then this HA gene, which is lacking a coding~region of globular head region of HA molecule, is integrated into a vector and expressed in animal cell such as CV-1 cells. Then the antigenic activity of the stem region polypeptides can be detected by binding activity to C179 type antibody. The example of stem region polypeptide should have a common concerned region for. stem region of HA molecute of H1N1 subtype and H2N2 subtype in its molecule and have the ability of~
inducing 0179 type antibody. As the example of the stem region polypeptide, a polypeptide having a TGZRN_polypeptide sequence represented by SEQ ID No. 1 in the sequence listing and a GITNRVNSVIEK polypeptide sequence represented by SEQ ID No. 2 in the sequence listing and having an antigenicity wherein the f5 configuration of these sequence is substantially same as that natural HA molecule of H1N1 and H2N2 subtypes can be obtained, isolated and used.
The example of stem region polypeptide may be the polypeptide having deletion, substitution, addition, insertion, inversion, or replacement of amino acid, and it doesn't alter the antigenicity and C179 type antibody inducible activity. It may be the polypeptide deleting some part of C terminal and/or N
terminal of stem region polypeptide or having a signal polypeptide of HA molecule at C terminal of stem region polypeptide or some part of globular head region in the stem region polypeptide.
when such a polypeptide is used as a vaccine, its antigenicity can be elevated by selecting an appropriate carrier. Examples of the carrier include albumin and polyamino acids. The vaccine of the present invention can be administered by the conventional active immunization method. More .specifically, it can be administered in such an amount as to give an immunogenicity-effective for the prevention or treatment one or more times by a method suitable for the preparation. The vaccine may be formulated into a pharmaceutical preparation by a conventional method. It may further contain an adjuvant for improvlng immune response.
The antibody, which recognizes a site common to the stem regions in HA molecules of the H3N2 subtype of human influenza A
virus, can be prepared as a monoclonal antibody in the following manner. A mammal such as mouse, guinea pig or rabbit is immunized with an antigen. As the antigen, viral particles selected from among those of the H3N2 subtype may be used.
Alternately, the mammal can be immunized with an HA molecule obtained from these viruses, an HA polypeptide prepared by using the genetic recombination technology, a recombinant polypeptide containing the recognition site of the antibody, namely, the antigen site of the stem region of an HA molecule therein or a synthetic polypeptide containing the antigen site of the stem region of an HA molecule therein. Next, spleen cells obtained from the animal thus immunized are fused with myeloma cells.
From the hybridomas thus obtained, cells which produce an antibody having the characteristics (D) to (F) as will be specified below are selected and incubated to thereby give the target antibody.
(D) It has an avidity for virus of H3N2 subtype.
(E) It has none avidity for viruses of the H1N1 and H2N2 subtypes, and influenza B viruse strains.
(F) It recognizes HA molecules of the H3N2 subtype, does not inhibit the haemagglutination activity for which the globular head region of the HA molecule is responsible.
These hybridomas are prepared in accordance with above description. As a mouse to be immunized, a Balb/c mouse and an F1 mouse obtained by mating a Balb/c mouse with another mouse of a different series may be used. The immunization is effected, for example, thrice within 2 to 5 months by using 100 to 1000 HAU/animal of viral par~icles as an antigen. The feeding of the mouse and the collection of its spleen cells are carried out in a conventional manner.
As the myeloma cells, SP2/0-Agl4, p3x63Ag8U.l, p3x63Ag8 or p3x63-Ag8.653 may be suitably employed. The spleen cells and the myeloma cells are mixed together.at a ratio of from 1 . 1 to 10 . 1. The fusion is effected by maintaining the mixture of these cells at 35 to 37°C in a phosphate buffer solution (pH 7.2 -7.4) containing NaCl (about 0.850 , dimethyl sulfoxide [10 - 20~

(v/v)] and polyethylene glycol of a molecular weight of 1000 to 6000 for 1 to 5 minutes. B'y using an HAT medium, cells growing thereon are selected as fused cells. The fused cells are cloned by repeating the limiting dilution procedure at least thrice.
The hybridomas are incubated by a method commonly used for incubating animal cells. Thus the antibody of the present invention can be obtained in the medium. Alternately, the hybridomas may be transplanted into the peritoneal cavity of a nude mouse or a Balb/c mouse treated with pristane and grown therein. As a result, the antibody of the present invention can be accumulated in the ascites. Namely, 0.5 to 1 mg of pristans is inoculated into the peritoneal cavity of the mouse. Two to 3 weeks thereafter, 5 x 106 to 1 x 10' hybridomas are transplanted into the peritoneal cavity of the animal. Then the ascites, which is usually accumulated after 7 to 10 days, is taken out.
The monoclonal antibody contained in the culture and the ascites may be purified by a conventional method.
The monoclonal antibody thus obtained recognizes the stem regions of HA molecules of the H3N2 subtype. Now the properties of this antibody will be described in greater detail.
(g) The results of the staining test indicate that the antibody recognizes MDCK cells infected with the H3N2 subtype but does not recognize MDCK cells infected with the H1N1 subtype or H2N2 subtype.
(h) The results of the immunoprecipitation test indicate that the antibody recognizes HA molecules of the H3N2 subtype but does not recognize an HA molecule of the HlNl and H2N2 subtypes.
(i) In the haemagglutination test, the antibody does not inhibit the hemagglutination activities of the H1N1, H2N2 and H3N2 subtypes.
~8 (j) The antibody recognizes a common conserved region characteristic of the stem regions of HA molecules of the H3N2 subtype, which is specified by analyzing genes coding for the HA
molecules, but does not recognize a common conserved region characteristic of the stem region of an HA molecule of the H1N1 and H2N2 subtypes.
As common conserved regions in HA molecules of H3N2 subtype, the TGMRN polypeptide sequence represented by the SEQ
ID No. 3 in the sequence listing and the QINGKLNR(L/V)IEK
polypeptide sequence represented by the SEQ ID No. 4 in the sequence listing in the stem regions in the HA molecules of the H3N2 subtype, which have been found out by the present inventors, can be cited. Fig. 2 is a schematic view of the tertiary structure of an HA molecule [Wiley et al., Nature, 289, 373 - 378 (1981)] and shows the position of the common conserved regions in the HA molecules of H3N2 subtype. As Fig. 2 shows, these polypeptide sequences, represented by the A' region and the B' region in the figure, are close to each other at the center of the stem region of the HA molecule. A monoclonal antibody AI3C, which is an example of the antibody which binds the conserved regions and is prodced by Hybridoma AI3C (FERM
BP-4516), recognizes A' region (the TGMRN polypeptide sequence represented by the SEQ ID No. 3 in the sequence listing) and B' region (the QINGKLNR(L/V)IEK polypeptide sequence represented by the SEQ ID No. 4 in the sequence listing] in the stem region of this HA molecule.
The monoclonal antibody AI3C can bind specifically to the stem regions of HA molecules of H3N2 subtype (hereinafter this type antibody is referred to simply as AI3C type antibody).
Accordingly, the polypeptide capable of inducing the AI3C type antibody is usable as a vaccine for influenza. Namely, the Iq prevalence of influenza caused by the H3N2 subtype can be prevented and treated by using a polypeptide, which has an ant,igenicity substantially same as the stem regions of HA
molecules of the H3N2 subtype, as an immunogen. Examples of the immunogenic polypeptide include HA molecules prepared from the H3N2 subtype and an HA po:Lypeptide constructed by the genetic recombination technology. However, the globular head region of HA molecule is easy to become antigenic epitope and most frequently undergoes antigen mutation. So, a stem region polypeptide is more effective as an antigen polypeptide which can induce AI3C type antibody.
The stem region polypeptide having an antigenicity which is substantially same as that of the stem region of HA molecule of H3N2 subtype is obtained by enzymatic digestion and deletion of a globular head region of HA molecule or an HA polypeptide.
For example, the stem. region polypeptide can be prepared by limitedly digesting HA molecules purified from viral particles of the H3N2 subtype with a. protease. Alternately, the stem region polypeptide prepared by treating each of viral particles, a split vaccine obtained by inactivating viral particles, or <in extract obtained by t_eating viral particles with a surf actant with a protease may be used. As the r~rotease to be used herein, proteinase which can digest the globular head region in HA molecules without causing the loss of the antigenicity of the stem region are desirable. As an example of the proteinase usable in thE: present invention, Proteinase K may be cited. By using a proteinase which is comparable to this Proteinase K in the achievement of the digestion results, the stem region polypeptide of the present invention can be prepared. It is also ' .possible to combine a proteinase with a peptidase and conduct the treatment with the peptidase after the completion of the treatment with the proteinase.
Since HA molecules exist in the form of rigid trimers in a solution, they are hardly digested with a protease. Accordingly HA molecules can be efficiently treated with the protease in the presence of a modifier such as guanidine hydrochloride or urea. The modifier may be used at such a concentration as to allow the digestion by the protease without causing irreversible denaturation of the target stem region polypeptide. When urea is used as the modifier, the digestion with the protease may be effected in the presence of from 0.1 to 8 M, preferably from 1 to 3 M of urea. This protease-treatment can be performed by using a resin such as Sepharose on which the protease has been immobilized. After the completion of the reaction, the protease-immobilized resin can be easily eliminated by centrifugation. The modifier and low molecular weight matters in the reaction mixture can be eliminated by dialysis. Thus protease-treated HA molecules can be prepared. The molecular weight of the protease=treated HA molecules can be measured by gel electrophoresis. Further, the target stem region polypeptide can be confirmed by measuring the avidity of the protease-treatment product for AI3C type antybody and its haemagglutination activity.
The stem region polypeptide obtained by the protease-treatment is a polypeptide having an antigenicity substantially the same as that of the stem region in HA molecule (an avidity for AT3C type * Trade-mark 2~

antibody) and lacking the biological activity of the globular head region thereof (a hemagglutination activity). It consists of a polypeptide part originating in the'HAl~stem region in HA molecule and another polypeptide part originating in HA2 therein.
In this point, this polypeptide essentially differs from the above-mentioned vaccine of H. Glathe et. al.
which consists of a polypeptide originating in HA2 alone.
The stem region polypeptide having an antigenicity which is substantially same as that of the stem region of HA molecule of H3N2 subtype is obtained by genetic recombination or by chemical synthesis. For example it is possible to get the polypeptide as follows. HA gene is prepared from a viral RNA of H3N2 subtype, and a gene encoding a globular head region is deleted from HA
gone by using some restriction enzyme or~using PCR method. Then this HA gene, which is lacking a coding region for globular head region of HA molecule, is integrated into a vector and expressed in animal cell such as CV-1 cells. Then the antigenic activity of these stem region polypeptides can be detected by binding activity to AI3C type antibody. The example of stem region polypeptide should have a common conserved region for stem region of HA molecute of H3N2 subtype in its molecule and have the ability of inducing AI3C type antibody. As the example of the stem region polypeptide, a polypeptide having a TGMRN
polypeptide sequence represented by SEQ ID No. 3.in the sequence listing and a QINGKLNR(L/v)IER polypeptide sequence represented .
by SEQ ID~No. 4 in the sequence listing and exhibiting an antigenicity wherein the configuration of these sequence is substantially same as that natural HA molecule of H3N2 subtype can be obtained, isolated and used.

The example of stem region polypeptide may be the ~~olypeptide having deletion, substitution, addition, insertion, inversion, or replacement of amino acid, and it doesn't alter the antigenicity and AI3C type antibody inducible activity. It may be the polypeptide deleting some part of C terminal and/or N
terminal of stem region polypeptide or having a signal polypeptide of HA. molecule at C terminal of stem region polypeptide or some part of globular head region in the stem region polypeptide.
When such a polypeptide is used as a vaccine, its antigenicity can be elevated by selecting an appropriate carrier. Examples of the carrier include albumin and polyamino acids. The vaccine of the present invention can be administered by the conventional active immunization method. More specifically, it can be administered in such an amount as to give an immunogenicity effective for the prevention or treatment one or more times by a method suitable for the preparation. The vaccine may be formulated into a pharmaceutical preparation by a conventional method. It may further contain an adjuvant for improving immune response.
The dose of the stem region polypeptide of this invention to be administered depends on, for example, the properies of the vaccine employed, the concentration of the polypeptide in a preparation and the administration route. Usually it may be administered to an adult in a dose of from l,ilg to 100 mg, preperably from 10 ~g to 10 mg.
Now, the antiinfluenza virus antibodies employed.
in the present invention will be described by .
reference to the following Referential Examples.

