KR101768600B1 - Universal influenza virus canine vaccine composition - Google Patents

Abstract

The present invention relates to a generalized influenza virus vaccine composition using an HA2 helical domain of hemagglutinin protein as an influenza surface protein, and a pharmaceutical composition for preventing or treating an influenza infectious disease. The polypeptide represented by SEQ ID NO: 3 of the present invention and the polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 are capable of mass production in E. coli and capable of effectively producing a neutralizing antibody against various influenza subtypes, Since it can enhance the immunity induction effect of existing influenza vaccine, it can be widely used as a general-purpose vaccine against influenza subtype and new variant influenza.

Description

BACKGROUND OF THE INVENTION [0001] The present invention relates to a universal influenza virus vaccine composition,

The present invention relates to a generalized influenza virus vaccine composition using an HA2 helical domain of hemagglutinin protein as an influenza surface protein, and a pharmaceutical composition for preventing or treating an influenza infectious disease.

Influenza is an acute febrile illness caused by influenza virus respiratory infections. Influenza viruses are classified into A, B, and C types according to the difference in surface structure proteins, and there are slight differences in host, epidemiology, and clinical features depending on each. Influenza viruses are subtypes of hemagglutinin (HA) and neuraminidase (NA) glycoproteins exposed on the surface as spherical viruses with a diameter of 80-120 nm. Subtypes are classified mainly by type A influenza. Currently, 16 types of HA from H1 to H16 and 9 types of NA from N1 to N9 are found. Even if calculated, a total of 144 types (for example, H1N1 and H1N2) . Influenza causes new and large new epidemics every year through antigen mutations. Antigen variants are antigenic shifts (eg H3N2 → H2N2), which are replaced by new HA or NA and which are subtyped, and point mutations of existing HA and NA genes, There is an antigenic drift. Antigen urine is a cause of seasonal epidemics because it occurs almost every year, mainly for influenza A and B.

Among HA and NA surface antigens, immunization against HA in particular is associated with influenza prevention and disease severity. Therefore, the most important component of the influenza vaccine is that neutralizing antibodies produced in the body against hemagglutinin play a crucial role in preventing influenza virus infection. An influenza vaccine contains an inactivated vaccine and a live vaccine. The inactivated vaccine is made by purifying the virus cultured in an embryonated egg and deactivating it with formazan. A split vaccine in which the virus envelope is pulverized with a whole virus vaccine or ether using the inactivated virus as a whole, a subunit vaccine in which hemagglutinin and neuramidase components are purified, and the subunit vaccine. Live vaccine live attenuated influenza vaccine (LAIV) has been developed and used. Because antiviral vaccines cause adverse effects in children, they are currently not used globally, and only in some countries. On the other hand, component vaccines such as split vaccines or sub-vaccines are the most safe and effective. In addition, a vaccine containing an adjuvant such as MF-59 or a virosome vaccine to form viral-like vesicles has been developed and used in some countries to enhance the immune response. Antibodies against specific subtypes or influenza viruses obtained through natural infection or vaccination do not form protective antibodies against influenza viruses of other or subtype types and problems that do not show sufficient immunogenicity against new variants in one antigen have. Because influenza viruses cause big and small mutations every year, the epidemic is changing every year, and therefore it is difficult to expect the protection effect of the vaccine that was inoculated in the previous year.

Globally, influenza virus is a serious and continuing threat to human health. Each year, 3 to 5 million people are infected with serious symptoms, 500,000 people die, and a seasonal influenza pandemic could potentially kill millions. Antagonists to the virus surface glycoprotein, neuraminidase, have been widely used for the treatment of influenza infections, but their efficacy has been drastically reduced by drug resistant virus strains. Vaccines are the most effective way to prevent influenza virus infection, but as mentioned above, the protective efficacy of the vaccine is not optimized for high-risk groups such as those with poor immunity, such as children and the elderly. In addition, immunization after vaccination typically responds specifically to new strains, but because the influenza virus is changing rapidly, the vaccine should be produced almost every year. The determination of the antigenic composition of the vaccine is based on the anticipation of a strain that will become popular in the new year. Therefore, if the vaccine variant is different from the prevalent variant, the vaccine is ineffective. As a result, vaccines with new preventive and therapeutic effects that can protect against influenza viruses are in desperate need.

