KR102394946B1 - Universal canine influenza virus vaccine composition - Google Patents

Abstract

The present invention relates to a canine influenza virus vaccine composition, and more particularly, prevention of canine influenza virus infection using a fusion protein comprising the short helical domain of hemagglutinin (HA2SH) of influenza virus and the ectodomain of M2 protein (M2e) It relates to a vaccine composition for use. The fusion protein can be expressed and purified in large quantities in E. coli, and can effectively produce neutralizing antibodies against canine influenza viruses having several subtypes. In addition, the fusion protein can effectively induce immunity by exhibiting an excellent antibody-inducing effect, and thus can be utilized as a universal vaccine capable of inducing defense against influenza virus subtypes and novel mutated influenza viruses.

Description

Universal canine influenza virus vaccine composition {UNIVERSAL CANINE INFLUENZA VIRUS VACCINE COMPOSITION}

The present invention relates to a canine influenza virus vaccine composition, and more particularly, prevention of canine influenza virus infection using a fusion protein comprising the short helical domain of hemagglutinin (HA2SH) of influenza virus and the ectodomain of M2 protein (M2e) It relates to a vaccine composition for use.

There are various subtypes of influenza virus that causes respiratory diseases accompanied by chills, fever, and muscle pain, which are proteins that determine the immunogenicity of the virus, such as hemagglutinin (HA) and neuraminidase (NA). ) is due to the diversity of For example, for influenza A, there are 16 types of HA and 9 types of NA, and mutations thereof occur frequently. Therefore, in order to develop a vaccine, there is a burden of predicting an epidemic strain in advance and producing a vaccine, and despite the prediction of an epidemic strain, there are several problems, such as a marked decrease in vaccine efficacy due to the occurrence of a new mutant.

As a method to solve this problem, the development of a universal vaccine for influenza virus using a site with high conservation of amino acid residues present on the surface protein of the influenza virus is being studied. For example, "mini-HA" using a hemagglutinin protein having a highly conserved epitope demonstrated its versatility by showing viral efficacy in an experiment performed with an H1 subtype protein as an antigen (Impagliazzo A et al., Science 349.6254 (2015): 1301-1306).

Recently, a canine influenza virus was newly isolated and identified, and it is known that the canine influenza virus causes acute respiratory disease and has clinical symptoms such as severe cough, fever, and nasal discharge. Prevention of canine influenza virus with a vaccine is important because the possibility of secondary infection and the fatality rate vary. However, the development of a canine influenza virus vaccine worldwide is insignificant at present, and it is very difficult to manufacture an influenza vaccine as the virus continues to evolve.

As described above, there have not been many studies on broadly neutralizing monoclonal antibodies (bNAbs) that can neutralize various influenza virus subtypes at once, and in particular, studies on the development of canine influenza virus vaccines are insignificant. Therefore, there is an urgent need for a new canine influenza vaccine that can be used as a broad-spectrum vaccine against various canine influenza variants.

Impagliazzo, Antonietta, et al. "A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen." Science 349.6254 (2015): 1301-1306.

Accordingly, the present inventors have studied HA2SH and M2e, which show high conservation among various influenza viruses, for the development of a universal influenza virus vaccine. It was confirmed that the present invention was completed.

Accordingly, it is an object of the present invention to provide a fusion protein trimer comprising HA2SH and M2e that can be utilized as a universal vaccine by showing effects on various canine influenza virus subtypes.

It is also an object of the present invention to provide a vaccine composition for preventing canine influenza virus infection and a pharmaceutical and/or veterinary composition for preventing or treating canine influenza virus infection disease, comprising the fusion protein trimer as an active ingredient.

In order to achieve the above object, the present invention provides a fusion protein trimer comprising the short helical domain of hemagglutinin (HA2SH) of influenza virus and the ecto domain of M2 protein (M2e).

In addition, the present invention provides an expression vector comprising a nucleotide encoding the fusion protein trimer.

In addition, the present invention provides a vaccine composition for preventing canine influenza virus infection comprising the fusion protein trimer as an active ingredient.

In addition, the present invention provides a pharmaceutical and / or veterinary composition for the prevention or treatment of canine influenza virus infection disease comprising the fusion protein trimer as an active ingredient.

The fusion protein trimer according to the present invention can be expressed and purified in large quantities in E. coli, and can effectively produce neutralizing antibodies against canine influenza viruses having several subtypes. In addition, the fusion protein trimer can effectively induce immunity by exhibiting an excellent antibody-inducing effect. Therefore, the fusion protein trimer can be used as a universal vaccine capable of preventing not only canine influenza subtypes, but also new mutated canine influenza, and pharmaceutical and/or veterinary compositions having excellent therapeutic effects on canine influenza virus infection diseases.