Referential example A 2121 ~ ~ 9 Preparation of viruses:
Virus strains of the H1N1 subtype used included A/PR/8/34, A/Bangkok/10/83, A/Yamagata/120/86, A/Osaka/930/88, A/Suita/1/89 and A1/FM/1/47 were used. Virus strains of the H2N2 subtype used :included A/Okuda/57, A/Adachi/2/57, A/Kumamoto/1/65, A/Kaizuka/2/65 and A/Izumi/5/65 were used. Virus strains of the H3N2 subtype, used included A2/Aichi/2/68, A/Fukuoka/C29/85, .?~/Sichuan/.2/87, A/Ibaraki/1/90, A/Suita/1/90 and A/Kitakyushu/159/93 were used. A strain of influenza B virus used was B/Nagasaki/1/87. Each strain was inoculated into the allantoic cavity of an embryonated hen egg aged 11 days, incubated at 34°C for 4 days and then harvested.
2. Preparation of monoclonal antibodies:
(1) Balb/c mice were immunized with two doses of A/Okuda/57 strain (320 HAU) prepared in the above Refential example A-1, which had been suspended in Freund's complete adjuvant before use, via intraperitoneal injection one month apart. One month thereafter, the mice were boosted by intraperitoneally injecting a suspension of the same antigen (320 HAU) in PBS. Three days thereafter, the spleen of each animal was taken out and thus spleen cells were prepared.
Mouse myeloma cells were prepared by incubating p3x63Ag8 cells in a DME medium containing 10~ of fetal bovine serum for 2 days after passage and then washing with physiological saline before cell fusion. The spleen cells were mixed with the myeloma cells at a ratio by cell count of 1 . 5. After centrifuging and removing the supernatant, the precipitated cell clusters were thoroughly loosened and then added to 1 ml of a mixture [polyethylene glycol 4000 (2 g), MEM (2 ml), and dimethyl ~ulfoxide] under stirring. After maintaining at 37°C for 5 minutes,- MEM was slowly added thereto so as to adjust the total amount to 10 ml. After the mixture was centrifuged, the supernatant was removed and the cell clusters were gently loosened. 30 ml of a normal medium (PRMI-1640 containing 10% of fetal bovine serum) was added thereto and the cells were slowly suspended with the use of a measuring pipet.
The suspension was pipetted into a 96-well incubation plate and incubated in an incubator containing 5~ of COz at 37°C for 24 hours. Then HAT medium was added thereto and the incubation was continued for 10 to 14 days. Subsequently, a part of the culture supernatant was sampled and subjected to hybridoma screening.
(2) To obtain a monoclonal antibody undergoing a cross :reaction between influenza A virus subtypes, the above-mentioned culture supernatant, which had not been diluted, was used as a ;primary antibody and a staining test on MDCK cells infected with the three subtypes (H1N1, H2N2 and H3N2) was effected. The staining test was carried out in accordance with the above-mentioned method described in Journal of Clinical Microbiology.
Specifically, the MDCK cells infected with the human influenza virus subtype strains (H1N1: A/Yamagata/120/86, H2N2:
A/Okuda/57, H3N2: A/Fukuoka/C29/85) were rinsed with PBS (pH
7.4) on 96-well microtiter plates (Falcon 3072; manufactured by Becton Dickinson Labware) and fixed with absolute ethanol at room temperature for 10 minutes. Then these cells were continuously treated with 4 antibodies [the above-mentioned culture supernatant containing the monoclonal antibody, rabbit anti-mouse immunoglobulin G serum (manufactured by Organon Teknika) diluted 1000-fold, goat anti-rabbit immunoglobulin G

serum (manufactured by Organon Teknika) diluted 500-fold, and ~eroxidase-rabbit anti-peroxidase complex (manufactured by C>rganon Teknika) diluted 1000-fold, each for 40 minutes, and the cells thus treated were washed with PBS. Finally, the peroxidase reaction was effected by the method of Graham and Rarnovsky [see Histochem. Cytochem., 14, 291 - 302 (1966)] with the use of 0.01 HZOz and 0.3 mg/ml of 3,3'-diaminobenzidine tetrahydro-chloride in PBS. The stained cells were observed under an ordinary light microscope to sort antibodies recognizing .respectively the H1N1 subtype-infected MDCK cells and the H2N2 subtype-infected MDCK cells. Next, the cells in the wells where the production of these antibodies had been confirmed were taken out and treated by the limiting dilution thrice to thereby clone the target cells. The hybridoma strain thus cloned was named Hybridoma C179, while the monoclonal antibody produced thereby was named monoclonal antibody C179.
The Hybridoma C179 has been deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology under the accession number PERM BP-4517.
(3) 5 x 106/animal of the above-mentioned hybridomas were intraperitoneally administered to Balb/c mice treated with pristane. Ten to 21 days thereafter, the ascites of a mouse having ascites cancer thus induced was sampled and centrifuged at 3000 rpm for 5 minutes to thereby remove solid components and give an ascites fluid. This fluid contained about 5 mg/ml of the monoclonal antibody C179 (hereinafter referred to simply as C179). After purifying with Protein A-Sepharose 4B (manufactured by Pharmacia), C179 was confirmed as an antibody of the IgG2a type.

,f 2121559 3.Properties of monoclonal antibody:
(1) A 100-fold dilution of the ascites fluid as described in the above Referential example A-2-(3) was diluted stepwise and the staining test as described in the above Referential example A-2-(2) was effected to examine the antigen recognizing characteristics of C179. The H1N1 subtype strains used included A/PR/8/34, A/Bangkok/10/83, A/Yamagata/120/86, A/Osaka/930/88, A.1/FM/1/47 and A/Suita/1/89. The H2N2 subtype strains used included A/Okuda/57, A/Adachi/2/57, A/Kumamoto/1/65, A/Kaizuka/2/65 and A/Izumi/5/65. The H3N2 subtype strains used included A/Aichi/2/68, A/Fukuoka/C29/85, A/Sichuan/2/87, A/Ibaraki/1/90, A/Suita/1/90 and A/Kitakyushu/159/93. Further, B/Nagasaki/1/87 was used as an influenza B virus strain.
C179 recognized all of the H1N1 subtype and H2N2 subtype strains but did not recognize the H3N2 subtype strains and the influenza~virus B strain.
(2) The neutralization activity of the antibody was determined by effecting the above-mentioned influenza virus rapid focus reduction neutralization test in accordance with the description of Arch. Virol., 86, 129 - 135 (1985) and Microbiol.
Immunol., 29, 327 - 335 (1985). The ascites fluid of the above Referential example A-2-(3) was used as an antibody, to which was added thrice by volume as much a receptor destroying enzyme (RDE: manufactured by Takeda Chemical Industries, Ltd.) solution before the use. After reacting at 37°C for 18 hours, the RDE was inactivated by heating at 56°C for 45 minutes. Finally, a 16-fold dilution of the ascites fluid was prepared and subjected as a test sample to the determination as will be described hereinbelow.
Namely, 10'/well of MDCK cells were pipetted into 96-well microplates. On the next day, the abovementioned antibody (16-2~l ~~21~~9 fold dilution) diluted in 4 steps was mixed with the equal amount of the suspension of each virus strain of 30 focus-forming units/well prepared in the above Referential example A-3-(1), and the mixture was kept at 37°C for 1 hour. Then 25 ul of this mixture was pipetted into the wells of the microtiter plates containing the above-mentioned MDCR cells and kept at 37°C for 30 minutes. Then the solution in each well was removed and the well was rinsed with PBS. Next, MEM containing 0.50 of t:ragacanth gum (manufactured by Wako Pure Chemical Industries, htd.) and 5 ug/ml of trypsin was added thereto. After being kept at 37°C for 20 to 24 hours, the solution added above was removed and each well was rinsed with PBS. Then the cells were fixed by treating with absolute ethanol at room temperature for 10 minutes. Then these cells were dried and stained in accordance with the staining test as described in the above Example 2-(2).
After the completion of the staining, the cells were rinsed with tap water and dried. Then the stained foci were counted under a .Light microscope.
C179 inhibited the focus formation of all of the H1N1 subtype and H2N2 subtype strains and had a potent virus neutralization activity. On the other hand, it exerted no effect on the focus formation by the H3N2 subtype strains and the :influenza B virus strain. The plaque reduction neutralization test gave similar results.
(3) The haemagglutination inhibition (HI) activity of the antibody was examined by the following method. The antibody (32-fold dilution) which had been treated with RDE in the same manner as the one described in the above Refent.ial example A-3-(2) was~diluted stepwise and mixed with each virus strains (16 HAU) as described in the above Referntial example A-3-(1) to effect a reaction at room temperature for 30 minutes. After adding avian erythrocytes and well mixing, the effect of the _intibody on the haemagglutination activity of each virus strain was examined. It was found that the haemagglutination activity of none of the virus strains was affected by C179.

(4) The fusion inhibition activity of the antibody was determined by the above method as described in Nature, 300, 658 -- 659 (1982) with a few slight modifications. Namely, monolayer cultures of CV-1 cells were infected with each of the virus strains as described in the above Referntial example A-3-(1). 24 hours after the inoculation, the cells were washed twice with I)MEM and then kept at 37°C in DMEM containing 10 ug/ml of t:rypsin for 15 minutes. Subsequently, the cells were washed twice with DMEM and kept at 37°C in the ascites fluid of the above Refential example 2-(3) diluted with DMEM for 30 minutes.
~Chereafter, the cells were treated for 2 minutes at 37°C with a i:usion medium (RPMI free from NaZCO~, containing 0.2~ bovine serum albumin, 10 mM MES and 10 mM HEPES) adjusted to pH 5Ø
Then the cells were washed twice with DMEM to remove the fusion medium, and then kept at 37°C for 3 hours in DMEM containing 2~
of fetal bovine serum. Next, the cells were fixed with absolute methanol and subjected to Giemsa's staining. Then the formation of polykaryons was examined under a light microscope.
C179 inhibited the polykaryon formation by all of the H1N1 and H2N2 subtype strains but did not inhibit the formation by t:he H3N2 subtype strain and the influenza B virus strain.
As discussed above, C179 is an antibody which specifically recognizes the H1N1 and H2N2 subtypes, inhibits membrane fusion of viruses and exhibits a neutralization activity. Table 1 summarizes these results, 2q Table 1 Antibody Vl.ruS titers Fusion of C179 measured by Staining' NeutralizationbHI inhibitions A/PR/8/34 1, 638, 512 <32 -f-A/Bangkok/10/83 1, 638, 512 <32 -+-A/Yamagata/120/86409. 600 1, 024 <32 +

A/Osaka/930/88 409.600 5t2 <32 +

A/Sui ta/1/89 409, 600 I, 024 <32 +

A1/FM/1/47 409.600 512 <32 +

A/Okuda/57 1, 638, 1, 024 <32 +

AlAdachi/2/57 1, 638. 1, 024 <32 +

A/Kumamoto/1/65 409. 600 l, 024 <32 +

A/Ka i zuka/2/65409. 600 2, 048 <32 A/Izumi/5/65 409, 600 l, 024 <32 +

A2/Aichi/2/68 <104 <16 <32 -A/Fukuoka/C29/85<100 <16 <32 -A/Sichuan/2/87 <I00 <16 <32 A/Ibaraki/1/90 <100 <I6 <32 -A/Suita/1/90 <100 <16 <32 -A/KitakyuShu/159/93 <16 <32 -<100 B

B/Nagasaki/1/87 <100 <16 <32 -' Staining test.
b Neutralization test.
Hemagglutination inhibition test.
d Fusion inhibition test.