Immunization against influenza virus is mediated mainly by neutralizing antibodies targeting hemagglutinin (HA). Identification of the antigenic position in hemagglutinin is based on the identification of an immunologically predominant hemagglutinin head domain (HA1) that mediates the entry of influenza antibodies into the sialic acid receptor and into the host cell, . Studies with anti-HA head domain monoclonal antibodies indicate that this antibody class blocks the attachment of the virus to the sialic acid on the host cell surface, thereby preventing the virus from entering the cell. However, because of high mutation rates and tolerance for antigenic changes in the hemagglutinin head domain, antibodies targeting hemagglutinin head are only effective against very similar variants. Therefore, the wider antibody targeting the receptor attachment site is not structurally understood. In contrast, antibodies that attach to the hemagglutinin stem domain (HA2) close to the cell membrane block the major structural rearrangement of HA essential for the fusion of virus and endosomal membranes of the host, Preventing it from entering into. The HA stem domain has less mutation than the head domain and has relatively conserved amino acid sequences in various influenza strains. However, due to the diversity of antigenic stimuli found in the hemagglutinin head domain, most single antibodies targeting this domain are known to generally neutralize only a single virus.

On the other hand, it was reported that influenza A (H1N1) was first reported in 2004 at the Greyhound dog breeding ground in Florida, USA. Since then, the outbreak of influenza in Texas, Alabama, and Arkansas has been recognized as a new epidemic of the disease. According to epidemiological studies of influenza in the United States, it is almost identical to the H3N8 strain of horse infections, suggesting that new influenza viruses would have been produced by the transfer of influenza from horses to dogs. In addition, the influenza virus infection in Korea was reported in Korea in 2008, and the sequence analysis of the virus confirmed that it was caused by the virus byan avian H3N2 influenza A virus. Since then, H3N2 serotype influenza virus has been continuously reported in Korea and China. In particular, H3N2 serotype influenza viruses from Korean cats and H3N2 viruses with 98% to 99.8% homology in all gene regions Influenza-like viruses were isolated. In particular, the H3N1 serotype influenza virus is thought to have been generated through the reassortment event between the H3N2 serotype influenza virus and the pH1N1 virus. The genetically hemagglutinin gene locus of the virus was derived from the H3N2 influenza virus and the other gene was derived from the pH1N1 virus. Thus, the H3N2 influenza virus has evolved continuously through a mechanism of antigenic drift and shift. As described above, the mechanism by which influenza of other animals is transmitted to a dog can be dynamically estimated in two ways. One is the possibility that the virus can spread by feeding the uncooked ducks and chickens by dogs to dog farms and the other is the possibility of direct contact or respiratory propagation of dogs infected with normal dogs and other animal influenza Possibility. It is speculated that the infected dog would have been exposed to other environments and contacted with other dogs to help establish the influenza virus. Because of the potential for secondary infections and the varying mortality rates of these influenza viruses, prevention with vaccines is important. However, the development of influenza virus vaccines worldwide is currently insignificant, and it is very difficult to manufacture influenza vaccines due to the constant evolution of viruses.

As a result, there have been no studies on broadly neutralizing monoclonal antibodies (bNAbs) that can neutralize various influenza virus subtypes at once, and studies on the development of influenza virus vaccines have been limited, There is a pressing need for a new influenza vaccine that can be used as a broad-spectrum vaccine for a variety of influenza variants.

It is an object of the present invention to provide a novel universal vaccine against influenza virus that can show effects on various types of influenza virus subtypes and can prepare for the emergence of new varieties of influenza virus strains.

It is therefore an object of the present invention to provide an influenza virus vaccine composition and a pharmaceutical composition comprising a single HA2 helical domain of influenza hemagglutinin or a trimerized HA2 helical domain thereof.

It is still another object of the present invention to provide an immunostimulant that enhances the immunity induction response of existing influenza vaccines.

It is another object of the present invention to provide a method for inducing influenza immunity.

To achieve the above object, the present invention provides an anti-influenza virus vaccine composition comprising a polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 or a polypeptide represented by SEQ ID NO: 3.

Also provided is a pharmaceutical composition for preventing or treating an influenza infectious disease comprising a polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 or a polypeptide represented by SEQ ID NO: 3.