1 shows the results of analyzing the sequences of the entire HA head domain and stem domain of influenza virus.
Figure 2 shows the results of analyzing the HA2 sequence of the influenza virus.
3 shows the results of analyzing the M2 protein sequence of influenza virus.
4 shows a schematic diagram of a fusion protein trimer (3XHA2SH-M2e) comprising HA2SH and M2e.
5 shows the E. coli expression vector pET28a-3XHA2HS-M2e.
6 shows the results of confirming the prepared 3XH1SH-M2e, 3XH3SH-M2e, 3XH5SH-M2e and H135SH-M2e on SDS-PAGE.
7A is a graph showing 3XH1SH-M2e purified by nickel-affinity chromatography.
7B is a graph showing 3XH3SH-M2e purified by nickel-affinity chromatography.
7c is a graph showing 3XH5SH-M2e purified by nickel-affinity chromatography.
7D is a graph showing H135SH-M2e purified by nickel-affinity chromatography.
8 shows the expression results of 3XH1SH-M2e, 3XH3SH-M2e, 3XH5SH-M2e and H135SH-M2e.
9 shows the antibody-inducing effect of 3XH1SH-M2e, 3XH3SH-M2e, 3XH5SH-M2e and H135SH-M2e through ELISA analysis.
10a to 10h show changes in body weight and survival rates after infection with influenza A virus (H1N1, H3N2, H5N1 and H5N8) of mice injected with 3XH1SH-M2e, 3XH3SH-M2e, 3XH5SH-M2e and H135SH-M2e.
11A to 11H show changes in body weight and survival rates after infection with influenza A virus (H1N1, H3N2, H5N1 and H5N8) in mice injected with 3XHA2SH-M2e (3XH1SH-M2e, 3XH3SH-M2e, 3XH5SH-M2e and H135SH-M2e); (Adjusted for H1N1 and H3N2 infected groups; 20% cut-off for weight loss after excluding the maximum weight gain and the maximum weight loss individual on the 7th day at dpi at n=8 (n=6)).

One aspect of the present invention provides a fusion protein trimer comprising the short helical domain of hemagglutinin (HA2SH) or a fragment thereof of influenza virus and the ectodomain (M2e) of M2 protein or a fragment thereof.

As used herein, the term "short helical domain of hemagglutinin (HA2SH)" refers to a short helical region among the stem domains (HA2) of hemagglutinin (HA), a surface protein of influenza virus, specifically As such, it refers to a region having an amino acid sequence that is conserved in common among various subtypes of influenza virus. In one embodiment of the present invention, amino acid residues 373-404 of the HA2SH fragment may be preferably used as the HA2SH included in the fusion protein.

As used herein, the term “ectodomain of M2 protein (M2e)” refers to the ectodomain of M2 protein, which is a membrane protein of influenza virus, specifically, a region having a conserved amino acid sequence between influenza virus subtypes in M2 protein. means In one embodiment of the present invention, 15 amino acid residues of the M2e fragment may be preferably used for M2e included in the fusion protein.

Here, HA2SH and M2e of the influenza virus specifically refer to proteins derived from influenza virus type A, and specifically, the type of influenza virus type A may be H1N1, H1N2, H2N2, H3N1, H3N2, H5N1 or H5N8. and, specifically, may be H1N1, H3N2 or H5N1.

In one embodiment of the present invention, the fusion protein trimer may have the following structural formula:

A ₁ -(L ₁ ) _n -A ₂ -(L ₂ ) _m -A ₃

Here, A ₁ is represented by HA2SH ₁ -(l ₁ ) _k -M2e ₁ , A ₂ is represented by HA2SH ₂ -(l ₂ ) _i -M2e ₂ , and A ₃ is HA2SH ₃ -(l ₃ ) _j -M2e ₃ ,

The HA2SH ₁ , HA2SH ₂ and HA2SH ₃ are each independently a short helical domain of hemagglutinin of any one influenza virus selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H5N1 and H5N8, or a fragment thereof,

The M2e ₁ , M2e ₂ and M2e ₃ are each independently an ectodomain of the M2 protein of any one influenza virus selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H5N1 and H5N8, or a fragment thereof,

The L ₁ , L ₂ , 1 ₁ , 1 ₂ and 1 ₃ are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond. In addition, n, m, k, i, and j are each independently an integer of 0 or 1.

In one embodiment of the present invention, the HA2SH ₁ , HA2SH ₂ and HA2SH ₃ may be each independently represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. In addition, SEQ ID NO: 7 may be coated with the nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 8 may be coated with the nucleotide sequence of SEQ ID NO: 11, and SEQ ID NO: 9 may be encoded with the nucleotide sequence of SEQ ID NO: 12.