In the above Table 1, each number represents the dilution ratio of the ascites fluid of the Refential example A-2-(3), a staining titer is expressed in the maximum dilution ratio of the ascites fluid whereby cells can be stained in the staining test, while a neutralization activity is expressed in the maximum dilution ratio of the ascites fluid whereby the formation of foci can be suppressed up to a level corresponding to one half of the focus count in the control lot wherein no antibody is added. Symbol + means that polykaryon formation is completely inhibited by a 1000-fold dilution of the ascites fluid, while symbol - means that polykaryon formation is not inhibited even by using a 10-fold dilution of the ascites fluid. A 32-fold dilution of the ascites fluid shows no HI activity.
4_ Determination of epitope:
(1) It was determined by immunoprecip.itation that the protein recognized by C179 was HA molecules. Specifically, MDCK
cells were infected with an H2N2 subtype strain A/Okuda/57 via adsorption for 30 minutes and then incubated in MEM wherein methionine was replaced with 10 uCi of ['SS]methionine for 24 hours to thereby label the infected cells. Next, the cells were harvested~and suspended again in an RIPA buffer solution [50 mM
Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1~ Nonide~ P-40, 1~
deoxycholate and 0.1~ SDS]. After removing the insoluble matters by centrifuging, a supernatant was obtained. Then this supernatant was mixed with C179 and kept at 4°C for 1 hour.
Protein A-Sepharose CL4H beads were added thereto and kept at room temperature for 2 hours to thereby allow the beads to adsorb the immunoprecipitate. These beads were collected, washed * Trade-mark ~~21559 times with an RIPA buffer solution and boiled to thereby liberate the protein binding to C179. Then this protein was electrophoresed on an SDS-12.5 polyacrylamide gel. The gel was faxed, soaked in a 1 M sodium salicylate solution and dried to effect autoradiography. The labeled protein binding to C179 was thus identified with the HA molecule of A/Okuda/57 based on its electrophoretic pattern. The H1N1 subtype strains, other ~I2N2 subtype strains and the H3N2 subtype strain were also tested in the same manner. It was found that C179 under'aent immunoprecipitation spec=ifically together with all of the H1N1 and H2N2 subtype strains but showed no avidity on the HA
molecule of the H3N2 subtype.
(2) In the presence of C179, MDCK cells infected with the H1N1 subtype or the H2N2 subtype were incubated to thereby give an antigen variant having no sensitivity to 0179. More specifically, A/Suita/1/89 of the H1N1 subtype and A/Izumi/5/65 of the H2N2 subtype were used each as a parent strain. MDCK
<:ells infected with each of these virus strains were incubated in the presence of C179. Thus variants capable of growing in the presence of C179 were separately isolated in a pure state from plaques of the MDCK cells. A variant of A/Suita/1/89 was named A/Suita/1/89(R) while a variant of A/Izumi/5/65 was named A/Izumi/5/65(R). These two variants had no reactivity with C179 both in the staining test and in the neutralization test. Each of/these variants was a mild infection strain having a low plaque forming ability, having no pathogenicity to mice used as test animals and capable of growing only in cultured cells.
(3) In order to specify the antigen recognition site of the antibody, a HA gene was analyzed.
(a) Synthesis of pr:i.mers: Primers 5 to 26 were synthesized with a DNA synthesizer, freed from the protective group and purified by ion exchange HPLC (TSK Gel, DEAE-2SW Column). After 3esalting with Sep-pack C18, about 50 erg portions of DNAs were obtained.
(b) MDCK cells infected with A/Suita/1/89 were harvested and guanidine isothiocyanate was added thereto. The mixture was repeatedly sucked and discharged 5 times with the use of a syringe to thereby dissolve the cells. After the completion of the dissolution, the cell extract was layered over a cesium chloride solution and ultracentrifuged. The precipitate on the bottom of a centrifuging tube was dissolved in a buffer solution, treated with phenol and chloroform, and precipitated from ethanol. The RNA thus recovered was used as a sample of virus genome RNA. Next, cDNAS were synthesized by using the primer 5 and the cDNAs thus synthesized were amplified by the PCR method with the use of. the primers 5 and 6. The cDNAs thus amplified were next separated by agarose gel electrophoresis to thereby elute a cDNA band of 1.7 kbp corresponding to the FiA
gene. This cDNA was further amplified by the PCR method with the use of the primers 5 and 6. To the amplified fragment was added 10% (w/v) of polyethylene glycol in 60% (v/v) of a 2.5 M NaCl solution. After centrifuging, a purified precipitate fraction was obtained.
Next, the base sequence of the gene thus purified was determined by the dideoxy method with the use of a thermal cycler as described in the above-mentioned Bio-Techniques wherein primers 7 to 14 which were sequencing primers for the FI1N1 subtype labeled with (~-"p~ were employed. More specifa.cally,.2 pmol of a primer was annealed with 1 pmol of the purified fragment by heating to 95°C for 3 minutes and then quenching. After adding Taq polymerase, the mixture was kept at 72°C for 10 minutes in a buffer solution containing deoxynucleotide and dideoxynucleotide, thus effecting a :~olymerase extension reaction. To complete the extension reaction, the reaction mixture was transferred into the thermal c;ycler, where a cycle of heating at 90°C for 1 minute, at 55°C
f:or 2 minutes and at 72°C for 3 minutes was repeated 10 times.
After the completion of the cycling, the reaction mixture was heated to 95°C for 3 minutes in the presence of formamide, quenched in ice and then electrophoresed on an 8~ denatured polyacrylamide gel. After the completion of the electrophoresis, t;he gel was dried and exposed with the use of an X-ray film.
Then the base sequence was read out to thereby determine the base sequence of the whole HA gene represented by the SEQ ID No.
27 in the sequence listing.
(c) The base sequence of the HA gene of A/Suita/1/89(R) was analyzed in accordance with the method as described in the above Referencial example A-4-(3)-(b). Thus the base sequence of the whole HA gene was determined and compared with the HA gene of the parent strain. As a result, it was found out that the HA
gene of the variant underwent nucleotide replacement at three positions. More precisely, G of the base No. 627, G of the base No. 736 and C of the base No. 1018 in the HA gene of the parent strain mutated respectively into A, A and A. When an HA molecule was cleaved with a protease at one site, its viral infectivity was activated. After the cleavage, the larger polypeptide was called HA1 while the smaller one was called HA2. These polypeptides were bound to each other via an S-S bond. This mutation was accompanied by amino acid replacements at the 189-, 225- and 318-positions i.n HA1. Amino acid residues at the 189-and 225-positions were located in a highly variable region and the replacement at the 318-position (Thr -~ Lys; ACA ~ AAA on the nucleotide level) was responsible for the C179 nonreactivity of the variant. In the present specification, amino acid position .in HA molecule are assigned in accordance with the H3 numbering method as described in Virus, 11, 257 - 266 (1961).
(d) The base sequences of HA genes of A/Izumi/5/65 and A/Izumi/5/65(R) were analyzed in accordance with the method as described in the above Refential example A-4-(3)-(b), except that primers 15 to 23 which were sequencing primers for the H2N2 :subtype were used. The base sequence of the HA gene of A/Izumi/5/65 is represented by the SEQ ID No. 28 in the sequence listing. The HA gene of this variant underwent nucleotide replacement at one position. Namely, T of the base No. 1197 in the HA gene of the parent strain mutated into A. This mutation was accompanied by an amino acid replacement at the 52-position of HA2. This replacement at the 52-position (val ~ Glu; GTA
~SAA on the nucleotide level) was responsible for the C179 nonreactivity of the variant.
(e) In order to specify the amino acid sequence around the 318-position of HA1 and the amino acid sequence around the 52-position of HA2 of the HA molecule of each of A/PR/8/34, A/Bangkok/10/83, A/Yamagata/120/86 and A/Osaka/930/88 of the H1N1 subtype, A/Okuda/57, A/Adachi/2/57, A/Kumamoto/1/65 and A/Kaizuka/2/65 of the H2N2 type and A2/Aichi/2/68, A/Fukuoka/C29/85, A/Sichuan/2/87, A/Ibaraki/1/90 and A/Suita/1/90 of the H3N2 subtype, a part of each HA gene was sequenced.
In the case of the strains of the H1N1 subtype, cDNA of the RNA genome of each virus was synthesized in accordance with the method as described in the above Referential example A-4-(3)-(b) and this cDNA was amplified by PCR with the use of the primers 9 and 13. By using the DNA fragment thus obtained as a template, the base sequence was determined by the dideoxy method with the use of a thermal cycler and the primers 11 and 12.
In the case of the strains of the H2N2 subtype, cDNA of the RNA genome of each virus was synthesized in accordance with the method as described in the above Referential example A-4-(3)-(b) and this cDNA was amplified by PCR with the use of the primers I7 and 21. By using the DNA fragment thus obtained as a template, the base sequence was determined similarly by the dideoxy method with the use of the primers 19 and 20.
In the case of the strains of the H3N2 subtype, cDNA of the RNA genome of each virus was synthesized in accordance with the method as~described in the above Referential example A-4-(3)-(b) .and this cDNA was amplified by PCR with the use of the primers 24 and 26. By using the DNA fragment thus obtained as a template, the base sequence was determined similarly by the dideoxy method with the use of the primers 25 and 26.
In the H1N1 and H2N2 subtypes, the TGLRN polypeptide sequence at the 318- to 322-positions in the HA1 region (A
region.) represented by the SEQ ID No. 1 in the sequence listing and the GITNKVNSVIEK golypeptide sequence at the 47- to 58-positions in the HA2 region (B region) represented by the~SEQ ID
No. 2 in the sequence listing are conserved. In the H3N2 subtype, on the other hand, the TGMRN polypeptide sequence at the 318- to 322-position in the HA1 region (A' region) represented by the SEQ ID No. 3 in the sequence listing and the QINGFCI~NR(L/v)IEK polypeptide sequence at the 47- to 58-positions in the HA2 region (B' region) represented by the SEQ ID No. 4 in the sequence listing are conserved. The A region differs from the A' region by one amino acid, while the B region differs from the B' region by 5 or 6 amino acid residues. The differences among these regions are responsible for the difference in the antigen recognition of the antibody. Thus the antibody could not ~1215~9 :react with the H3N2 subtype in the serological and fusion inhibition tests.
As Fig. 1 shows, the TGLRN polypeptide sequence of the A
region represented by the SEQ ID No. 1 in the sequence listing and the GITNKVNSVIEK polypeptide sequence of the B region represented by the SEQ ID No. 2 in the sequence listing are close to each other at the center of the stem region of the HA
molecule. C179 recognizes both of these sequences and thus this site corresponds to the epitope of C179. C179 binds to the stem region of the HA molecule and thus inhibits the membrane fusion action of the HA molecule and neutralizes the virus.
H1N1 subtype: The sequence of the base Nos. 1017 to 1031 of the HA gene of the A/Suita/1/89 represented by the SEQ ID No. 27 in the sequence listing codes for the A region, while the sequence of the base Nos. 1191 to 1226 thereof codes for the B
region. The SEQ ID No. 29 in the sequence listing shows a part of the HA gene of A/PR/8/34, wherein the sequence of the base Nos. 76 to 90 codes for the A region while the sequence of the base Nos. 250 to 285 codes for the B region. The SEQ ID No. 30 in the sequence listing shows a part of the HA gene of A/Bangkok/10/83, wherein the sequence of the base Nos. 76 to 90 codes for the A region while the sequence of the base Nos. 250 to 285 codes for the B region. The SEQ ID No. 31 in the sequence listing shows a part of the HA gene of A/Yamagata/120/86 wherein the sequence of the base Nos. 76 to 90 codes for the A region while the sequence of the base Nos. 250 to 285 codes for the B region. The SEQ ID No. 32 in the sequence listing shows a part of the HA gene of A/Osaka/930/88 wherein the sequence of the base Nos. 76 to 90 codes for the A region while the sequence of the base Nos. 250 to 285 codes for the B
region.

21~155~
H2N2 subtype: The sequence of the base Nos. 1007 to 1021 of :he HA gene of the A/Izumi/5/65 represented by the SEQ ID No. 28 in the sequence listing codes for the A region, while the sequence of the base Nos. 1181 to 1216 thereof codes for the B
region. The SEQ ID No. 33 in the sequence listing shows a part of the HA gene of A/Okuda/57, wherein the sequence of the base Nos. 94 to 108 codes for the A region while the sequence of the base Nos. 268 to 303 codes for the B region. The SEQ ID No. 34~
:in the sequence listing shows a part of the HA gene of A/Adachi/2/57, wherein the sequence of the base Nos. 103 to 117 codes for the A region while the sequence of the base Nos. 277 to 312 codes for the B region. The SEQ ID No. 35 in the sequence :Listing shows a part of the HA gene of A/Kumamoto/1/65, wherein the sequence of the base Nos. 104 to 118 codes for the A region while the sequence of the base Nos. 278 to 313 codes for the B
region. The SEQ ID No. 36 in the sequence listing shows a part ~~f the HA gene of A/Kaizuka/2/65, wherein the sequence of the base Nos. 88 to 102 codes for the A region while the sequence of the base Nos. 262 to 297 codes for the B region.
H3N2 subtype: The SEQ ID Nos. 37, 38, 39, 40 and 41 in the sequence listing respectively show a part of HA genes of .A2/Aichi/2/68, A/Fukuoka/C29/85, A/Sichuan/2/87, A/Ibaraki/ 1/90 and A/Suita/1/90. In each case, the sequence of the base Nos. 84 to 98 codes for the A' region while the sequence of the base Nos. 258 to 293 codes for the B' region.
As Fig. 2 shows, the TGMRN polypeptide sequence of the A' region represented by the SEQ ID No. 3 in the sequence listing and the QINGKLNR(L/V)IER polypeptide sequence of the B' region represented by the SEQ ID No. 4 in the sequence listing are close to each other at the center of the stem region of the HA
molecule.

Preventive effect on influenza virus:
In order to examine the preventive effect of C179, an influenza virus infection test was carried out by using mice.
One ml/animal of a C179 solution (1 mg/ml in PBS) was _Lntraperitoneally administered to 10 Balb/c mice. After 1 day, 25 ~1 of a 1000-fold dilution of A1/FM/1/47 (4000 HAU) of the H1N1 subtype was intranasally administered. As a control, 12 mice were inoculated with the virus alone.
As Fig. 3 shows, 8 mice in the control group died (two mice after 5 days, five after 6 days and one after 8 days). Other surviving mice in this group were extremely weakened. In ~~ontrast, the mice administered with C179 showed no abnormality and all remained healthy even after 14 days.
Fig. 3 is a graph showing the survival ratios of the C179-administered group and the control group wherein the ordinate .indicates the survival ratio while the abscissa indicates the time (days) after the infection with the virus.
Refrential example B
1. Preparation of viruses:
A strain of H5N3 subtype used was A/whistling swan/
Shimane/476/83. A strain of H6N6 subtype used was A/whistling swan/Shimane/37/80. A strain of H7N7 subtype used was A/turfted duck/Shimane/124R/80. A strain of H8N4 subtype used was A/turky/
Ontario/6118/68. A strain of H10N7 subtype used was A/chicken/
Germany"N"/49. Each strain is a stock of the Research Institute for Microbial Diseases. A/chicken/ Germany"N"/49 has the amino acid sequences represented respectively by SEQ ID No. 3 and SEQ
ID No. 4 in the HA molecule, but other strain lack these sequences.