The present invention also provides an immunostimulant comprising the polypeptide of SEQ ID NO: 3.

The present invention also relates to a method for producing a polypeptide comprising the steps of: administering a polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 or a polypeptide represented by SEQ ID NO: 3 to a subject other than human; Lt; RTI ID = 0.0 > influenza < / RTI > immunity.

The polypeptide represented by SEQ ID NO: 3 of the present invention and the polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 are capable of mass production in E. coli and capable of effectively producing a neutralizing antibody against various influenza subtypes, Since it can enhance the immunity induction effect of existing influenza vaccine, it can be widely used as a general-purpose vaccine against influenza subtype and new variant influenza.

FIG. 1 shows the amino acid sequence of the hemagglutinin HA1 head domain using ESPript 3.0. FIG.
FIG. 2 shows the result of analyzing the structure-based hemagglutinin HA2 stem domain amino acid sequence using ESPript 3.0 and its terpolymer domain type. FIG.
3 is a diagram showing an E. coli expression vector pET28a-3XHA2HD.
Fig. 4 shows the result of confirming a single HA2 helical domain (a) and its trimerization helical domain (b) using SDS-PAGE.
FIG. 5 is a graph showing the results of ELISA test for the effect of forming a single HA2 helical domain (HA2HD) and a trimerized HA2 helical domain (3XHA2HD).
FIG. 6 shows the binding effect of a trimerized HA2 helical domain (3XHA2HD) -induced antibody against influenza subtypes pH1N1, cH3N2, hH3N2, aH5N1 and dH7N9 (2F3, 4D9: a single antibody clone of 3xHA2HD)
FIG. 7 is a graph showing the results of single and mixed immunodeficiency evaluation of the trimerized HA2 helical domain and the result of confirming the immunity enhancement effect (HD: Long + short Helical domain).

The present invention provides an influenza virus vaccine composition comprising a polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 or a polypeptide represented by SEQ ID NO: 3.

The polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 of the present invention and the polypeptide of SEQ ID NO: 3 are capable of effectively forming a neutralizing antibody against various subtypes of influenza virus H1, H3, H5, H7 and H9 As a polypeptide, an immune response against an influenza virus can be induced by acting as an antigen in an individual and inducing an antigen-antibody reaction.

Hereinafter, the present invention will be described in detail.

The polypeptide of the present invention represented by residues 379 to 474 of SEQ ID NO: 1 or the polypeptide represented by SEQ ID NO: 3 includes not only the same polypeptide but also polypeptides whose amino acid is substituted by conservative substitution and 80 to 99% Homology, preferably 85 to 99%, and more preferably 90 to 99%.

The term " conservative substitution " refers to the replacement of one class of amino acids with another of the same class. Conservative substitutions do not alter the structure or function of the polypeptide, or both. (E.g., Met, Ala, Val, Leu), neutral hydrophilic (e.g., Cys, Ser, Thr), acidic (e.g., Asp, GIu) , Basic (e.g., Asn, Gln, His, Lys, Arg), disruptors (e.g., Gly, Pro) and aromatics (e.g., Trp, Tyr, Phe) .

The term " antigen " is an antigenic component capable of causing an immune function among constituents of a virus, and is preferably a protein expressed by a virus. In certain embodiments, the polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: 3 is derived from the hemagglutinin stem domain in which the influenza virus is expressed, and when administered to a subject induces an immune response Lt; / RTI > Such antigens can be prepared from the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 2 by methods well known in the art.

The term " vaccine " refers to the induction of an antigen-antibody reaction in an individual for the purpose of preventing or treating a common influenza virus.

The " polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 " refers to a single HA2 helical domain of the full-length sequence of influenza hemagglutinin.

The above-mentioned " polypeptide represented by SEQ ID NO: 3 " refers to a trimerized HA2 helical domain of influenza hemagglutinin. Wherein the trimerized HA2 helical domain comprises a fragment 1 represented by the sequence 379-480 of SEQ ID NO: 3, a fragment 2 represented by the sequence 373-480, and a fragment 3 represented by the sequence 373-480 , Fragments 1 to 3 of such a terpolymer have the same or conservative substitution as hereinbefore described, or 80 to 99% sequence homology thereof, preferably 85 to 99%, more preferably 90 to 99% All of the polypeptides having homology can be included.