At this time, the HA2SH ₁ , HA2SH ₂ and HA2SH ₃ are equal to each other, or HA2SH ₁ , Any one or more of HA2SH ₂ and HA2SH ₃ is different from each other, or the HA2SH ₁ , Both HA2SH ₂ and HA2SH ₃ may be different from each other. For example, the HA2SH ₁ , Both HA2SH ₂ and HA2SH ₃ may be represented by SEQ ID NO: 7, all of which are represented by SEQ ID NO: 8, or both may be represented by SEQ ID NO: 9. In addition, the HA2SH ₁ is represented by SEQ ID NO: 7, the HA2SH ₂ may be represented by SEQ ID NO: 8, and HA2SH ₃ may be represented by SEQ ID NO: 8. In addition, the HA2SH ₁ is represented by SEQ ID NO: 7, the HA2SH ₂ may be represented by SEQ ID NO: 8, and HA2SH ₃ may be represented by SEQ ID NO: 9. Here, SEQ ID NO: 7 is H1N1 influenza virus HA2SH or a fragment thereof, SEQ ID NO: 8 is H3N2 influenza virus HA2SH or a fragment thereof, and SEQ ID NO: 9 is H5N1 influenza virus HA2SH or a fragment thereof.

In one embodiment of the present invention, the M2e ₁ , M2e ₂ and M2e ₃ may each independently be represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In addition, SEQ ID NO: 13 may be coated with the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 14 may be coated with the nucleotide sequence of SEQ ID NO: 17, and SEQ ID NO: 15 may be encoded with the nucleotide sequence of SEQ ID NO: 18.

At this time, the M2e ₁ , M2e ₂ and M2e ₃ are equal to each other, or M2e ₁ , Any one or more of M2e ₂ and M2e ₃ is different from each other, or the M2e ₁ , Both M2e ₂ and M2e ₃ may be different from each other. For example, the M2e ₁ , Both M2e ₂ and M2e ₃ may be represented by SEQ ID NO: 13, both may be represented by SEQ ID NO: 14, or both may be represented by SEQ ID NO: 15. In addition, the M2e ₁ is represented by SEQ ID NO: 13, M2e ₂ may be represented by SEQ ID NO: 14, and M2e ₃ may be represented by SEQ ID NO: 14. In addition, the M2e ₁ is represented by SEQ ID NO: 13, M2e ₂ may be represented by SEQ ID NO: 14, and M2e ₃ may be represented by SEQ ID NO: 15. Here, SEQ ID NO: 13 is H1N1 influenza virus M2e or a fragment thereof, SEQ ID NO: 14 is H3N2 influenza virus M2e or a fragment thereof, and SEQ ID NO: 15 is H5N1 influenza virus M2e or a fragment thereof.

As used herein, the term “fusion protein” includes HA2SH and M2e, and specifically refers to a fusion protein monomer represented by A ₁ , A ₂ and A ₃ . Specifically, A ₁ is represented by HA2SH ₁ -(l ₁ ) _k -M2e ₁ , A ₂ is represented by HA2SH ₂ -(l ₂ ) _i -M2e ₂ , and A ₃ is HA2SH ₃ -(l ₃ ) can be represented as _j -M2e ₃ . More specifically, in the fusion protein, when k, i and j are each an integer of 0, the A ₁ is represented by HA2SH ₁ -M2e ₁ , the A ₂ is represented by HA2SH ₂ -M2e ₂ , and the A ₃ may be represented as HA2SH ₃ -M2e ₃ . In addition, when k, i and j are each an integer of 1, A ₁ is represented by HA2SH ₁ -(l ₁ ) ₁ -M2e ₁ , and A ₂ is HA2SH ₂ -(l ₂ ) ₁ -M2e ₂ and A ₃ may be represented by HA2SH ₃ -(l ₃ ) ₁ -M2e ₃ . In this case, k, i, and j may be the same, or at least one of k, i, and j may be different from each other. In addition, A ₁ , A ₂ , and A ₃ may be represented as meaning a fusion protein monomer including HA2SH and M2e, as in the structure of HA2SH-M2e.

In this case, HA2SH ₁ and M2e ₁ of A ₁ may be HA2SH of the same influenza virus and a fragment thereof, or M2e and a fragment thereof. Specifically, both HA2SH ₁ and M2e ₁ may be HA2SH and a fragment thereof of the influenza virus of H1N1, or M2e and a fragment thereof. Meanwhile, HA2SH ₁ and M2e ₁ of A ₁ may be HA2SH and fragments thereof of different influenza viruses, or M2e and fragments thereof. Specifically, the HA2SH ₁ may be H1N1 influenza virus HA2SH and fragments thereof, and M2e ₁ may be H3N2 influenza virus M2e and fragments thereof.

In addition, HA2SH ₂ and M2e ₂ of A ₂ may be HA2SH of the same influenza virus and a fragment thereof, or M2e and a fragment thereof. Specifically, both HA2SH ₂ and M2e ₂ may be HA2SH and a fragment thereof of an influenza virus of H3N2, or M2e and a fragment thereof. Meanwhile, HA2SH ₂ and M2e ₂ of A ₂ may be HA2SH and fragments thereof of different influenza viruses, or M2e and fragments thereof. Specifically, the HA2SH ₂ may be H3N2 influenza virus HA2SH and fragments thereof, and M2e ₂ may be H5N1 influenza virus M2e and fragments thereof.