21215~~
Each strain was inoculated into the allantoic cavity of an embryonated hen egg aged 11 days, incubated at 34°C for 4 days and then harvested.
2. Preparation of monoclonal antibodies:
(1) Balb/c mice were immunized with two doses of A2/
Aichi/57 strain (320 HAU) prepared in the above Referential example A-1, which had been suspended in Freund's complete adjuvant before use, via intraperitoneal injection one month apart. One month thereafter, the mice were boosted by intraperitoneally injecting a suspension of the same antigen (320 HAU) in PBS. Three days thereafter, the spleen of each animal was taken out and thus spleen cells were prepared.
Mouse myeloma cells were prepared by incubating p3x63Ag8 cells in a DME medium containing 10~ of fetal bovine serum for 2 days after passage and then washing with physiological saline before cell fusion. The spleen cells were mixed with the myeloma cells at a ratio by cell count of 1 . 5. After centrifuging and removing the supernatant, the precipitated cell clusters were thoroughly loosened and then added to 1 ml of a mixture [polyethylene glycol 4000 (2 g), MEM (2 ml), and dimethyl sulfoxide] under stirring. After maintaining at 37°C for 5 minutes, MEM was slowly added thereto so as to adjust the total amount to 10 ml. After the mixture was centrifuged, the supernatant was removed and the cell clusters were gently loosened. 30 ml of a normal medium (PRMI-1640 containing 10$ of fetal bovine serum) was added thereto and the cells were slowly suspended with the use of a measuring pipet.
The suspension was pipetted into a 96-well incubation plate and incubated in an incubator containing 5~ of COZ at 37°C for 24 hours. Then HAT medium was added thereto and the incubation was continued for 10 to 14 days. Subsequently, a part of the culture supernatant was sampled and subjected to hybridoma screening.
(2) To obtain a monoclonal antibody undergoing a cross reaction between H3N2 subtype and H10N7 subtype, the above-mentioned culture supernatant, which had not been diluted, was used as a primary antibody and a staining test on MDCK cells infected with the three subtypes (H3N2, H10N7 and H1N1) was effected.~The staining test was carried out in accordance with the above-mentioned method described in Referential example A-2-(2). Specifically, the MDCK cells infected with the influenza virus subtype strains (H3N2: A2/Aichi/2/68, H10N7:
A/chicken/ Germany"N"/49, H1N1: A/PR/8/34) were rinsed with PBS
(pH 7.4) on 96-well microtiter plates (Falcon 3072) and fixed with absolute ethanol at room temperature for 10 minutes. Then these cells were continuously treated with 4 antibodies [the above-mentioned culture supernatant containing the monoclonal antibody, rabbit anti-mouse immunoglobulin G serum diluted 1000-fold, goat anti-rabbit immunoglobulin G serum diluted 500-fold, and peroxidase-rabbit anti-peroxidase complex diluted 1000-fold, each for 40 minutes, and the cells thus treated were washed with PBS. Finally, the peroxi_dase reaction was effected by the method of Graham and Karnovsky with the use of 0.01 HzOz and 0.3 mg/ml of 3,3'-diaminobenzidine tetrahydrochloride in PBS. The stained cells were observed under an ordinary light microscope to sort antibodies recognizing respectively the H3N2 subtype-infected MDCK cells and the H10N7 subtype-infected MDCK cells. Next, the cells in the wells where the production of these antibodies had been confirmed were taken out and treated by the limiting dilution thrice to thereby clone the target cells. The hybridoma strain thus cloned was named Hybridoma AI3C, while the monoclonal antibody produced thereby was named monoclonal ~-1 antibody AI3C.
The Hybridoma AI3C has been deposited with Fermentation Research Institute, Agency of Industrial Science and Technology under the accession number FERM Bp-4516.
(3) 5 x 106/animal of the above-mentioned hybridomas were intraperitoneally administered to Balb/c mice treated with pristane. Ten to 21 days thereafter, the ascites of a mouse having ascites cancer thus induced was sampled and centrifuged at 3000 rpm for 5 minutes to thereby remove solid components and dive an ascites fluid. This fluid contained about 5 mg/ml of the monoclonal antibody AI3C (hereinafter referred to simply as AI3C). AI3C was purified with Protein A-Sepharose 4B.
3. Properties of monoclonal antibody:
(1) A 100-fold dilution of the ascites fluid as described =~n the Referential examp=Le B-2-(3) was diluted stepwise and the staining test as described in the above Referential example A-2-(2) was effected to examine the antigen recognizing <:haracteristics of AI3C. The H1N1 subtype strains used included A/PR/8/34, A/Bangkok/10/83, A/Yamagata/120/86, A/Osaka/930/88, A/Suita/1/89 and A1/FM/1/47. The H2N2 subtype strains used included A/Okuda/57, A/Adachi/2/57, A/Kumamoto/1/65, A/Kaizuka/2/65 and A/Izumi/5/65. The H3N2 subtype strains used included A/Aichi/2/68, A/Fukuoka/C29/85, A/Sichuan/2/87, A/Ibaraki/1/90, A/Suita/1/90 and A/Kitakyushu/159/93. Further, B/Nagasaki/1/87 was used as an influenza B virus strain and the :strains described in the Referential example B-1 were used.
AI3C recognized all of the H3N2 subtype and A/chicken/
Germany"N"/49 but did not recognize the H1N1 subtype strains, H2N2 subtype strains, the influenza virus B strain, and other subtype strains.
(2) The HI activity of the antibody was examined by the following method. The antibody (32-fold dilution) which had been treated with RDE in the same manner as the one described in the above Referential example A-3-(2) was diluted stepwise and mixed with each virus strains (16 HAU) as described in the above Referential example B-1 and B-3-(1) to effect a reaction at room i~emperature for 30 minutes. After adding avian erythrocytes and well mixing, the effect of the antibody on the haemagglutination activity of each virus strain was examined. It was found that the haemagglutination activity of none of the virus strains was affected by AI3C.
4. Determination of epitope:
It was determined by immunoprecipitation that the protein recognized by AI3C was HA molecules. Specifically, MDCK cells were infected with an H3N2 subtype strain A2/Aichi/2/68 via adsorption for 30 minutes and then incubated in MEM wherein methionine was replaced with 10 uCi of ('SS]methionine for 24 hours to thereby label the infected cells. Next, the cells were harvested~and suspended again~in an RIPA buffer solution [50 mM
Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1~ Nonidet P-40, 1~
deoxycholate and 0.1~ SDS]. After removing the insoluble matters by centrifuging, a supernatant was obtained. Then this supernatant was mixed with AI3C and kept at 4°C for 1 hour.
Protein A-Sepharose CL4B beads were added thereto and kept at room temperature for 2 hours to thereby allow the beads to adsorb the immunoprecipitate. These beads were collected, washed times with an RIPA buffer solution and boiled to thereby liberate the protein binding to AI3C. Then this protein was 2~.2~~5:9 electrophoresed on an SDS-12.5 polyacrylamide gel. The gel was fixed, soaked in a 1 M sodium salicylate solution and dried to effect autoradiography. The labeled protein binding to AI3C
was thus identified with the HA molecule of A2/Aichi/2/68 based on its electrophoretic pattern. The H1N1 subtype strains, H2N2 subtype strains, other H3N2 subtype strains, and strains described in above Reference B-1 were also tested in the same manner. It was found that AI3C underwent immunoprecipitation specifically together with all of the H3N2 subtype strains and A/chicken/Germany"N"/49 but showed no avidity on the HA molecule of the other subtypes.
[Brief Description of the Drawings]
[Fig. 1]
Fig. 1 is a schematic view of the tertiary structure of a IiA molecule and shows the position of common conserved regions .in HA molecules of H1N1 and H2N2 subtypes.
[Fig. 2]
Fig. 2 is a schematic view of the tertiary structure of a 13A molecule and shows the position of common conserved regions :in HA molecules of H3N2 subtype.
[Fig. 3]
Fig. 3 is a graph showing the survival ratio of a group infected with influenza virus.
[Fig.4]
Fig. 4 is a graph showing the survival ratio of a group infected with influenza virus.
[Fig. S]
Fig. 5 is a graph showing the average body weight loss of a group infected with influenza virus.
[Fig. 6]
Fig. 6 is a graph showing the survival ratio of a group 'C~-q-~~21~59 infected with influenza virus.
[Examples]
To further illustrate the present invention in greater detail, and not by way of limitation, the following Examples will be given.
Example 1 Construction of the stem region polypeptide:
(1) Synthesis of primers: Primers 27 to 30 were synthesized with a DNA synthesizer, freed from the protective group and purified by ion exchange HPLC (TSK Gel, DEAF-2SW Column). After desalting with Sep-pack C18, about 50 Ng portions of DNAs were obtained.
Primers 27 and 28 have the sequences of 5'-terminal of HA gene of H2N2 subtype, and primers 29 and 30 have the complimentary sequences of 3'-terminal of one. The base sequences of primers 27 to 30 are represented respectively by the SEQ ID Nos. 42 to 45.
(2) MDCK cells infected with A/Okuda/57 were harvested and guanidine isothiocyanate was added thereto. The mixture was repeatedly sucked and discharged 5 times with the use of a syringe to thereby dissolve the cells. After the completion of the dissolution, the cell extract was layered over a cesium chloride solution and ultracentrifuged. The precipitate on the bottom of~a centrifuging tube was dissolved in a buffer solution, treated with phenol and chloroform, and precipitated from ethanol. The RNA thus recovered was used as a sample of virus genome RNA. Next, cDNAs were synthesized by using the primer 5 and the cDNAs thus synthesized were amplified by the PCR method with the use of the primers 27 and 29. The cDNAs thus amplified were next separated by agarose gel electrophoresis to 2121~~9 thereby elute a cDNA band of 1.8 kbp corresponding to the HA
gene. This cDNA was further amplified by the PCR method with the use of the primers 28 and 30. To the second amplified fragment of 1.8 kbp was added 20~ (w/v) of polyethylene glycol in 60~
(v/v) of a 2.5 M NaCl solution. After centrifuging, a purified precipitate fraction was obtained.
(3) The base sequence of HA gene for A/Okuda/57 was analyzed by the methods described in Referential example A-4-(3)-(b),(d). The base and amino acid sequences of it are represented by the SEQ ID No. 46. In the sequence represented by the SEQ ID No. 46, the sequence of the base Nos. 1 to 5 originates in primer 28, the sequence of base Nos. 6 to 48 is the non-coding regions, the sequence of base Nos. 49 to 93 is the coding region for signal polypeptide, the sequence of base Nos. 94 to 231 is the coding region for the stem region of N-terminal domain of HA molecule, the sequence of base Nos. 232 to 873 is the coding region for the globular head region of HA molecule, the sequence of base Nos. 874 to 1734 is the coding region for the stem region of C-terminal domain of HA
molecule, the sequence of base_Nos. 1735 to 1775 is the non coding region, and the sequence of base Nos. 1776 to 1783 originates in primer 30.
(4) Construction of the plasmids.
(a) The terminals of the 1.8 kbp DNA fragment prepared in Referential example 1-(2) was treated by T4 DNA polymerase for creating blunt ends. It was ligated with a plasmid pHSG299 (manufactured by Takara Shuzo Co. Ltd.,) digested with restriction enzyme SmaI by T4 DNA ligase. E. coli ,TM109 was transformed with the ligated sample and some kanamycin resistant transformants were gotten. A plasmid pH2-299 which containing HA
gene was prepared from one of these transformants. E. coli JM109 harboring the plasmid pH'2-299 was named Escherichia coli JM109/pH2-299 and has been deposited with National Institute of Hioscience and Human- Technology, Agency of Industrial Science and Technology under the accession number FERM P-13431.
(b} A plasmid pEF-BOS/neoA, which is a shuttle vector for mammalian cell and E: coli, was constructed by A 2:6 kbp BamHI
DNA fragment from pMAMneo-s (manufactured by Clontech Lab. Inc.}
inserting into AatII site of a plasmid pEF-BOS (Nucleic Acids Research, 18; 5322 (1990)] .
Then the 1.8 kbp NheI DNA fragment from pH2-299 and pEF-HOS/neoA digested with restriction enzyme Xbal were ligated by T4 DNA ligase. E. coli JM109 was transformed with the ligated sample and some ampicillin resistant transformants was gotten. A
plasmid which containing the HA gene was prepared from one of these transformants and named pEHNaH2. E. coli JM109 harboring the plasmid pEBNaH2 was named Escherichia call JM109/pEBNaH2.
{c) Primers 31 and 32, represented respectively by the SEQ
ID Nos. 47 and 48, were synthesized by using DNA synthesizer and purified with HPLC (TSK gel, DEAF-2SW column} and Sep-pak C18.
The primer 31 has a complementary sequence to the sequence of the base Nos. 207 to 231 in the SEQ ID No. 46. The primer 32 has a sequence to sequence of the base Nos. 874 to 899 (but base No.
876 is changed A to C) in the SEQ ID No.~46. The amplification of 3.8 kbp DNA fragment which is lacking the region coding for the g7,obulat head region of gA molecule from pH2-299 was tried by PCR method using these primers.
The PCR reaction was performed with 50 pmol of primer 3l, 50 pmoi primer 32 and 0.5 pmol of pH2-299 prepared from Escherlchla coll°
JM109/pH2-299 (FERM F-13431) as template. The reaction was performed for 25 cycJ.es with each cycle consisting of l minute at 90°C, 2 minutes at 55°c, 3 minutes at 72°c. And a 3.8;
kbp 4'7 fragment was amplified. Then this fragment was phosphorylated by T4 kinase, treated with T4 DNA polymerase for creating blunt ends, and ligated by T4 DNA lipase to make plasmid. E. coli ~~-1M109 was transformed with the ligeted palsmid and some kanamycin resistant transformante were gotten. A plasmid prepared from one of these transformante was named p299H25n-c, that was containing the FiA gene which was lacking the region coding for the globular head region (the base Nos. 232 to 873 in the SEQ ID No. 46) and having the coding region for the stem region of N-terminal domain of HA molecule and C terminal domain of HA molecule joined. A 1.1 kbp DNA fragment containing the gene coding for the stem region polypeptide was prepared from p299H2Sn-c by digestion of restriction enzyme NheI. The base seguence for this fragment and the amino acid sequence of the stem region polypeptide translated from this DNA fragment were represented respectively by the SEQ ID No. 49 and SEQ ID No. 50 in the sequence listing. A plasmid that had the gene coding for the stem region polypeptide was constructed by ligation of the l.lkbp NheI fragment from p299H2Sn-c and pEF-HOS/neoA digested with XbaI with T4 DNA lipase. E. coli JM109 was transformed with the ligated sample and some ampicillin resistant transformats were gotten. A plasmid containing the gene coding for the stem region polypeptide was named pENH2dH01, and E_ coli Jhil09 harboring the plasmid pENH2dH01 was named Escherichia coli JM109/pENH2dH01 and has been deposited with National Institute of Bioscience and Human- Technology, Agency of Industrial science and Technology under the accession number FERM BP-4190.