The polypeptide represented by SEQ ID NO: 3 can be encoded by the nucleotide sequence of SEQ ID NO: 2.

The " influenza virus " is classified as an influenza virus type of A, B and C. The term " influenza virus subtype " as used herein refers to influenza A virus variants characterized by a combination of hemagglutinin (H) and a neuramidase (N) virus surface protein. According to the present invention, influenza virus subtypes are characterized by their H numbers, for example, by "influenza virus comprising H3 subtype HA", "H3 subtype influenza virus" or "H3 influenza" And combinations of N numbers, for example, "influenza virus subtype H3N2" or "H3N2 ". The term "subtype " specifically includes all individual" strains "in each subtype resulting from an ordinary mutation, including natural isolates and artificial mutants or rearrangements, and exhibits different pathological profiles. Such strains may also be referred to as the various "isolates" of viral subtypes. Thus, the terms "strain" and "isolate" may be used interchangeably.

The vaccine of the present invention can effectively induce the formation of neutralizing antibodies against various influenza virus subtypes and is preferably capable of inducing the formation of antibodies against at least one influenza virus subtype selected from the group consisting of H1, H3, H5, H7 and H9 And the antibodies so formed can bind to various subtypes of hemagglutinin of influenza viruses and more preferably bind subtypes such as pH1N1, cH3N2, hH3N2, aH5N1, dH7N9.

Accordingly, the present invention relates to an anti-influenza virus vaccine composition comprising a polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 or a polypeptide represented by SEQ ID NO: 3, wherein the virus is selected from the group consisting of H1, H3, H5, H7 and H9 Lt; RTI ID = 0.0 > influenza < / RTI > virus subtype.

The present invention also provides a vaccine composition wherein the vaccine is a general-purpose vaccine.

The term " universal vaccine " refers to a vaccine having a broad range of applications that can neutralize a variety of influenza viruses at one time, including the influenza virus subtypes and novel variants thereof.

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

The term "subunit vaccine" is a vaccine prepared by extracting only an antigen component capable of causing an immune function among components of a virus, thereby minimizing adverse effects by inducing immune formation only at an antigenic site necessary for viral defense.

The term "synthetic vaccine" refers to a vaccine comprising peptides produced only by chemically synthesizing an antigen or antigenic determinant of a virus or by recombinant DNA technology.

The term " genetic engineering vaccine "may be a modification or deletion of a specific gene that causes virulence of the virus.

The vaccine of the present invention can be used as a mixed or complex vaccine which can be mixed with inactivated cells or antigens used in the production of other vaccines used for preventing influenza to prevent other diseases including influenza together. The term "combined vaccine " refers to a vaccine that uses different antiviral vaccines together, and the term" combination vaccine " refers to a vaccine that uses both a vaccine and a bacterial vaccine.

In addition, the vaccine composition of the present invention may further comprise at least one member selected from the group consisting of a solvent, an adjuvant, and an excipient. Examples of the solvent include physiological saline or distilled water. Examples of the immunity enhancer include Freund's incomplete or complete adjuvant, aluminum hydroxide gel, and vegetable and mineral oil. Examples of excipients include aluminum phosphate, But are not limited to, rocksides or aluminum potassium sulphate (alum), and may further include any of the known materials used in the manufacture of vaccines well known to those skilled in the art.

The vaccine composition of the present invention can be prepared in oral or parenteral formulations, preferably in the form of a parenteral preparation, injectable solution, and can be administered orally, intraperitoneally, intramuscularly, intraperitoneally, intravenously, subcutaneously, eidural) route of administration.

The present invention also provides a pharmaceutical composition for preventing or treating an influenza infectious disease comprising a polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 or a polypeptide represented by SEQ ID NO: 3.

The polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 of the present invention or the polypeptide represented by SEQ ID NO: 3, when administered to an individual, induces an immune response by forming an antibody in the individual, thereby preventing or treating influenza infectious diseases Can be usefully used.

The term " influenza virus disease ", as used herein, refers to a condition caused by the presence of an influenza virus, e. G. Influenza A or B virus in a cell or subject, or a condition resulting from invasion of a cell or subject by influenza virus Quot; In certain embodiments, the term refers to respiratory illness caused by influenza virus, and may preferably be at least one selected from the group consisting of sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea.