In addition, HA2SH ₃ and M2e ₃ of A ₃ may be HA2SH and a fragment thereof of the same influenza virus, or M2e and a fragment thereof. Specifically, both HA2SH ₃ and M2e ₃ may be HA2SH and a fragment thereof of the influenza virus of H5N1, or M2e and a fragment thereof. Meanwhile, HA2SH ₃ and M2e ₃ of A ₃ may be HA2SH and fragments thereof of different influenza viruses, or M2e and fragments thereof. Specifically, the HA2SH ₃ may be HA2SH and a fragment thereof of the H5N1 influenza virus, and the M2e ₂ may be M2e of the H1N1 influenza virus and a fragment thereof.

In this case, A ₁ , A ₂ and A ₃ may be the same as each other, at least one of A ₁ , A ₂ and A ₃ may be different from each other, or A ₁ , A ₂ , and A ₃ may all be different from each other. For example, the A ₁ , Both A ₂ and A ₃ may be represented by SEQ ID NO: 19, both may be represented by SEQ ID NO: 20, or both may be represented by SEQ ID NO: 21. In addition, the A ₁ is represented by SEQ ID NO: 19, and A ₂ may be represented by SEQ ID NO: 20, and A ₃ may be represented by SEQ ID NO: 20. In addition, the A ₁ is represented by SEQ ID NO: 19, and A ₂ may be represented by SEQ ID NO: 20, and A ₃ may be represented by SEQ ID NO: 21.

Here, SEQ ID NO: 19 may include H1N1 influenza virus HA2SH and fragments thereof, or M2e and fragments thereof, and SEQ ID NO: 20 may include H3N2 influenza virus HA2SH and fragments thereof, or M2e and fragments thereof and SEQ ID NO: 21 may include H5N1 influenza virus HA2SH and fragments thereof, or M2e and fragments thereof. In addition, SEQ ID NO: 19 may be coated with the nucleotide sequence of SEQ ID NO: 22, SEQ ID NO: 20 may be coated with the nucleotide sequence of SEQ ID NO: 23, and SEQ ID NO: 21 may be encoded with the nucleotide sequence of SEQ ID NO: 24.

In one embodiment of the present invention, the fusion proteins A ₁ , A ₂ and A ₃ each independently include a monomer represented by one or more amino acid sequences selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 can do. The fusion protein is identical to the amino acid sequence, or amino acids thereof are substituted by conservative substitution, or 90 to 99% sequence homology, preferably 95 to 99% sequence homology, more preferably 98 to 99% sequence homology. It may include all fusion proteins having % sequence homology.

As used herein, the term "fusion protein trimer" refers to a form in which fusion protein monomers including HA2SH and M2e are repeatedly combined three times, specifically, the A ₁ -(L ₁ ) _n -A ₂ -(L ₂ ) _m -A ₃ Refers to a fusion protein trimer having a structure. A fusion protein trimer formed from HA2SH and M2e having amino acid sequences that are conserved in common among various subtypes of influenza virus can effectively form neutralizing antibodies against various subtypes of canine influenza virus. The fusion protein trimer can induce an immune response against canine influenza virus by inducing an antigen-antibody response by acting as an antigen in the individual.

The fusion protein trimer may be represented by the structural formula of A ₁ -(L ₁ ) _n -A ₂ -(L ₂ ) _m -A ₃ (wherein A ₁ , A ₂ and A ₃ are as defined above), Specifically, when n and m are each an integer of 1, the fusion protein trimer may be represented by A ₁ -A ₂ -A ₃ , and when n and m are each an integer of 1, the fusion protein trimer may be represented as A ₁ -(L ₁ ) ₁ -A ₂ -(L ₂ ) ₁ -A ₃ . In addition, when n is an integer of 0 and m is an integer of 1, the fusion protein trimer may be represented by A ₁ -A ₂ -(L ₂ ) ₁ -A ₃ , wherein n is an integer of 1 and m When this is an integer equal to 0, the fusion protein trimer may also be represented as A ₁ -(L ₁ ) ₁ -A ₂ -A ₃ . As described above, A ₁ , A ₂ and A ₃ may be the same as each other, at least one of A ₁ , A ₂ and A ₃ is different from each other, or A ₁ , A ₂ and A ₃ are all different from each other. can

In addition, the fusion protein trimer may be represented by the structure of 3XHA2SH-M2e, and specifically, it may be represented by 3XH1SH-M2e, 3XH3SH-M2e, 3XH5SH-M2e and H135SH-M2e. Herein, the 3XH1SH-M2e (SEQ ID NO: 25, SEQ ID NO: 33 or SEQ ID NO: 41) is a fusion protein comprising all of A ₁ , A ₂ and A ₃ H1N1 influenza virus HA2SH and fragments thereof, or M2e and fragments thereof refers to a trimer, wherein the 3XH3SH-M2e (SEQ ID NO: 26, SEQ ID NO: 34 or SEQ ID NO: 42) is that all of A ₁ , A ₂ and A ₃ are H3N2 influenza virus HA2SH and fragments thereof, or M2e and fragments thereof It means a fusion protein trimer comprising. In addition, the 3XH5SH-M2e (SEQ ID NO: 27, SEQ ID NO: 35 or SEQ ID NO: 43) is a fusion protein comprising all of A ₁ , A ₂ and A ₃ of H5N1 influenza virus HA2SH and fragments thereof, or M2e and fragments thereof means a trimer. On the other hand, the H135SH-M2e (SEQ ID NO: 28, SEQ ID NO: 36 or SEQ ID NO: 44) includes HA2SH and fragments thereof of H1N1 influenza virus, or M2e and fragments thereof, _{and A 2 is} H3N2 of influenza virus It includes HA2SH and fragments thereof, or M2e and fragments thereof, and A ₃ refers to a fusion protein trimer including HA2SH and fragments thereof of influenza virus of H5N1, or M2e and fragments thereof.