(5) Expression of polypeptides:
The plasmid pENH2dH01 containing the gene coding for the stem region polypeptide was prepared from Escherichia coli JM109/pENH2dH01 and the plasmid pEBNaH2 containing FiA gene was prepared from Escherichia coli ,7M1091pEBNaH2.
Trypsin treated CV-1 cells (5x106 cells) were washed with 20m1 10% FCS-MEM in one time, and 20m1 PBS in two times, and suspended in lml PBS. The 0.8m1 part of it and the plasmid pENH2dH01 (30N~g) were put into a cuvette for Genepulser~' (manufactured by BioRad), and the cuvette was set into Genepulser~'. The cells and plasmid were treated in 250V, 960 OFD
by Genepulser~". After the sample. was put at 0°C for 10 minutes, the cells were suspended in 30m1 10% FCS-MEM and 5m1 each was cultured in a dish (6cm) for two days.
The CV-1 cells transformed with the plasmid pENH2dH01 were washed with PBS (pH7.4) and fixed with absolute ethanol at room temperature for 10 minutes. Focus staining was done by successive treatment of the cells with C179 (1:1000), rabbit anti-mouse immunoglobulin G serum (1:1000), goat anti- rabbit immnuoglobulin G serum (1:500), and peroxidase-rabbit anti-peroxidase (PAP) complex (1:1000). Each treatment was 40 minutes long and was followed by a washing with PBS. The peroxide reaction was developed for about 5 minutes by the methed.of Graham and Karnousky in which 0.01% H~OZ and 0.3 mg of 3,3'-diaminobenzidene tetrahydrochloride per ml in PBS were used.
The CV-1 cells transformed with pENH2dH01 were stained by immunostaining with C179. So the expressed the stem region polypeptide had normal structure of high dimension for the stem region of HA molecule in spite of lacking of the globular head region of HA molecule. As this polypeptide is lacking the globular head region of HA. molecule which is apt to become antigenic determinants and to arise antigenic mutation, it will be able to become the antigen that induce the antibodys recognizing the stem region of HA molecule and counteracting 4q 21'~1~a9 both H1N1 subtype and H2N2 subtype influenza viruses, like C179 type antibody. So this stem region polypeptide is useful for the influenza vaccine.
Similarly, the CV-1 cells transformed with pEBNaH2 were stained by immunostaining method with C179, so the expressed p~olypeptide also had normal structure of high dimension for the stem region of HA molecule.
Example 2 Construction of the stem region polypeptide:
(1) Synthesis of primers: Primers 33 to 35 were synthesized with a DNA synthesizer, freed from the protective group and purified by ion exchange HPLC (TSK Gel, DEAE-2SW Column). After desalting with Sep-pack <:18, about 50 ug portions of DNAs were obtained.
Primers 33 has the sequences of 5'-terminal of HA gene of H3N2 subtype, and primers 34 and 35 have the complimentary sequences of 3'-terminal of one. The nucleotide sequences of primers 33 to 35 are represented respectively by t:he SEQ ID Nos. 51 to 53.
(2) MDCK cells infected with A2/Aichi/2/68 were harvested and guanidine isothiocyanate was added thereto. The mixture was repeatedly sucked and discharged 5 times with the use of a syringe to thereby dissolve the cells. After the completion of the dissolution, the cel:L extract was layered over a cesium chloride solution and ultracentrifuged. The precipitate on the bottom-of a centrifuging tube was dissolved in a buffer solution, treated with phenol and chloroform, and precipitated from ethanol. The RNA thus recovered was used as a sample of ~rirus genome RNA. Next, cDNAs were synthesized by using the primer 5 and the cDNAs thus synthesized were amplified by the PCR method with the use of the primers 33 and 34. The cDNAS thus amplified were next separated by agarose gel electrophoresis to thereby elute a cDNA band of 1.8 kbp corresponding to the HA
gene. This cDNA was further amplified by the PCR method with the use of the primers 33 and 35. To the second amplified fragment of 1.8 kbp was added 20% (w/v) of polyethylene glycol in 60%
(v/v) of a 2.5 M NaCl solution. After centrifuging, a purified precipitate fraction was obtained.
(3) The hase sequence of HA gene for A2/Aichi/2/68 was analyzed by the methods described in Referential example A-4-(3)-(b),(d). The base and amino~acid sequences of it are represented by the SEQ ID No. 54 in the sequence listing. In the sequence No. 54, the sequence of the base Nos. 1 to 8 originates in primer 33, the sequence of base Nos. 9 to 36 is the non coding regions, the sequence of base Nos. 37 to 84 is the coding region for signal polypeptide, the sequence of base Nos. 85 to 246 is the coding region for the stem region of N-terminal domainof HA molecule, the sequence of base Nos. 247 to 903 is the coding region for the globular head region of HA molecule,.
the sequence of base Nos. 904 to 1734 is the coding region for the stem region of C-terminal domain of HA molecule, the sequence of base Nos. 1735 to 1769 is the non coding region, and the sequence of base Nos. 1770 to 1777 originates in primer 35.
(4) Construction of the plasmids:
(a) The terminals of the 1.8 kbp DNA fragment prepared in example 2-(3) was treated by T4 DNA polymerase for creating blunt ends. It was ligated with a plasmid pUC118 (manufactured by Takara Shuzo Co. Ltd.,) digested with HincII by T4 DNA
ligase. E. coli JM109 was transformed with the ligated sample and some ampicillin resistant transformants were gotten. A
plasmid which containing HA gene was prepared from one of these transformants and named pU118H3xxn. E. coli JM109 harboring the plasmid pI1118H3xxn was named Escherichia coli ,TM109/pU118H3xxn and has been deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology under the accession number FERM P-13567.
(b) Primers 36 and 37, represented respectively by the SEQ
ID Nos. 55 and 56, were synthesized by using DNA synthesizer and purified with HPLC (TSK gel, DEAF-2Sw column) and Sep-pak C18.
The primer 36 has a complementary sequence to the sequence of the base Nos. 227 to 246 in the SEQ ID No. 54. The primer 3? has a sequence to sequence of the base Nos. 904 to 923 in~the SEQ ID
rto. 54. The amplification of 4.3 kbp DNA fragment which was lacking the region coding for the globular head region of HA
molecule from pU11BH3xxn was tried by PCR method using these primers. The PCR reaction was performed with 50 pmol of primer 36, 50 gmol primer 37 and 0.05 pmol of pU118H3xxn prepared from Escherichia coli JM109/pU118H3xxn (FFRM P-13567) as template. The reaction was performed for 25 cycles with each cycle consisting of 1 minute at 90°C, 2 minutes at 55°c, 3 minutes at 72°c.
And a 4.3 kbp fragment was amplified. Then this fragment was phosphorylated by T4 kinase, treated with T4 DNA ploymerase for creating blunt ends, and ligated by T4 DNA ligase to make plasmid. E. co.Li JM109 was transformed with the ligeted palsmid and some ampicillin resistant transformante were gotten. A
plasmid prepared from one of these transformante was named p118H3dH01, that was containing the HA gene which was lacking the region coding for the globular head region (the base Nos.
247 to 903 in the SEQ ID No.54) and having the coding region for the stem region of N-terminal domain of HA molecule and C
terminal domain of HA molecule joined. A 1.1 kbp DNA fragment containing the gene coding for the stem region polypeptide was prepared from p118H3dH01 by digestion of Nhel and Xbal. The nucleotide sequence for this Lragment and the amino acid sequence of the stem region polypeptide translated from this DNA
fragment were represented respectively by the SEQ ID No. 57 and SEQ ID No. 58 in the~sequence listing. A plasmid that had the gene coding for the stem region polypeptide was constructed b.y ligation of the l.lkbp Nhel fragment from p118H3dH01 and pEF-HOSlneoA digested With XbaI with T4 DNA lipase. E_ coli 3M109 was transformed with the ligated sample and some ampicillin resistant transformats were gotten. A plasmid prepared from one of these transformants was named pENH3dH01 that was containing the gene coding for the stem region polypeptide, and E. toll JMlo9 harboring the plasmi.d pENH3dH01 was named Escherichia toll JM109/pENH3dH01 and has been deposited with National Institute of Bioscience and Human- Technology, Agency of Industrial and technology under the accession number FERM BP-4518_ (5) Expression of the stem region polypeptide:
The plasmid pENH3dH01 containing the gene coding for the stem region polypeptide was prepared from Escherichia toll J'M109/pENH3dFi0I (FERM BF-4518). ' Trypsin treated CV-1 cells (5x10' cells) were washed with 20m1 10$ FCS-MEM in one time, and 20m1 PBS in two times, and suspended in lml PBS. The 0.8m1 part of it and the plasmid pENH3dH01. (30~g) were put into a cuvette for Genepulsern', and the cuvette was set into Genepulser=". The cells and plasmid were treated in 250V, 960 /OFD by GenepulserT". After. the sample was put at 0°C for 10 minutes, the cells were suspended in 30m1 10%
FCS-MEM and Sml each was cultured in a dish (6cm) for two days.
The Cv-1 cells transformed with the plasmid pENE33dH01 were washed with PBS (pH7.4) and fixed with absolute. ethanol at roam temperature for IO minutes. Focus staining Was done by successive treatment of the cells with AI3C {1:1000), rabbit anti-mouse immunoglobulin G serum (1:1000), goat anti-rabbit immnuoglobulin G serum (1:500), and peroxidase-rabbit anti-peroxidase {PAP) complex (1:1000). Each treatment was 40 minutes long and was followed by a washing with PBS. The peroxide reaction was developed for about 5 minutes by the methed of Graham and Karnousky in which 0.01 HZOz and 0.3 mg of 3.3-diaminobenzidene tetrahydrochloride per ml in PBS were used.
The CV-1 cells transformed with pENH3dH01 were stained by immunostaining with AI3C_ So the expressed the stem region polypeptide peptides had normal structure of high dimension for the stem region of HA molecule of H3N2 subtype in spite of lacking of the globular head region of HA molecule. This polypeptide is lacking the globular head region of HA molecule which is apt to became antigenic determinants and to arise antigenic, mutation, it will be able to become the antigen that induce the antibodys recognizing the stem-region of HA molecule of H3N2 subtype influenza viruses, like AI3C type antibody. So this stem region polypeptide is useful for the influenza vaccine.
Example 3 Preparation of antigen polypeptide;
(1) Preparation of HA molecules Viral particles (40 mg) of A/Yamagata/32J89 prepared in Referential example A-1 ware suspended in 27 ml of 5 mM Tris-HC1 (pH 8.~). After adding 3 ml of 20~ NP-40, the mixture was maintained at 37°C for 30 minutes. Then it was centrifuged and the supernatant was collected and filtered through a 0.8 ~.cm filter unit (Millex*'PF: manufactured by * Trade-mark 2121~~~
Millipore). Subsequently the filtrate was loaded on an ion exchange membrane (memSep DEAE: manufactured by Millipore) and washed with the same buffer. Further, HA molecules were eluted with the same buffer containing 1 M of NaCl.
(2) Treatment of HA molecule with proteinase In an N-ethylmorpholine buffer solution (pH 7.5), the HA molecules (2.6 wg) prepared in the above Example 3-(1) were digested with 4-pmol portions of lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.), V8 protease (manufactured by Sigma Chemical Co.) and chymotrypsin (manufactured by Boehringer) at 37°C for 1 hour.
The HA molecules (2.6 ~,g) prepared in the above Example 3-(1) were denatured by maintaining at 42°C in the presence of 2 M of urea for 1 hour. Next, these molecules were digested with 4-pmol portions of lysyl endopeptidase, V8 protease, chymotrypsin, subtilisin (manufactured by Boehringer), proteinase K
(manufactured by Boehringer), pronase (manufactured by Boehringer) and thermolysin (manufactured by Boehringer) in a 50 mM tris hydrochloride buffer solution (pH 7.6) at 37 C for 12 hours and then dialyzed against PBS.
A portion of each digestion mixture was collected and the digested fragments were analyzed by the dot-blot method with the use of C179 and SDS
polyacrylamide gel electrophoresis.
The dot-blot method was effected in the following manner.