The individual is preferably individual, and the term " dog " herein may include animals of known dogs without limitation.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dosage level will vary depending on the species and severity, age, sex, Activity, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including simultaneously used drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The present invention also provides an immunostimulant comprising the polypeptide of SEQ ID NO: 3.

The term " immune enhancer " refers to an agent that increases, enhances, enhances, or improves the immune response caused by an antigen-antibody reaction in an individual, and the effect is positively increased and expanded in comparison with the existing immune effect .

The polypeptide represented by SEQ ID NO: 3 of the present invention can be used in combination with a known influenza vaccine to prevent the influenza virus infection of a new subtype influenza virus The induction effect can be enhanced.

Accordingly, the present invention provides an immunostimulating agent, wherein the polypeptide enhances an immune-inducing response of a CIV (canine influenza virus) vaccine.

The present invention also provides a method for producing a polypeptide comprising the steps of: administering to a dog a polypeptide represented by residues 379 to 474 of SEQ ID NO: 1 or a polypeptide represented by SEQ ID NO: 3; Lt; RTI ID = 0.0 > influenza < / RTI > immunity.

The dog means a canine animal that has already been infected or infected with influenza virus. The composition of the present invention can be administered to canine animals to treat canines infected with various influenza virus subtypes or variants. More specifically, the canine refers to all kinds of canines that can spread the virus between canines.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Preparation of single and terpolymers of influenza hemagglutinin HA2 helical domains

In order to identify the sequence of influenza hemagglutinin (HA) that can be used as a general antigen, a structure-based protein sequence analysis comparison program (Clustal W & ESPript 3.0) was used to identify the hemagglutinin The six amino acid sequences of the protein were compared and analyzed to determine HA2 helical domains with conserved sequences between subtypes. Sequence analysis results of such structure-based influenza hemagglutinin HA2 head and stem domains are shown in FIG. 1 and FIG. Through the above-described sequence analysis, an HA2 helical domain having an amino acid sequence conserved in hemagglutinin was identified and was prepared as the following antigen.

Hemagglutinin protein (567 amino acids) of Influenza A virus (A / California / 04/2009 (H1N1)) used in the experiment was synthesized in Bioneer Co., and the nucleotide sequence thereof is shown in SEQ ID NO: 4. An HA2 helical domain (residue 379-474) having an amino acid sequence conserved commonly in hemagglutinin (A / California / 04/2009 (H1N1), NCBI: FJ966082.1) shown in SEQ ID NO: And subcloned at the XhoI site and overexpressed using Escherichia coli BL21 (DE3) RIL strain.

In order to prepare a trimerized HA2 helical domain, fragment 1 used amino acid residues 379-480 of HA2 helical domain, fragment 2 of 373-480 and fragment 3 of 373-480 using NdeI and BamHI, BamHI and HindIII and HindIII and XhoI Subcloning was performed so that pET28a had 6 histidines at the N- and C-terminus of the protein and was overexpressed using Escherichia coli BL21 (DE3) Star strain. Each protein was inoculated into Escherichia coli and cultured at 291K for 16 hours with 0.2 mM IPTG at 0.6-0.8 at OD 600nm. The used E. coli expression vector pET28a-3XHA2HD is shown in Fig.

His- tagged single HA2 domain or trimerized HA2 helical domain was purified by nickel-affinity chromatography and eluted with a buffer containing 20 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 5% (v / v) glycerol Solution to obtain a purified single HA2 domain and trimeric HA2. The results are shown in Fig.

As shown in Fig. 4, a single HA2 helical domain and its trimerized helical domain were identified on SDS-PAGE and confirmed to have a purity of 96% or more by nickel-affinity chromatography. The nucleotide sequence for expressing the trimerized HA2 helical domain is shown in SEQ ID NO: 2, and the 351 amino acid sequence thus prepared is shown in SEQ ID NO: 3. The single HA2 helical domains and their trimerized helical domains prepared below were used as antigens to confirm whether they were useful as an influenza general-purpose vaccine.

Example 2. Identification of Antibody Production of a Single HA2 Helical Domain and a Trimerized HA2 Helical Domain

In order to confirm whether the monoclonal or trimerized HA2 helical domain antigen obtained in Example 1 effectively induced an antibody, its antibody production effect was confirmed.