In one embodiment of the present invention, the fusion protein trimer is SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 41, It may be represented by one or more amino acid sequences selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44. The fusion protein trimer is identical to the amino acid sequence, or an amino acid thereof is substituted by conservative substitution, 90 to 99% sequence homology, preferably 95 to 99% sequence homology, more preferably 98 It may include all of the fusion protein trimers having a sequence homology of from to 99%.

In this case, the SEQ ID NOs: 25 to 28 indicate a fusion protein trimer that does not contain a linker (wherein n and m each have an integer equal to 0), and SEQ ID NOs: 33 to SEQ ID NO: 36 include a linker (Wherein n and m each have an integer equal to 1) A fusion protein trimer is indicated, and SEQ ID NOs: 41 to 44 indicate a fusion protein trimer including a His-tag.

In one embodiment of the present invention, SEQ ID NO: 25 is the nucleotide sequence of SEQ ID NO: 29, SEQ ID NO: 26 is the nucleotide sequence of SEQ ID NO: 30, SEQ ID NO: 27 is the nucleotide sequence of SEQ ID NO: 31, and SEQ ID NO: 28 is the nucleotide sequence of SEQ ID NO: 32, SEQ ID NO: 33 is the nucleotide sequence of SEQ ID NO: 37, SEQ ID NO: 34 is the nucleotide sequence of SEQ ID NO: 38, SEQ ID NO: 35 is the nucleotide sequence of SEQ ID NO: 39, the sequence Number 36 is the nucleotide sequence of SEQ ID NO: 40, SEQ ID NO: 41 is the nucleotide sequence of SEQ ID NO: 45, SEQ ID NO: 42 is the nucleotide sequence of SEQ ID NO: 46, SEQ ID NO: 43 is the nucleotide sequence of SEQ ID NO: 47, SEQ ID NO: 44 may be encoded by the nucleotide sequence of SEQ ID NO: 48.

As used herein, the term "conservative substitution" means that an amino acid of one class is replaced by another amino acid of the same class. Conservative substitutions do not alter the structure or function of the polypeptide. Classes of amino acids for the purpose of conservative substitutions are hydrophobic (eg Met, Ala, Val, Leu), neutral hydrophilic (eg Cys, Ser, Thr), acidic (eg Asp, Glu). , basic (eg Asn, Gin, His, Lys, Arg), conformational disrupters (eg Gly, Pro) and aromatic (eg Trp, Tyr, Phe) can

As used herein, the term “antigen” refers to a component capable of generating an immune function among the components of a virus, preferably a protein expressed by the virus. Specifically, when the fusion protein trimer is administered to a subject, the fusion protein trimer may act as an antigen inducing an immune response.

In one embodiment of the present invention, the HA2SH and M2e may be directly coupled or coupled through a linker, and the fusion protein may be coupled directly to each other or coupled through a linker.

The linker refers to an amino acid sequence of a certain length that connects HA2SH and M2e, or artificially connects between fusion protein monomers. Specifically, the linker may be a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond, and the amino acids are glycine, serine, alanine, proline, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, and threonine. , cysteine, asparagine, glutamine, lysine, arginine, histidine, may be at least one selected from the group consisting of aspartic acid and glutamic acid. More specifically, the linker may be composed of glycine and serine, lysine and leucine, or leucine and glutamic acid.

In addition, one aspect of the present invention provides an expression vector comprising a nucleotide encoding a fusion protein trimer.

The nucleotide may be any one selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, and the nucleotide may include a signal peptide or His-tag.

In addition, one aspect of the present invention provides a vaccine composition for preventing canine influenza virus infection comprising the fusion protein trimer as an active ingredient.

As used herein, the term “vaccine” means inducing an antigen-antibody response in an individual, preferably a dog, for the purpose of preventing or treating a common canine influenza virus.

In the present invention, the vaccine is a “universal vaccine” and refers to a vaccine having a wide application range capable of neutralizing various canine influenza viruses including canine influenza virus subtypes and novel variants thereof at once.

In the present invention, "dog" may include, without limitation, known canines. Specifically, it refers to a canine animal that is already infected or may be infected with an influenza virus. By administering the composition of the present invention to canines, it is possible to treat canines infected with various influenza virus subtypes or variants. More specifically, the canine animal refers to any kind of canine animal capable of transmitting a virus among canines.