CA 02121559 2003-12-23 a._.
1 ~.1 of the digestion mixture was loaded onto a nitrocellulose filter (manufactured by MSI) and dried.
The same procedure was repeated 5 times to thereby load 5 ~,l of the digestion mixture in total. Then blocking was carried out with the use of Blockace (manufactured by Snow Brand Milk Products Co.). Next, it was reacted with a S00-fold dilution of a C179 solution at room temperature for 1 hour. After washing with a tris hydrochloride buffer solution (pH 7.6) containing 0.02% of Tween*20, washing was further effected with a tris hydrochloride buffer solution (pH 7.6) for 10 minutes thrice.
Then it was reacted with a S00-fold dilution of an alkaline phosphatase-labeled goat anti-mouse immunoglobulin G solution (manufactured by Orgenics, Ltd.) at room temperature for 1 hour and washed in the same manner as the one described above. Finally, the alkaline phosphatase reaction was performed by using a solution of nitro blue tetrazolium 5-bromo-4-chloro-3-indolyl phosphate in carbon/sodium carbonate (pH 9.0) in tFie presence of 1 mM of MgClz.
As a result, it was found out that most of the HA
molecules remained undigested when treated with each of these proteases in the absence of urea. The HA
molecules, which had been denatured with urea, employ-ed as a substrate were not digested with V8 protease, thermolysin and pronase. When lysyl endopeptidase, chymotrypsin and subtilisin were used, the digestion proceeded excessively and the antigenicity for C179 completely disappeared. When proteinase R was used, * Trade-mark on the other hand, it was confirmed that the HA
molecules were digested. and polypeptide fragments.
having an avidity for C179 were formed.
(3) Preparation of stem region polypeptide To the HA molecules (250 a g/1400 ~ 1) prepared in Example 3-(lj were successively added 100 a 1 of 1 M
Tris-HC1 {pH 7.6) and 500 ul of 8 M urea and the resulting mixture was maintained at 42°C for 1 hour.
To this solution was added 2000 u1 of an immobilized Proteinase K gel and maintained at 37°C for 7 hours under shaking. After centrifuging, the reaction mixture thus obtained was dialyzed against PBS for 12 hours and thus the stem region polypeptide was obtained. The immobilized Proteinase K gel was prepared in the following manner.
4 mg of Proteinase K (manufactured by Boehringer) was dissolved in 1 ml of Hz0 and the pH value of the solution was adjusted to 5.0 with 0.1 N HC1. After adding 1 ml of ECH-Sepharose (manufactured by Pharmacia) and 1 ml of 0.2 M EDC (pH 5.0) thereto, the mixture was maintained at 4°C for 24 hours. This gel was washed with 10 m1 portions of PBS thrice to thereby give the immobilized Proteinase R gel.
(4) Properties of stem region polypeptide By using the stem region polypeptide of Example -(3) as a test sample, the antigenicity for C179 was examined by the ELISA method. Namely, a diluted solution of the stem region polypeptide~was added to a microtiter plate (Maxi Sorp; manufactured by Nunc) and ~snmobilized at 37°C for 90 minutes. Then blocking was 5~7 effected by using Block Ace (manufactured by Snow Brand Milk Products). Then these cells were continuously reacted with 2 antibodies [10 mg/ml C179 solution diluted 200-fold, and peroxidase-labeled goat anti-mouse immunoglobulin G
solution (manufactured by Cappel) diluted 500-fold]
each for 90 minutes and the cells thus treated were washed with PBS. Finally, the peroxidase reaction was effected by using 0.03 HzOz and 1 mg/ml of o-phenylenediamine dihydrochloride in citric acid/
phosphoric acid (pH 5.?_). The amount of the antigen was calculated from the absorbance of the reaction mixture at 492 nm. As a standard, HA molecules described in Example 3--(1) were used.
As the result of the ELISA method, it has been proved that this. stem region polypeptide has an antigenicity comparable to that of HA molecules.
The haemagglutination activity (HA value) of the stem region polypeptide was determined in the following manner. On a U-shaped 96-well microtiter plates (Falcon 3911: manufactured by Becton Dickinson Labware), the sample solution was diluted with PBS in two steps. Then the same amount of a 0.5~ avian erythrocyte suspension was added thereto and the mixture was stirred well. After reacting at room temperature for 1 hour, agglutination of the erythrocytes was observed. The highest dilution ratio showing agglutination was taken as the HA value.
The HA value of the stem region polypeptide was less than 1/1000 of the HA value of HA molecules.
Thus it has been clarified that the stem region polypeptide prepared by the treatment with the protease has an antigenicity comparable to that of HA
molecules and the haemagglutination activity originating in the globular head region has substantially disappeared.
This polypeptide can easily serve as an antigen determinant and the globular head region, which is liable to undergo antigen mutation, has been digested therefrom. Thus it is usable as a vaccine capable of specifically recognizing the stem region of the H1N1 ,and H2N2 subtypes and inducing an antibody neutralizing the virus.
Example 4 Preparation of antigen polypeptide:
(1) Preparation of HA molecules viral particles (40 mg) of A/-Kitakyushu/159/93 prepared in Referential example A-1 ware suspended in 27 ml of 5 mM Tris-HC1 (pH 8..~).~ After adding 3 ml of 20~ NP--40, the mixture was maintained at 37°C for 30 minutes. Then it was centrifuged and the supernatant was collected and filtered through a 0.8 Er,m filter unit (Millex PF: manufactured by Millipore). Subsequently the filtrate was loaded on an ion exchange membrane (memSep DEAE* manufactured by Millipore) and washed with the same buffer. Further, HA molecules were eluted with the same buffer containing 1 M of NaCl.
(2) Treatment of HA molecule with proteinase In an N-ethylmorpholine buffer solution (pH 7.5), the HA molecules (2.6 ~.g) prepared in the above * Trade-mark 5q 212:1559 Example 4-(1) were digested with 4-pmol portions of lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.), V8 protease (manufactured by Sigma Chemical Co.) and chymotrypsin (manufactured by Boehringer) at 37'C for 1 hour.
The HA molecules (2.6 ug) prepared in the above Example 4-(1) were denatured by maintaining at 42°C in the presence of 2 M of urea for 1 hour. Next, these molecules were digested with 4-pmol portions of lysyl endopeptidase, V8 protease, chymotrypsin, subtilisin (manufactured by Boehringer), proteinase K
(manufactured by Boehringer), pronase (manufactured by Boehringer) and thermolysin (manufactured by Boehringer) in a 50 mM tris hydrochloride buffer.
solution (pH 7.6) at 37 C for 12 hours and then dialyzed against PBS.
A portion of each digestion mixture was collected and the digested fragments were analyzed by the dot-blot method with the use of AI3C and SDS
polyacrylamide gel electrophoresis.
As a result, it was found out that most of the HA
molecules remained undigested when treated with each of these proteases in the absence of urea. The HA
molecules, which had been denatured with urea, employ-ed as a substrate were not digested with V8 protease, thermolysin and pronase. When lysyl endopeptidase, chymotrypsin and subtil:isin were used, the digestion proceeded excessively and the antigenicity for AI3C
completely disappeared. When proteinase R was used, on the other hand, it was confirmed that the HA
molecules were digested. and polypeptide fragments.
having an avidity for AI3C were formed.
(3) Preparation of stem region polypeptide To the HA molecules (250 a g/1400 ul) prepared in Example 4-( 1 ) .were successively added 100 ,u 1 of 1 M
Tris-HC1 (pH 7.6) and 500 u1 of 8 M urea and the resulting mixture was maintained at 42°C for 1 hour.
To this solution was added 2000 ~cl of an immobilized Proteinase K gel and maintained at 37°C for 7 hours under shaking. After centrifuging, the reaction mixture thus obtained was dialyzed against PBS for 12 hours and thus the stem region polypeptide was obtained.
(4) Properties of stem region polypeptide By using the stem region polypeptide of Example 4-(3)~ as a test sample, the antigenicity for AI3C was examined by the ELISA method. Namely, a diluted solution of the stem region polypeptide~was added to a microtiter plate (Maxi Sorp; manufactured by Nunc) and immobilized at 37°C for 90 minutes. Then blocking was effected by using Block Ace (manufactured by Snow Brand Milk Products). Then these cells were continuously reacted with 2 antibodies [10 mg/ml AI3C solution diluted 200-fold, and peroxidase-labeled goat anti-mouse immunoglobulin G
solution (manufactured by Cappel) diluted 500-fold]
each for 90 minutes and the cells thus treated were washed with PBS. Finally, the peroxidase reaction was effected by using 0.03 HZOz and 1 mg/ml of o-phenylenediamine dihydrochloride in citric acid/
phosphoric acid (pH 5.2). The amount of the antigen was calculated from the absorbance of the reaction mixture at 492 nm. As a standard, HA molecules described in Example 4-(1) were used.
As the result of the ELISA method, it has been proved that this. stem region polypeptide has an antigenicity comparable to that of HA molecules.
'rhe haemagglutination activity (HA value) of the stem region polypeptide was determined in the following manner. On a U-shaped 96-well microtiter plates (Falcon 3911: manufactured by Becton Dickinson :Gabware), the sample solution was diluted with PBS in two steps. Then the same amount of a 0.5~ avian ~arythrocyte suspension was added thereto and the mixture was stirred well. After reacting at room temperature for l hour, agglutination of the erythrocytes was observed. The highest dilution ratio showing agglutination was taken as the HA value.
The HA value of the stem region polypeptide was less than 1/1000 of the HA value of HA molecules.
Thus it has been clarified that the stem region polypeptide prepared by the treatment with the protease has an antigenicity comparable to that of HA
molecules and the haemagglutination activity originating in the globular head region has substantially disappeared.
This polypeptide can easily serve as an antigen determinant and the globular head region, which is liable to undergo antigen mutation, has been digested therefrom. Thus it is usable as a vaccine capable of spec if ically recognizing the stem region of H3N2 subtype and inducing an antibody neutralizing the virus.
Example 5 Preventive effect on influenza virus From Escherichia coli JM109/pENH2dH01 (FERM BP-4190), a plasmid pENH2dH01 having, integrated thereinto, a gene codes for a polypeptide lacking the globular head region of A/Okuda/57 (H1N1) HA molecule was prepared.
Trypsin treated CV-1 cells (5x106 cells) were washed with 20m1 10~ FCS-MEM in one time, and 20m1 PBS in two times, and suspended in lml PBS. The o.8m1 part of it and the plasmid pENH3dH01 (30mg) were put into a cuvette for Genepulser~', and the cuvette was set inta GenepulserT"'. The cells and plasmid were treated in 250v, 960 mFD by Genepulser~'. After the sample was put at 0°C for 10 minutes, the cells were suspended in 60m1 10~
FCS-MEM and Sml each was cultured in a dish (6cm), On the third' day of the incubation, the expression of the polypeptide was confirmed by a staining test with the use of C179. Cells in which the polypeptide had been expressed were treated with PBS
containing trypsin and then harvested by centrifugation. The cells thus harvested were suspended in PBS and intraperitoneally administered:.to female BALB/c mice aged 4 weeks as a vaccine. in a dose of 1 x 105/animal. Two weeks thereafter, the second immunization was carried out in the same manner. As a control, CV-1 cells which had not been transformed by pENH2dH01 were used. These control cells were also intraperitoneally administered twice t.o 10 mice in a dose of 1 x 1051cells/animal. One week after the final immunization, 25 a 1 ( 8 x 10° FFU) of A1/FM/1/47 (H1N1) was intranasally administered to the mice. Subsequently, the life or death of the animals was checked everyday.
Fig. 4 shows the results. As Fig. 4 shows, 7 mice among 10 of the test group (black circle) .immunized with the CV-1 cells with the expression of the antigen polypeptide survived 15 days after the inoculation of the highly toxic strain A1/FM/1/47. In wontrast, 9 mice among 10 of the control group (black triangle) died.
Fig. 4 shows the survival ratios of the test (antigen polypeptide-administered) group and the control group wherein the ordinate refers to the survival ratio while the abscissa refers to the time (days) after the infection with the virus.
Thus it has been clarified that the antigen p~olypeptide lacking the globular head region of HA
molecules can serve as a vaccine for the virus of the H1N1 subtype, though it per se origins in the H2N2 subtype.
This polypeptide can easily serve as an antigen determinant and the globular head region, which is liable to undergo antigen mutation, has been digested therefrom. Thus it is usable as a vaccine capable of specifically recognizing the stem region of the H1N1 and H2N2 subtypes and inducing an antibody neutralizing the virus.

E;~ample 6 21215 5 9 Preventive effect on influenza virus:
By using the stem polypeptide described in the Example 3 as a test samp:Le, the preventive effect on the infection with influenza virus was examined. The stem region polypeptide was suspended in PBS and :intraperitoneally administered to female Balb/c mice.
aged 4 weeks in a dose of 10 ug/0.5 ml/animal. The .animals were immunized thrice in total by repeating the intraperitoneal administration in the same does at intervals of 1 week. To a control group, PBS alone was administered. Ten days after the final immunization, the animals were intranasally inoculated with 25 ~.l (2.0 x 10' FFtl) per animal of Al/FM/1/47 (H1N1) virus. Then the life and death of the animals were observed and changes in the body weight of surviving mice were monitored.
As Fig. 5 shows the average body weight loss of the mice immunized with the stem region polypeptide was significantly lower than that of the control group. As Fig. 6 shows, further, 5 mice among 11 in the control group died within 7 days after the inoculation with the virus, while 8 mice among 10 immunized with the stem region polypeptide survived for 14 days after the inoculation, thus showing a survival ratio 14 days after the inoculation with the virus of 80~.
On the other hand, the survival ratio of the control group 14 days after the inoculation was 55~.

Fig. 5 is a graph showing the body weight changes of the stem region polypeptide-administered group and the control group wherein the ordinate indicates the average body weight of the surviving mice of each group while the abscissa indicates the time (days) after the inoculation with the virus. Fig. 6 is a graph showing the survival ratios of the stem region polypeptide-administered group and the control group wherein the ordinate indicates the survival ratio of each group while the abscissa indicates the time (days) after the inoculation with the virus.
Thus it has been clarified that the antigen polypeptide lacking the globular head region of HA
molecules can serve as a vaccine for the influenza virus.
[Effects of the Invention]
The present invention provides an immunogenic polypeptide capable of producing an antibody, which binds specifically to the stem region in HA molecule of the subtypes of human influenza A virus, and a gene coding for this polypeptide.
The above-mentioned polypeptide can be supplied in a large amount through genetic engineering techniques and is not affected by any changes in antigenicity due to changes in the globular head region in HA molecule. Thus it is particularly useful as a vaccine for preventing influenza.

sequence Listing SEQ ID N0:1 LENGTH: 5 TYPE: amino acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: peptide FLAGMENT TYPE: internal fragment SEQUENCE DESCRIPTION:SEQ ID N0:1:
Thr Gly Leu Arg Asn SEQ ID N0:2 LENGTH: 12 TYPE: amino acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: peptide FLAGMENT TYPE: internal fragment SE~aUENCE DESCRIPTION:SEQ ID N0:2:
Gl;y Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys SEQ ID N0:3 LENGTH: 5 TYl'E:amino acid STIIANDEDNESS:single TOPOLOGY: l i near 6~1 MO' ';ULE TYPE: peptide FLAGMENT TYPE: internal fragment SEQUENCE DESCRIPTION:SEQ ID N0:3:
Thr Gly Met Arg Asn SEQ ID N0:4 LENGTH: 12 TYPE: amino acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: peptide FLAGMENT TYPE: internal fragment FEATURE:
LOCATION:9 NAME/KEY:VaI or Leu SEQUENCE DESCRIPTION:SEQ ID N0:5:
Gln Ile Asn Gly Lys Leu Asn Arg Xaa Ile Glu Lys SEQ ID N0:5 LENGTH: 19 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID NO:5:

6g ~~.21559 SE~ ID N0:6 LENGTH: 21 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID NC:6:

SEQ ID N0:7 LENGTH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:7:

SEQ ID N0:8 LENGTH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOL,OGY:linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:8:
GCCCC;ACTAC AATTGGGGAA ATG 23 SEQ ID N0:9 LENGTH: 24 6q TY- 'nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:9:

SEQ ID N0:10 LENGTH: 24 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTfON:SEQ ID N0:10:

SEQ ID N0:11 LENGTH: 22 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:11:

SEQ ID N0:12 LENGTH: 24 TYPE: nucleic acid STRANDEDNESS:single T0~ ',OGY: I i near MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:12:

SEQ ID N0:13 LENGTH: 24 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:13:

SEQ ID N0:14 LENGTH: 24 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:14:

SEQ ID N0:15 LENGTH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acrid (synthetic DNA) SE ?NCE DESCRIPTION:SEQ ID N0:15:

SEQ ID N0:16 LENGTH: 20 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:16:

SEQ ID N0:17 LENGTH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:17:

SEQ ID N0:18 LENGTH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N():18:

~2 SEQ ID N0:19 LENGTH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:19:

SEQ ID N0:20 LENGTH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:20:

SEQ ID N0:21 LENGTH: 24 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:21:

SEQ ID N0:22 LE TH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:22:
CATAC'TTGGG ATAATCATAC GTC 23 SEQ ID N0:23 LENGTH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOhOGY:linear MOLECULE TYPE: Other nucleic ac: id (synthetic DNA) SEQUI?NCE DESCRIPTION:SEQ ID N0:23:
GCCA~.'TTATG CTACAGTAGC AGG 23 SEQ 'D N0:24 LENGTH: 24 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUI?NCE DESCRIPTION:SEQ ID N0:24:
GATC;~GATTG AAGTGACTAA TGCT 24 SEQ ID N0:25 LENGTH: 24 TYPE: nucleic acid ST NDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic ac: id (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:25:

SEQ :D N0:26 LENGTH: 23 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:26:

SEQ ID N0:27 LENGTH: 1754 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/Suita/1/89 SEQUENCE DESCRIPTION:SEQ :
ID N0:27 CTACAGATGC AGACACAATATGTATAC;GCTACCATGCGAA CAACTCAACC GACACTGTTG120 TTGCCGGATG GATCTTAGGAAACCCA(~AATGCGAATCACT GTTTTCTAAG GAATCATGGT300 2121~~9 CC ~ATTGC AGAAACACCAAACTCCGAGA ATGGAACATGTTACCCAGGGTATTTCGCCG 360 ACTATGAGGAACTGAGGGAG~CAATTGAGTT CAGTATCATCATTCGAGAGATTCGAAATAT 420 CCCAT'AATGGGAAAAGCAGTTTTTACAGAA ATTTGCTATGGCTGACGGGGAAGAATGGCT 540 TGTAC;CCAAATCTGAGCAAGTCCTATGTGA ACAACAAAGAGAAAGAAGTCCTTGTACTAT 600 GGGGT'GTTCATCACCCGTCTAACATAG(~GGACCAAAGGGCCATCTATCATACAGAAAATG 660 CTTAT'GTCTCTGTAGTGTCTTCACATTATA GCAGGAGATTCACCCCAGAAATAGCAAAAA 720 GACCC;AAAGTAAGAGGTCAAGAAGGAA(~AATTAACTACTACTGGACTCTGCTGGAACCCG 780 GGGAC;ACAATAATATTTGAGGCAAATG(~AAATCTAATAGCGCCATGGTATGCTTTCGCAC 840 ACATTTGGAC~ATATAATGCAGAATTGTTGG TTCTACTGGAAAATGAAAGGACTTTGGATT 1380 TTCA~'GACTCAAATGTGAAGAATCTGTATG AGAAAGTAAAAAGCCAATTAAAGAATAATG 1440 SEQ lD N0:28 LENGTH: 1728 TYPE: nucleic acid ~6 ST VDEDNESS:double TOPOL,OGY:linear MOLECULE TYPE:cDNA to genomic RNA
ORIGINAL SOURCE:
ORGANISM:A/Izumi/5/65 SEQUENCE DESCRIPTION:SEQ ID N0:28:

GAGG(~GGGACCAGATATGCA TTGGATACCATGCCAATAATTCCACAGAAAAGGTCGACAC 120 AATTCTAGAGCGGAATGTCA CTGTGAC'('CATGCCAAGGACATCCTTGAGAAGACCCACAA 180 CGGATGGCTCCTTGGAAATC CAGAATG'1'GATAGGCTTCTAAGGGTGCCAGAATGGTCCTA 300 TGAA(~AATTGAAACATCTCC TCAGCAGCGTAAAACATTTCGAGAAAGTAAAGATTCTGCC 420 CAAA(sATAGATGGACACAGC ATACAAC,4ACTGGAGGTTCAAAGGCCTGCGCAGTGTCAGG 480 CCAT(~CTAATGATGAGGCAG AACAAAGAGCATTGTACCAGGAAGTGGGAACCTATGTTTC 660 GAAT(1GACTAGGAAGTAGAA TGGAATTCTCTTGGACCCTCTTGGATGTGTGGGACACCAT 780 AAAT'fTTGAGAGCACTGGTA ATCTAGTTGCACCAGAGTATGGATTCAAAATATCGAAAAG 840 TA"T~TCAAGAACCTGTATGATAAAGTCACAATGCAGCTGAGAGACAACG TCAAAGAACT 1440 SEQ ID N0:29 LENGTH: 442 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/PR/8/34 SEQUENCE DESCRIPTION:SEQ :
ID N0:29 AATGAAAGGA CTCTGGATTTCC

SEQ ID N0:30 LENGTH: 424 TYPE: nucleic acid STRANDEDNESS:double TO '.OGY: 1 i near MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/Bangkok/ 10/83 SEQUENCE DESCRIPTION:SEQ ID N0:30:

AATG

SEQ ID N0:31 LENGTH: 424 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/Yamagata /120/86 SEQUENCE DESCRIPTION:SEQ ID N0:31:

GT' ",TGATG GATTTCTGGA CATTTGGACA TATAATGCAG AATTGTTGGT CCTACTGGAA 420 AATG

SEQ ID N0:32 LENGTH: 429 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/Osaka/930/88 SEQUENCE DESCRIPT(ON:SEQ :
fD N0:32 AAATT'AAGGA TGGTTACAGGACTAAGGAAC ATCCCATCCATTCAATCCAG AGGTTTGTTT 120 AATGA.AAGG

SEQ ID N0:33 LENGTH: 400 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA to genomic RNA
ORIGINAL SOURCE:
ORGANISM:A/Okuda/57 g0 z~z155~
SEA ,NCE
DESCRIPTION:SEQ
ID N0:33:

SEQ ID N0:34 LENGTH: 409 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/Adachi/2/57 SEQUENCE DESCRIPTION:SEQ :
ID N0:34 SEQ ID N0:35 LENGTH: 410 TYPE: nucleic acid ST' !DEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/Kumamoto/1/65 SEQUENCE DESCRIPTION:SEQ :
ID N0:36 TGAATGCCCC AAATATGTA,4AATCGGAGAAACTGGTCTTA GCAACAGGAC TA,4GGAATGT120 SEQ ID N0:36 LENGTH: 394 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/Kaizuka/2/65 SEQUENCE DESCRIPTION:SEQ ID N0:36:

GTAAA.ATCGG AGAAATTGGTCTTAGCAACA GGACTAAGGAATGTTCCCCA GATTGAATCA 120 TCCAC'PCAAA AGGCATTTGATGGAATCACC AACAAGGTAAATTCTCTGAT TGAAAAGATG 300 g2 TT~ 4CAAAA AGATGGAAGA CGGGTTTCTA GATG 394 SEQ ID N0:37 LENGTH: 329 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A2/Aichi/2 /68 SEQUENCE DESCRIPTION:SEQ ID N0:37:

GCCTATTCGG CGCAATAGCAGGTTTCAT'AGAAAATGGTTG GGAGGGAATG ATAGACGGTT180 AGAAATTCCA TCAAATCGAAAAGGAATTC

SEQ ID N0:38 LENGTH: 334 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA to genomic RNA
ORIGINAL SOURCE:
ORGANISM:A/Fukuoka/C29/85 SEQUENCE DESCRIPTION:SEQ ID N0:38:

g3 GC' 1TTCGGCGCAATAGCAGGTTTCATAGAAAATGGTTG GGAGGGAATG GTAGACGGTT180 SEQ ID N0:39 LENGTH: 329 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/Sichuan/2/87 SEQUENCE DESCRIPTION:SEQ :
ID N0:39 SEQ ID N0:40 LENGTH: 334 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA to genomic RNA
ORIGINAL SOURCE:
ORGANISM:A/Ibaraki/1/90 2121~5~
SE 'NCE
DESCRIPTION:SEQ
ID N0:40:

SEQ ID N0:41 LENGTH: 329 TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA genomic to RNA

ORIGINAL SOURCE:

ORGANISM:A/Suita/1/ 90 SEQUENCE DESCRIPTION:SEQ ID N0:41:

ATGACAAACC CTTTCAAAATGTA\AACAGGATCACATATGG GGCATGTCCC AGATATGTTA60 AGAAATTCCA TCAAACCGAAAAGGAATT'C 329 SEQ ID N0:42 LENGTH: 30 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOr ~ULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:42:

SEQ ID N0:43 LENGTH: 30 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SERUENCE DESCRfPTION:SEQ ID N0:43:

SEQ ID N0:44 LENGTH: 29 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:44:

SEQ ID N0:45 LENGTH: 30 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:45:

CG-"TAGCAG AAACAAGGGT GTTTTTAATT 30 SEQ ID N0:46 LENGTH: 1783 ' TYPE: nucleic acid STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYPE:cDNA o nomic A
t ge RN

ORIGfNAL SOURCE:

ORGANISM:A/Okuda/5 7 SEQUENCE DESCRIPTION:SEQID NC1:46:

TA

GCAAAACA ATG GCC ATT TATCTC ATT CTCCTG TTC ACAGCA GTG AGA GGG

Met Ala Ile Ile TyrL,euIle LeuLeu Phe ThrAla Val Arg Gly 15 -~10 -5 GAC C;AG ATA TGC GGA TACCAT GCC AATAAT TCC ACAGAG AAG 138 ATT

Asp Gln Ile Cys Gly TyrHis Ala AsnAsn Ser ThrGlu Lys Ile CTA -Val A.sp Thr Ile Glu ArgAsn Val ThrVal Thr HisAla Lys Leu GAC A,TC CTT GAG ACC CATAAC GGA AAGTTA TGC AAACTA AAC 228 AAG

Asp Ile Leu Glu Thr HisAsn Gly LysLeu Cys LysLeu Asn Lys CTT

Gly Ile Pro Pro Glu LeuGly Asp CysSer Ile AlaGly Trp Leu CCA

Leu Leu Gly Asn Lys CysAsp Arg LeuLeu Ser ValPro Glu Pro 21~1~~9 65 7p 75 ArgSer Tyr Ile LeuGlu Lys GluAsn Pro ArgAsp Gly Leu Cys TyrPro Gly Ser PheAsn Asp TyrGlu Glu LeuLys His Leu Leu SerSer Val Lys HisPhe Glu LysVal Lys IleLeu Pro Lys Asp ArgT'rpThr Gln HisThr Thr ThrGly Gly SerArg Ala Cys Ala GTGTCT GGT AAT CCATCA TTT TT'CAGG AAC ATGGTC TGG CTG ACA 543 ValSer Gly Asn ProSer Phe PheArg Asn MetVal Trp Leu Thr AAGGAA GGA TCA GATTAT CCG GTTGCC AAA GGATCG TAC AAC AAT 5gg LysGlu Gly Ser AspTyr Pro ValAla Lys G!ySer Tyr Asn Asn ThrSer Gly Glu GlnMet Leu IleIle Trp GfyVal His His Pro ATTGAT GAG ACA GAACAA AGA ACATTG TAC CAGAAT GTG GGA ACC 67g IleAsp Glu Thr GluGln Arg ThrLeu Tyr GlnAsn Val Gly Thr TyrVal Ser Val GlyThr Ser ThrLeu Asn LysArg Ser Thr Pro GAAATA GCA ACA AGGCCT AAA GTGAAT GGA CAAGGA GGT AGA ATG 76g gg Gl~~le Ala ThrArg Pro Lys ValAsn Gly Gln Gly Arg Met Gly ATA

GluPhe Ser TrpThr Leu Leu AspMet Trp Asp Thr Asn Phe Ile GAGAGT ACT GGTAAT CTA ATT GCACCA GAG TAT GGA AAA ATA g5g TTC

Glu Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile SerLys Arg Gly SerSer GlyIle Met LysThr Glu Gly ThrLeu GAGAAC TGT GAG ACCAAA TGCCAA ACT CCTTTG GGA GCA ATAAAT g4g GluAsn Cys Glu ThrLys CysGln Thr ProLeu Gly Ala lleAsn ThrThr Leu Pro PheHis AsnVal His ProLeu Thr Ile GlyGlu 290 2~5 300 CysPro Lys Tyr ValLys SerGlu Lys LeuVal Leu Ala ThrGly CTAAGG AAT GTT CCCCAG ATTGAA TCA AGAGGA TTG TTT GGGGCA 10$3 LeuArg Asn Val ProGln IleGlu Ser ArgGly Leu Phe GlyAla IleAla Gly Phe IleGlu GlyGly Trp GlnGIy Met Val AspGly TrpTyr Gly Tyr HisHis SerAsn Asp GlnGly Ser Gly TyrAIa GC'~ AC AAA GAA TCC ACT CAA AAG GCA TTT GAT GGA ATC ACC AAC 1218 Ala Asp Lys Glu Ser Thr Gln Lys Ala Phe Asp Gly Ile Thr Asn AAG G'rA AAT TCT GTG ATT GAA AAG ATA AAC ACC CAA TTT GAA GCT 1263 Lys Val Asn Ser Val Ile Glu Lys Ile Asn Thr Gln Phe Glu Ala ValGly Lys GluPhe Gly AsnLeu Glu Lys ArgLeu Glu AsnLeu AsnLys Lys MetGlu Asp GlyPhe Leu Asp ValTrp Thr TyrAsn AlaGlu Leu LeuVal Leu MetGlu Asn Glu ArgThr Leu AspPhe HisAsp Ser AsnVal Lys AsnLeu Tyr Ser LysVal Arg MetGln LeuArg Asp AsnVal Lys GluLeu Gly Asn GlyCys Phe GluPhe TyrHis Lys CysAsp Asp GluCys Met Asn SerVal Lys AsnGly ThrTyr Asp TyrPro Lys TyrGlu Glu Glu SerLys Leu AsnArg AsnGlu Ile LysGly Val LysLeu Ser Ser MetGly Val TyrGln 212~~~

IleLeu Ala Ile Tyr AlaThr ValAla GlySer Met Ser Leu Ala IleMet Met Ala Gly IleSer PheTrp ValCys Ser Asn Gly Ser CTGCAG TGC AGG ATC TGCATA TGATTATAAG TCATTTTATA TTAAAAACA

A

LeuGln Cys Arg (le CysIle SEQ ID N0:47 LENGTH: 25 TYPE: nucleic acid STRANDEDNESS:single TOPOhOGY:linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:47:

SEQ 1D N0:48 LENGTH: 26 TYPE: nucleic acid STRANDEDNESS:single TOPOhOGY:linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQU):;NCE DESCRIPTION:SEQ ID N():48:

~12~559 N0:

LENGTH: 35 TYPE: nucleic acid STRAP1DEDNESS:double TOPO(.OGY:linear MOLECULE YPE:cDNA genomic T to RNA

ORIGINAL OURCE:
S

OR(~ANISM :A/Okuda/57 SEQUE?NCE ESCRIPTION:SEQ ID N0:49:
D

CTAG(:AAAAGCAGGGGTTATACCATAGAAA ACCAAAAGCAAAACAATGGC CATCATTTAT60 CTCA'PTCTCCTGTTCACAGCAGTGAGA(sGGGACCAGATATGCATTGGATA CCATGCCAAT120 AATT(;CACAGAGAAGGTCGACACAATTCTA GAGCGGAACGTCACTGTGAC TCATGCCAAG180 GACA'~CCTTGAGAAGACCCATAACGGAAAG TTATGCAAACTAAACGGATC CGGGATCATG240 AAAACAGAAGGAACACTTGAGAACTGT(~AGACCAAATGCCAAACTCCTTT GGGAGCAATA300 AATA(;AACATTACCTTTTCACAATGTCCAC CCACTGACAATAGGTGAGTG CCCCAAATAT360 AGAG(tATTGTTTGGGGCAATAGCTGGTTTT ATAGAAGGAGGATGGCAAGG AATGGTTGAC480 GGTT(iGTATGGATACCATCACAGCAAT(~ACCAGGGATCAGGGTATGCAGC AGACAAAGAA540 GTTC~'GATGGAAAATGAGAGGACACTT(~ACTTTCATGATTCTAATGTCAA GAATCTGTAT780 AGCA1'GGGGGTTTATCAAATCCTTGCCATT TATGCTACAGTAGCAGGTTC TATGTCACTG1020 ATCTGCATATGATTATAAGTCATTTTATAA TTAAAAACACCCT'PGTTTCT GCTAG 1135 q2 SEQ ID N0:50 LENG~'H : 348 TYPE: amino acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUI~,NCE DESCftIPTION:SEQ ID N0:50:
Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp le Leu Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys Glu Thr (,ys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro Phe flis Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr 80 g5 90 Val hys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro (Jln Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile (~lu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser T'hr Gln Lys Ala Phe Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Ile Asn Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Gly Asn Leu Glu Lys Arg Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val '~ys Asn Leu Tyr Ser Lys Val Arg Met Gln Leu Arg Asp Asn Val ~ys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp ,4sp Glu Cys Met Asn Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala Ile Tyr Ala Thr Val Ala Gly Ser Met Ser Leu Ala Ile Met Met Ala Gly Ile Ser Phe Trp Val Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile SEQ ID N0:51 LENGTH: 30 ~'~"°E:naclaic acid STRANDEDNESS:single TOPOLOGY: l i near MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:5I:

SEQ ID N0:52 LENGTH: 29 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:52:

SEQ ID N0:53 LENGTH: 30 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic a;.id (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:53:
CGGC'fAGCAG AAACAAGGGT GTTTTTAATT 30 SEQ ID N0:54 LENGTH: 1777 TYPE: nucleic acid STR.AyDEDNESS:double ~~i1 C~ h TO ~ '~OGY: l i near MOLECULE TYPE:cDNA to genomic RNA
ORIGINAL SOURCE:
ORGANISM:A2/Aichi/2/68 SEQUENCE DESCRIPTION:SEQ ID N0:54:
GATCTA(~AAG CAAAGCAGGG GATAATTCTA TTAATC 36 ATG AAC~ ACC ATC ATT GCT TTG AGC TAC ATT TTC TGT CTG GCT CTC 8I
Met Ly~~ Thr Ile ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu-GGC CA;a GAC CTT CCA GGA AAT GAC AAC AGC ACA GCA ACG CTG TGC 126 Gly Gl1 Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys 1 5 l0 CTG GGA C.AT CAT GCG GTG CCA AAC GGA ACA CTA GTG AAA ACA ATC 171 Leu Gly His fiis Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr .Asp Asp Gln (le Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr Gly Lys Ile Cys Asn Asn Pro His Arg fle Leu Asp Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys .Asp Val Phe Gln Asn Glu Thr Trp Asp Leu Phe Val GIu CGC AGC AAA GCT TTC AGC AAC TG'1' TAC CCT TAT GAT GTG CCA GAT 396 Arg Ser Lys Ala Phe Ser Asn Cys Tyr 1'ro Tyr Asp Val Pro Asp ' 2121559 Tyr AlaSer Leu Arg SerLeu Val Ala SerSer GlyThr Leu Glu TTT ATCACT GAG GGT TTCACT TG(fACT GGGGTC ACTCAG AAT GGG 486 Phe IleThr Glu Gly PheThr Trp Thr GlyVal ThrGln Asn Gly Gly SerAsn Ala Cys LysArg Gly Pro GlySer GlyPhe Phe Ser Arg LeuAsn Trp Leu ThrLys Ser Gly SerThr TyrPro Val Leu AAC G'I'GACT ATG CCA AACAAT GAC AATTTT GAC AAA CTATAC ATT 621 Asn V;31Thr Met Pro AsnAsn Asp AsnPhe .AspLys LeuTyr fle 165 1'~0 Iy5 Trp GlyIle His His ProSer Thr AsnGln Glu Gln ThrSer Leu Tyr ValGln Ala Ser GlyArg Val ThrVal Ser Thr ArgArg Ser CAG CAAACT ATA ATC CCGAAT AT'CGGGTCC AGA CCC TGGGTA AGG

Gln GlnThr Ile Ile ProAsn Ile GlySer Arg Pro TrpVal Arg GGT CTGTCT AGT AGA ATAAGC ATC TATTGG ACA ATA GTTAAG CCG

Gly L.euSer Ser Arg IleSer I;e TyrTrp Thr Ile ValLys Pro GGA CACGTA CTG GTA ATTAAT AGT AATGGG AAC CTA ATCGCT CCT

ctrl . 2121559 ~°'y Asp Val Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro CGG (~GT TAT TTC AAA ATG CGC ACT GGG AAA AGC TCA ATA ATG AGG 891 Arg (~ly Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg TCA (~AT GCA CCT ATT GAT ACC TGT ATT TCT GAA TGC ATC ACT CCA 936 Ser Asp Ala Pro Ile Asp Thr Cys Ile Ser Glu Cys Ile Thr Pro AAT (~GAAGCATT CCC AATGAC AAG CCC TTTCAA AAC GTAAAC AAG 981 Asn Gly Serfle Pro AsnAsp Lys Pro PheGln Asn ValAsn Lys ATC ,4CATATGGA GCA TGCCCC AAG TAT GTTAAG CAA AACACC CTG 1026 Ile 'ThrTyrG1y Ala CysPro Lys Tyr ValLys Gln AsnThr Leu AAG 'TTGGCAACA GGG ATGCGC AAT GTA CCAGAG AAA CAAACT AGA

Lys Leu AlaThr Gly MetArg Asn Val ProGlu Lys GlnThr Arg GGC CTA TTCGGC GCA ATAGCA GGT TTC ATACAA AAT GGTTCG GAG

Gly Leu PheGly Ala IleAla Gly Phe IleClu ,4snGlyTrp G1u GGA ATG ATAGAC GGT TGGTAC GGT TTC AGGCAT CAA AATTCT GAG

Gly Met IleAsp Gly TrpTyr Gly Phe ArgHis Gln AsnSer Glu GGC ACA GGACAA GCA GCAGAT CTT AAA AGCACT CAA GCAGCC ,ATC

Gly Thr GlyGln Ala AlaAsp Leu Lys SerThr Gln AlaAla Ile CAC CAA ATCAAT GGG AAATTG AAC AGG GTAATC G,4GAAGACG AAC

Asp Gln IleAsn Gly LysLeu Asn Arg ValIle Clu LysThr Asn .

G AAA TTCCAT CAA ATCGAA AAG CAA TTCTCA GAA GTAGAA GGG

Glu Lys PheHis Gln I(eGlu Lys Glu PheSer Glu ValGlu Gly AGA ATT CAGGAC CTC GAGAAA TAC GTT GAAGAC ACT AAAATA GAT

Arg Ile GlnAsp Leu GluLys Tyr Val GluAsp Thr LysIle Asp Leu Trp SerTyr Asn AlaGlu Leu Leu ValAla Leu GluAsn Gln His Thr IleAsp Leu ThrAsp Ser Glu MetAsn Lys Leuf'heGiu AAA AC,AAGGAGG CAA CTGAGG GAA AAT GCTGAA GAG ATGGGC AAT 1476 Lys Thr ArgArg GIn LeuArg Gfu Asn AlaGlu Glu MetGly Asn GGT T(~CTTCAAA ATA TACCAC AAA TGT CACAAC GCT TGCATA GAG 1521 Gly Cys PheLys Ile TyrHis Lys Cys AspAsn Ala CysIle Glu TCA A~'CAGAAAT GGT ACTTAT GAC CAT GATGTA TAC AGAGAC GAA

Ser Ile ArgAsn G(y ThrTyr Asp His AspVal Tyr ArgAsp Glu GCA TTA AACAAC CGG TTTCAG AT(:AAA GGTGTT GAA CTGA

AG TCT

Ala Leu AsnAsn Arg PheGln Ile Lys GlyVal Glu LeuLys Ser GGA TAC AAAGAC TGG ATCCTG TC(~ATT TCCTTT GCC ' A

Gly Tyr LysAsp Trp IleLeu Trp Ile SerPhe Ala IleSer Cys TTT TTG CTTTGT GTT GTTTTG CTG GGC TTCATC ATG

TGGGCC TGC

Phe Leu LeuCys Val ValLeu Leu Gly PheIle Met rp Ala Cys T

Y~

m CAG A:~A GGC AAC ATT AGG TGC AAC ATT TGC ATT TGAGTGTATT AGTAATTAAA 1754 Gin Arg Gly Asn Ile Arg Cys Asn Ile Cys Ile SEQ ID N0:55 LENGTH: 20 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTfON:SEQ ID N0:55:

SEQ ID N0:56 LENGTH: 20 TYPE: nucleic acid STRANDEDNESS:single TOPOLOGY: linear MOLECULE TYPE: Other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:SEQ ID N0:56:

SEQ ID N0:57 LENGTH: 1110 TYPE:STRANDEDNESS:double TOPOLOGY: linear MOLECULE TYI'E:cDNA to genomic RNA
m 0' ~ I
NAL SOURCE:

ORGANISM: A2/Aichi/2/68 SEQUENCE
DESCRIPTION:SEQ
ID N0:57:

AATATTGATACCTGTATTTCTGAATG(;ATCACTCCAAATG GAAGCATTCCCAATGACAAG 300 CCC~'TTCAAAACGTAAACAAGATCACATATGGAGCATGCC CCAAGTATGTTAAGCAA~AC 360 ACC(;TGAAGTTGGCAACAGGGATGCGGAATGTACCAGAGA AACAAACTAGAGGCCTATTC 420 GGC(rCAATAGCAGGTTTCATAGAAAATGGTTGGGAGGGAA TGATAGACGGTTGGTACGGT 480 GCCATCGACCAAATCAATGGGAAATT(~AACAGGGTAATCG AGAAGACGAACGAGAAATTC 600 CAT(;AAATCGAAAAGGAATTCTCAGAAGTAGAAGGGAGAA TTCAGGACCTCGAGAAATAC 660 GTT(iAAGACACTAAAATAGATCTCTG(~TCTTACAATGCGG AGCTTCTTGTCGCTCTGGAG 720 AAT(;AACATACAATTGACCTGACTGACTCGGAAATGAACA AGCTGTTTGAAAAAACAAGG 780 TGT(~ACAACGCTTGCATAGAGTCAATc;AGAAATGGTACTT ATGACCATGATGTATACAGA 900 GAC(~AAGCATTAAACAACCGGTTTCAGATCAAAGGTGTTG AACTGAAGTCTGGATACAAA 960 GGG9.'TCATCATGTGGGCCTGCCAGAGAGGCAACATTAGGT GCAACATTTGCATTTGAGTG 1080 SEQ ID N0:58 LENGTH: 346 TYPE: amino acid STRANDEDNESS:single TOPOLOGY: l i near MOLECULE TYPE: peptide ._ 2121559 SE;pUENCE N0:58:
DESCRIPTION:SEQ
ID

MetLys Thr IleIle Ala LeuSer Tyr IlePhe Cys Leu Ala Leu GlyGln Asp LeuPro Gly AsnAsp Asn SerThr Ala Thr Leu Cys LeuGly His HisAla Val ProAsn Gly ThrLeu Val Lys Thr Ile ThrAsp Asp GlnIle Glu ValThr Asn AlaThr Glu Leu Val Gln SerSer Ser ThrGly Lys IleCys Asn AsnIle Asp Thr Cys fle SerGlu Cys IleThr Pro AsnGly Ser IlePro Asn Asp Lys Pro PheGln Asn ValAsn Lys IleThr Tyr GlyAla Cys Pro Lys Tyr ValLys Gln AsnThr Leu LysLeu Ala ThrGly Met Arg Asn Val ProGlu Lys ClnThr Arg GlyLeu Phe GlyAla Ile Ala Gly Phe IleGlu Asn GlyTrp Glu GlyMet Ile AspGly Trp Tyr Gly Phe ArgliisGln AsnSer Glu GlyThr Gly GlnAla Ala Asp Leu Lys SerThr Gln AlaAla Ile AspGln Ile AsnGly Lys Leu Asn Arg ValIle Glu LysThr Asn GluLys Phe HisGln Ile Glu Lys Glu PheSer Glu ValGlu Gly ArgIle Gln AspLeu Glu Lys 'tyrVal G( sp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Glu Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Arg Gly Asn fle Arg Cys Asn Ile Cys Ile l03

Claims (8)

1. An immunogenic artificial polypeptide having an antigenicity of the stem region of haemagglutinin molecule f human influenza A virus and lacking a globular head of said molecule, wherein said polypeptide is selected from the group consisting of:
(a) the polypeptide which contains as least a TGLRN
polypeptide sequence represented by the SEQ ID No. 1 in the sequence listing and a GITNKVNSVIEK polypeptide sequence represented by the SEQ ID No. 2 in the sequence listing; and (b) the polypeptide which contains at least a TGMRN
polypeptide sequence represented by the SEQ ID No. 3 in the sequence listing and a QINGKLNR(L/V)IEK
polypeptide sequence represented by the SEQ ID No. 4 in the sequence listing.

2. An Immunogenic artificial polypeptide as claimed in claim 1, characterized by being obtained by separating from haemagglutinin molecules of human influenza A virus which have been treated with a protease.

3. An immunogenic artificial polypeptide as claimed in claim 2, characterized by being obtained by separating from haemagglutinin molecules of human influenza A virus which have been treated with a proteinase K.

4. An immunogenic artificial polypeptide as claimed in claim 1, wherein said polypeptide is selected from a polypeptide having an amino acid sequence represented by the SEQ ID No. 50 and a polypeptide having an amino acid sequence represented by the SEQ ID NO. 58.

5. An isolated gene which codes for the immunogenic artificial polypeptide having an antigenicity of the stem region of haemagglutinin molecule of human influenza A virus and lacking a globular head region of said molecule, wherein said polypeptide contains at least a TGLRN polypetide sequence represented by the SEQ ID No. 1 in the squence listing and a GITNKVNSVIEK polypeptide sequence represented by the SEQ ID No. 2in the sequence listing.

6. An isolated gene as claimed in claim 5, wherein said gene has a DNA sequence represented by the SEQ ID No. 49.

7. An isolated gene which codes for the immunogenic artificial polypeptide having an antigenicity of the stem region of haemagglutinin molecule of human influenza A virus and lacking a globular head region of said molecule, wherein said polypeptide contains at least a TGMRN polypeptide sequence represented by the SEQ ID No. 3 in the sequence listing and a QINGKLNR(L/V)IEK
polypeptide sequence represented by the SEQ ID No. 4 in the sequence listing.

8. An isolated gene as claimed in claim 7, wherein said gene has a DNA sequence represented by the SEQ ID. No. 57.