First, pre-immune serum was taken and used as a negative control before each of these was injected into the rat. For primary immunization, 200ug of each purified or single-polymerized HA2 helical domain antigen was mixed with complete freund's adjuvant (Sigma) for intraperitoneal (IP) injection, and after 2 weeks, It was first boosted by mixing with sigma. After 1 week, the primary blood was collected and subjected to ELISA test and boosting and bleeding at intervals of 1 week. Blood was collected from the heart of the rat one week after the third boosting, and the final serum was separated after 4 weeks. ELISA tests were performed. In the ELISA test, 100 ng / well of the antigen was diluted to 1: 100-1: 100,000 with 1XPBS, and diluted 1: 10,000 with anti-mouse IgG-HRP (ABC5001) Respectively. Color development was detected at an optical density of 405 nm, and finally, immunological antibody production was confirmed, which is shown in FIG.

As shown in Fig. 5, a single HA2 helical domain showed an immuno-antibody-forming effect in all four rats. In addition, the trimerized HA2 helical domain exhibited much higher immunity and antibody production than the single domain in all four rats. These results indicate that both single HA2 helical domains and trimerized HA2 helical domains can effectively induce antibodies in the individual.

Example 3. Verification of universal vaccine availability of trimerized HA2 helical domain

It was confirmed that the recombinant trimerized HA2 helical domain showed excellent antibody-inducing effect, and thus it was confirmed whether it could be utilized as a general-purpose vaccine for various influenza antigens. More specifically, mouse monoclonal antibodies were prepared using purified trimerized HA2 helical domain antigens, and ascites from two clones were used Whether the recombinant hemagglutinin expressed in insects (pH1N1, cH3N2, hH3N2, aH5N1, dH7N9) could be detected was confirmed by Western blot. The results are shown in Fig.

As shown in FIG. 6, the monoclonal antibody of the trimerized HA2 helical domain binds to various subtypes of hemagglutinin, and the result shows that the trimerized HA2 helical domain can be used as a universal vaccine for various subtypes Respectively.

Example 4. Evaluation of mixed immunity efficacy of trimerized HA2 helical domain

Challenge experiments with CA04 (pH1N1) virus were performed after 4 weeks of canine immunization to confirm the effect of the trimerized HA2 helical domain and mixed immunization efficacy. CIV (canine influenza virus) vaccine, a commonly used influenza vaccine, was used as a vaccine for comparison and mixed immunization. Virus shedding was observed from day 1 to day 8, and the results of the experiment and the results of the virus excretion are shown in FIG.

As shown in FIG. 7, the trimerized HA2 helical domain exhibited excellent viral emission reduction effect alone against influenza H3N2, and was more effective when mixed with a conventional CIV vaccine. Therefore, it was confirmed that the trimerized HA2 helical domain is not only effective as a single influenza vaccine but also used as an immunity enhancer that can enhance the effect of CIV. In addition, the virus outbreak in the influenza H1N1 was observed, and it was confirmed that the trimerized HA2 helical domain excellently exerts a viral reduction effect in both the single and CIV mixtures, and thus has excellent immunity.

Considering that the H3N1 serotype infectious influenza virus is caused by a mutation caused by antigen migration and transmission occurring during recombination between H3N2 serotype influenza virus and pH1N1 virus, Domain combination can be effective against new subtypes that may be caused by various mutations through combination with existing vaccines.

These results indicate that the trimerized HA helical domain exhibits a remarkable effect of significantly reducing the virus release, and in particular, when mixed with a vaccine having a limited range of applications, it can expand and enhance the immunity inducing effect to other influenza .