The vaccine composition can induce the formation of antibodies against various canine influenza virus subtypes. The antibody thus formed may bind to any one subtype selected from the group consisting of various subtypes of influenza virus, preferably H1N1, H1N2, H2N2, H3N1, H3N2, H5N1, H5N8, H3N8, and combinations thereof. More preferably, the antibody formed as described above may bind to influenza virus subtypes of H1N1, H3N2, H5N1, H3N8 or H5N8.

As used herein, the term “influenza virus subtype” refers to a variant of influenza A virus in which hemagglutinin (H) and neuramidase (N) virus surface proteins are combined. The influenza virus subtype may have an influenza virus subtype having a combination of H number and N number (eg, H1N1, H1N2, H2N2, H3N1, H3N2, H5N1, H5N8, H3N8, etc.).

The vaccine composition may further include one or more selected from the group consisting of a solvent, an adjuvant and an excipient. Physiological saline or distilled water may be used as the solvent, and as the immune enhancing agent, Freund's incomplete or complete adjuvant, aluminum hydroxide gel, and vegetable and mineral oils may be used, and as the excipient can use aluminum phosphate, aluminum hydroxide or aluminum potassium sulfate. In addition, it may further include a known substance used in the manufacture of vaccines.

The vaccine composition may be prepared as an oral or parenteral formulation, and is preferably prepared as an injection solution, which is a parenteral formulation, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, nasally or epidurally (eidural). ) can be administered by route.

Another aspect of the present invention provides a pharmaceutical and/or veterinary composition for preventing or treating canine influenza virus infection disease comprising the fusion protein trimer as an active ingredient.

In one embodiment of the present invention, the canine influenza virus infection disease may be any one selected from the group consisting of sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea.

The pharmaceutical composition may be administered in a pharmaceutically or veterinarily effective amount. The term "pharmaceutically or veterinarily effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical and/or veterinary treatment, and effective dose levels vary depending on the severity, age, sex, infection Virus type, drug activity, sensitivity to drug, administration time, administration route and excretion rate, treatment period, factors including drugs used concurrently, and other factors well known in the medical field may be determined according to factors. The pharmaceutical and/or veterinary composition may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

Another aspect of the present invention may provide a method for inducing immunity against canine influenza virus, comprising administering the fusion protein trimer to a dog. When the fusion protein trimer is administered to a dog, it forms an antibody in the dog individual and induces an immune response, so it can be usefully used to prevent or treat canine influenza infection disease.

Another aspect of the present invention may provide a vaccination method comprising administering the fusion protein trimer to a dog.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1. Sequence identification of influenza hemagglutinin HA2 and M2e

In order to identify the sequence of influenza hemagglutinin (hereinafter referred to as 'HA') that can be used as a universal antigen, a structure-based protein sequencing comparison program (Clustal W & ESPript 3.0) was used. The above program was used to compare and analyze the sequences of six amino acid sequences of HA proteins having various subtypes, and determine the short helical domain (HA2SH) of HA2 with a sequence conserved between subtypes among the entire HA sequence. The results of sequence analysis of the entire head domain and stem domain of the structure-based influenza HA and the results of sequence analysis of HA2 are shown in FIGS. 1 and 2 .

In the same way, the sequences of 6 types of amino acid sequences of the M2 protein (matrix-2 protein) were compared and analyzed, and the ectodomain of the M2 protein (hereinafter referred to as 'M2e'), which has a sequence that is conserved between subtypes in the M2 protein, was determined. . The results of sequence analysis of the structure-based influenza M2 protein are shown in FIG. 3 . Through the sequence analysis as described above, the HA2 short helical domain having an amino acid sequence commonly conserved in HA and the M2e domain having an amino acid sequence commonly conserved in M2 protein were identified, which were prepared as antigens as in Example 2 below. did

Example 2. Preparation of influenza hemagglutinin HA2 short helical domain and M2e fusion protein trimer (3XHA2SH-M2e)

Example 2.1. Synthesis of HA protein and M2 protein

HA protein (567 amino acids) of influenza A virus (A/California/04/2009(H1N1)) used in the experiment, HA protein (588) of influenza A virus (A/canine/Korea/01/2007(H3N2)) Dog amino acids) and HA protein (568 amino acids) genes of influenza A virus (A/chiken/korea/es/2003(H5N1)) were synthesized by Bioneer. Their nucleotide sequences are the same as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

In addition, M2 protein (97 amino acids) of influenza A virus (A/California/04/2009(H1N1)) used in the experiment, M2 protein of influenza A virus (A/canine/Korea/01/2007(H3N2)) (97 amino acids) and M2 protein (97 amino acids) genes of influenza A virus (A/chiken/korea/es/2003(H5N1)) were synthesized by Bioneer. Their nucleotide sequences are the same as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

Example 2.2. Preparation of 3XHA2SH-M2e

To prepare 3XHA2SH-M2e (Fig. 4), fragment 1 is amino acid residues 373-404 of the HA2 short helical domain and 15 amino acid residues of the M2e domain, fragment 2 is amino acid residues 373-404 of the HA2 short helical domain and amino acid residues 373-404 of the M2e domain of 15 amino acid residues, and fragment 3 is amino acid residues 373-404 of the HA2 short helical domain and 15 amino acid residues of the M2e domain, respectively. It was subcloned so that six histidines were attached to the terminals (N-terminus and C-terminus), and overexpressed using Escherichia coli BL21(DE3) RIL strain. The used E. coli expression vector pET28a-3XHA2HS-M2e is shown in FIG. 5 . In addition, amino acid residues 373-404 of the HA2 short helical domain used are represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, and the amino acid residues of M2e used are SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: It is represented by the amino acid sequence of 15.

Each protein was inoculated into E. coli, 0.2 mM IPTG was added at OD600 nm between 0.5 and 0.6, and incubated at 290K for 18 hours. His-tagged 3XHA2SH-M2e was purified by nickel-affinity chromatography, followed by dialysis in a buffer containing 25 mM Tris-HCl (pH 7.5), 0.2 M NaCl, and 5% (v/v) glycerol. 3XHA2SH-M2e was obtained. 3XHA2SH-M2e (3XH1SH-M2e, 3XH3SH-M2e, 3XH5SH-M2e and H135SH-M2e) was confirmed on SDS-PAGE, and it was confirmed that it was purified to a purity of 96% or more by nickel-affinity chromatography (Fig. 6 and Fig. 7). In addition, the expression test results of 3XHA2SH-M2e are shown in FIG. 8 .

Example 3. Confirmation of antibody production effect of 3XHA2SH-M2e

In order to confirm whether the 3XHA2SH-M2e (3XH1SH-M2e, 3XH3SH-M2e, 3XH5SH-M2e, and H135SH-M2e) prepared in Example 2 effectively induces the antibody, their antibody-generating effect was confirmed.

Before antigen injection into mice, pre-immune serum was collected and used as a negative control. For primary immunization, 50 ug of each purified 3XHA2SH-M2e antigen was mixed with emulsion adjuvant (ISA 71 VG) (montinide) and intramuscularly injected (IM), and primary blood was collected 2 weeks later for ELISA Analysis was performed. Then, 50 ug of the protein antigen was mixed with an emulsion adjuvant (ISA 71 VG) (montinide) to perform boosting immunization, and 2 weeks later, the final serum was isolated and a final ELISA analysis was performed.

In the ELISA assay, 100 ng of antigen was used per well. In addition, immune serum was used by diluting 1:100 to 1:100,000 with 1x PBS, and for secondary confirmation, anti-mouse IgG-HRP (ABC5001) was diluted 1:5,000 and used. By detecting color development at an optical density of 405 nm, it was finally confirmed whether an immune antibody was generated. As a result, it was found that 3XHA2SH-M2e had an excellent antibody-generating effect in mice. Through these results, it was confirmed that 3XHA2SH-M2e can effectively induce the antibody (FIG. 9).

Example 4. Confirmation of the availability of 3XHA2SH-M2e as a universal vaccine

In Example 3, it was confirmed that 3XHA2SH-M2e exhibited an excellent antibody-inducing effect, and through this, it was confirmed whether it could be utilized as a universal vaccine capable of inducing protection against various influenza viruses.

For primary immunization, 50 ug of each purified 3XHA2SH-M2e antigen was mixed with emulsion adjuvant (ISA 71 VG) (montinide) and intramuscularly injected into mice, and after 2 weeks, 50 ug of antigen was administered with emulsion adjuvant (ISA 71 VG) ( montinide) and boosting immunization was performed. After 2 weeks, each of the influenza A viruses H1N1, H3N2, H5N1 and H5N8 was diluted in 1x PBS, and 30 ul of each of the mice was infected through nasal inoculation (IN). Thereafter, the effect of 3XHA2SH-M2e antigen inducing protective immunity was confirmed by measuring the change in body weight and survival rate of mice over 2 weeks ( FIGS. 10 and 11 ).

Through the above results, it was confirmed that the general-purpose vaccine composition according to the present invention is capable of inducing protection against various influenza viruses, and thus it was confirmed that it can be used as a canine influenza virus vaccine composition.