Therefore, the single HA2 helical domain of the present invention and its trimerized HA2 helical domain can act as antigens to effectively induce antibodies against various influenza influenza strains, and can be used for mixed immunization together with existing influenza vaccines, In the treatment of the disease.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Universal influenza virus canine vaccine composition <130> 1-18 p <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 566 <212> PRT <213> influenza virus <400> 1 Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn   1 5 10 15 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr              20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn          35 40 45 Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val      50 55 60 Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly  65 70 75 80 Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile                  85 90 95 Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe             100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe         115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp     130 135 140 Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 145 150 155 160 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro                 165 170 175 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val             180 185 190 Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu         195 200 205 Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser     210 215 220 Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 225 230 235 240 Glu Gly Arg Met Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys                 245 250 255 Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe             260 265 270 Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro         275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn     290 295 300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg                 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly             340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr         355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser     370 375 380 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His                 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe             420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn         435 440 445 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu     450 455 460 Lys Val Arg Ser Glu Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val                 485 490 495 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu             500 505 510 Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr         515 520 525 Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val     530 535 540 Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560 Gln Cys Arg Ile Cys Ile                 565 <210> 2 <211> 1056 <212> DNA <213> influenza virus <400> 2 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggcagccg acctgaagag cacacagaat gccattgacg agattactaa caaagtaaat 120 tctgttattg aaaagatgaa tacacagttc acagcagtag gtaaagagtt caaccacctg 180 gaaaaaagaa tagagaattt aaataaaaaa gttgatgatg gtttcctgga catttggact 240 tacaatgccg aactgttggt tctattggaa aatgaaagaa ctttggacta ccacgattca 300 aatgtgaaga acttatatga aaaggtaaga agccagctaa aaaacaatgc caaggaaatt 360 ggaaacggcg gatccgagca ggggtcagga tatgcagccg acctgaagag cacacagaat 420 gccattgacg agattactaa caaagtaaat tctgttattg aaaagatgaa tacacagttc 480 acagcagtag gtaaagagtt caaccacctg gaaaaaagaa tagagaattt aaataaaaaa 540 gttgatgatg gtttcctgga catttggact tacaatgccg aactgttggt tctattggaa 600 aatgaaagaa ctttggacta ccacgattca aatgtgaaga acttatatga aaaggtaaga 660 agccagctaa aaaacaatgc caaggaaatt ggaaacggca agcttgagca ggggtcagga 720 tatgcagccg acctgaagag cacacagaat gccattgacg agattactaa caaagtaaat 780 tctgttattg aaaagatgaa tacacagttc acagcagtag gtaaagagtt caaccacctg 840 gaaaaaagaa tagagaattt aaataaaaaa gttgatgatg gtttcctgga catttggact 900 tacaatgccg aactgttggt tctattggaa aatgaaagaa ctttggacta ccacgattca 960 aatgtgaaga acttatatga aaaggtaaga agccagctaa aaaacaatgc caaggaaatt 1020 ggaaacggcc tcgagcacca ccaccaccac cactga 1056 <210> 3 <211> 351 <212> PRT <213> influenza virus <400> 3 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile              20 25 30 Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr          35 40 45 Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile      50 55 60 Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr  65 70 75 80 Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp                  85 90 95 Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln             100 105 110 Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Gly Ser Glu Gln Gly         115 120 125 Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu     130 135 140 Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe 145 150 155 160 Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn                 165 170 175 Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn             180 185 190 Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His         195 200 205 Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys     210 215 220 Asn Asn Ala Lys Glu Ile Gly Asn Gly Lys Leu Glu Gln Gly Ser Gly 225 230 235 240 Tyr Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr                 245 250 255 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala             260 265 270 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn         275 280 285 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu     290 295 300 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 305 310 315 320 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn                 325 330 335 Ala Lys Glu Ile Gly Asn Gly Leu Glu His His His His His             340 345 350 <210> 4 <211> 1701 <212> DNA <213> influenza virus <400> 4 atgaaggcaa tactagtagt tctgctatat acatttgcaa ccgcaaatgc agacacatta 60 tgtataggtt atcatgcgaa caattcaaca gacactgtag acacagtact agaaaagaat 120 gtaacagtaa cacactctgt taaccttcta gaagacaagc ataacgggaa actatgcaaa 180 ctaagagggg tagccccatt gcatttgggt aaatgtaaca ttgctggctg gatcctggga 240 aatccagagt gtgaatcact ctccacagca agctcatggt cctacattgt ggaaacacct 300 agttcagaca atggaacgtg ttacccagga gatttcatcg attatgagga gctaagagag 360 caattgagct cagtgtcatc atttgaaagg tttgagatat tccccaagac aagttcatgg 420 cccaatcatg actcgaacaa aggtgtaacg gcagcatgtc ctcatgctgg agcaaaaagc 480 ttctacaaaa atttaatatg gctagttaaa aaaggaaatt catacccaaa gctcagcaaa 540 tcctacatta atgataaagg gaaagaagtc ctcgtgctat ggggcattca ccatccatct 600 actagtgctg accaacaaag tctctatcag aatgcagata catatgtttt tgtggggtca 660 tcaagataca gcaagaagtt caagccggaa atagcaataa gacccaaagt gagggatcaa 720 gaagggagaa tgaactatta ctggacacta gtagagccgg gagacaaaat aacattcgaa 780 gcaactggaa atctagtggt accgagatat gcattcgcaa tggaaagaaa tgctggatct 840 ggtattatca tttcagatac accagtccac gattgcaata caacttgtca aacacccaag 900 ggtgctataa acaccagcct cccatttcag aatatacatc cgatcacaat tggaaaatgt 960 ccaaaatatg taaaaagcac aaaattgaga ctggccacag gattgaggaa tatcccgtct 1020 attcaatcta gaggcctatt tggggccatt gccggtttca ttgaaggggg gtggacaggg 1080 atggtagatg gatggtacgg ttatcaccat caaaatgagc aggggtcagg atatgcagcc 1140 gacctgaaga gcacacagaa tgccattgac gagattacta acaaagtaaa ttctgttatt 1200 gaaaagatga atacacagtt cacagcagta ggtaaagagt tcaaccacct ggaaaaaaga 1260 atagagaatt taaataaaaa agttgatgat ggtttcctgg acatttggac ttacaatgcc 1320 gaactgttgg ttctattgga aaatgaaaga actttggact accacgattc aaatgtgaag 1380 aacttaatg aaaaggtaag aagccagcta aaaaacaatg ccaaggaaat tggaaacggc 1440 tgctttgaat tttaccacaa atgcgataac acgtgcatgg aaagtgtcaa aaatgggact 1500 tatgactacc caaaatactc agaggaagca aaattaaaca gagaagaaat agatggggta 1560 aagctggaat caacaaggat ttaccagatt ttggcgatct attcaactgt cgccagttca 1620 ttggtactgg tagtctccct gggggcaatc agtttctgga tgtgctctaa tgggtctcta 1680 cagtgtagaa tatgtattta a 1701

Claims (13)

Wherein the polypeptide comprises a fragment 1 represented by the sequence of 379-480 of SEQ ID NO: 1, a fragment 2 represented by the sequence of 373-480 of SEQ ID NO: 1, a 373-480 sequence of SEQ ID NO: 1, RTI ID = 0.0 &gt; 4 &lt; / RTI &gt; sequence.
2. The vaccine composition according to claim 1, wherein the polypeptide represented by SEQ ID NO: 3 is encoded by the nucleotide sequence of SEQ ID NO: 2.
delete delete delete 2. The vaccine composition according to claim 1, wherein the virus is at least one influenza virus subtype selected from the group consisting of H1, H3, H5, H7 and H9.
2. The vaccine composition according to claim 1, wherein the vaccine is a general-purpose vaccine.
2. The vaccine composition according to claim 1, wherein the vaccine is at least one selected from the group consisting of a subunit vaccine, a synthetic vaccine, and a genetic engineering vaccine.
Wherein the polypeptide comprises a fragment 1 represented by the sequence of 379-480 of SEQ ID NO: 1, a fragment 2 represented by the sequence of 373-480 of SEQ ID NO: 1, a 373-480 sequence of SEQ ID NO: 1, 480. 3. A pharmaceutical composition for preventing or treating an infectious disease of an influenza virus,
The pharmaceutical composition according to claim 9, wherein the influenza infectious disease is at least one selected from the group consisting of sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea. Composition.
SEQ ID NO: 3 as an active ingredient, said polypeptide comprising a fragment 1 represented by the sequence of 379-480 of SEQ ID NO: 1, a fragment 2 represented by the sequence of 373-480 of SEQ ID NO: 1, a 373- 4. The immunostimulating agent according to any one of claims 1 to 3, wherein the immunizing agent comprises a fragment 3 represented by SEQ ID NO: 480.
delete Comprising administering to a dog an effective amount of a polypeptide consisting of SEQ ID NO: 3, wherein the polypeptide comprises a fragment 1 represented by the sequence 379-480 of SEQ ID NO: 1, a fragment 2 represented by the sequence 373-480 of SEQ ID NO: , And a fragment 3 represented by the sequence 373-480 of SEQ ID NO: 1.

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