<110> Korea Research Institute of Bioscience and Biotechnology <120> INFLUENZA VIRUS VACCINE COMPOSITION <130> FPD/201808-0053 <160> 48 <170> KoPatentIn 3.0 <210> 1 <211> 1701 <212> DNA <213> Artificial Sequence <220> <223> hemagglutinin gene of A/California/04/2009(H1N1) <400> 1 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 accaacaaa g 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 aacttatatg 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 <210> 2 <211> 1764 <212> DNA <213> Artificial Sequence <220> < 223> hemagglutinin gene of A/canine/Korea/01/2007(H3N2) <400> 2 agcaaaagca ggggatactt tcattaatca tgaaaactgt tattgcttta agctacattt 60 tctgcctggc ttttggtcag aatcttccag gaaatgaaaa taatgctgca acactatgcc 120 tgggacatca tgcagtgccg aacgggacaa tagtgaaaac tatcacagac gatcaaattg 180 aggtgaccaa cgccaccgag ctagtccaaa actcctcaac agggaaaata tgcaacaatc 240 cccacaagat tcttgatggg agggactgca cactaataga tgccctacta ggggacccgc 300 actgtgatgt cttccaaaat gagacatggg acctttttgt ggaacgaagc aatgctttta 360 gcaattgtta cccttatgat gtaccagact atgcatccct tcgatccata gttgcatcat 420 caggcacatt ggagttcatc actgaaggtt tcacttggac aggagtaact cagaatggag 480 gaagcggtgc ttgcaaaagg ggacctgcta a tggtttctt cagtagattg aattggttaa 540 ctaagtcagg aaatacatat ccagtgttga atgtgactat gccaaacaat aacaatttcg 600 acaaattata catttgggga gttcatcacc caagcactaa tcaagaacaa accagcctgt 660 atattcaggc ctcaggaaga gtcacagtct ctaccaggag aagccaacag accataatcc 720 caaacattgg atctagaccc ttggtaaggg gccaatctgg cagaataagc gtatattgga 780 caatagtcaa acctggagac gtactggtaa taaacagtaa tggaaaccta atcgctcctc 840 gaggctactt caaaatgcgc attgggaaaa gctcaataat gagatcagat gcacctattg 900 acacctgcat ttccgaatgt atcactccga acgggagcat ccccaatgac aagcccttcc 960 aaaatgtaaa caagatcaca tacggagcat gtcccaaata tgttaagcaa aacaccttga 1020 aactggcaac aggaatgcgg aatgtccctg agaggcaaac cagaggcctg ttcggcgcaa 1080 tagcaggttt catagaaaat ggatgggaag ggatggtaga cggttggtat ggcttcaggc 1140 accaaaattc cgaaggtaca ggacaagcag cagaccttaa aagcactcag gcagccattg 1200 accagattaa tgggaaattg aacagagtga ttgaaaaaac gaatgagaag ttccatcaaa 1260 tcgaaaagga gttttccgaa gtagaaggga ggattcaaga ccttgagaga tacgttgaag 1320 acacaaaagt agatctttgg tcttacaatg ccgagcttct tgtt gcttta gaaaaccaga 1380 aaacaattga tttaactgat tcagaaatga acaaattgtt tgaaaagact aggaggcaat 1440 tgagggaaaa tgctgaagac atgggcaatg gctgcttcaa gatataccac aagtgtgaca 1500 atgcttgcat agaatcgatt agaaacggaa cttatgacca taacatatat agagatgagg 1560 cagtgaacaa tcggttccag atcaaaggtg ttgagctaaa gtctggatac aaagactgga 1620 tcttgtggat ttcctttgcc atatcatgct ttttgctttg tgttgtcttg ctgggtttca 1680 ttatgtgggc ctgccagaga ggcaacatta ggtgcaacat ttgcatttga gtgtactaat 1740 aattaaaaac acccttgttt ctac 1764 < 210> 3 <211> 1704 <212> DNA <213> Artificial Sequence <220> <223> hemagglutinin gene of A/chiken/korea/es/2003(H5N1) <400> 3 atggagaaaa tagtgcttct tcttgcaata gtcagtcttg ttaaaagtga tcagatttgc 60 attgga ctcgacagag caggttgaca caataatgga aaagaacgtc 120 actgttacac atgcccaaga catactggaa aagacacaca acgggaagct ctgcgatcta 180 gatggagtga agcctctaat tttaagagat tgtagtgtag ctggatggct cctcgggaac 240 ccaatgtgtg acgaattcat caatgtgccg gaatggtctt acatagtgga gaaggccaat 300 ccacccaatg acctctgtta cccagggaat ttcaacgact atgaagaact gaaacaccta 360 ttgagcagaa taaaccattt tgaaaaaatt cagatcatcc ccaaaagttc ttggtccgat 420 catgaagcct catcaggggt gagctcagca tgtccatacc agggaaggtc ctccttcttc 480 agaaatgtgg tatggcttat caaaaagaac agtgcatacc caacaataaa gagaagctac 540 aataatacca accaagaaga tcttttggta ctgtggggga ttcaccatcc aaatgatgcg 600 gcagagcaga caagactcta tcaaaaccca accacctata tttccgttgg gacatcaaca 660 ctaaaccaga gattggtacc aaaaatagct actagatcca aagtaaacgg gcaaagtgga 720 aggatggagt tcttctggac aattttaaaa ccgaatgatg caatcagctt tgagagtaat 780 ggaaatttca ttgctccaga atatgcatac aaaattgtca agaaagggga ctcagcaatt 840 atgaaaagtg aattggaata tggtaactgc aacaccaagt gtcaaactcc aatgggggcg 900 ataaactcta gtatgccatt ccacaacata caccctctca ccatcgggga atgccccaaa 960 tatgtgaaat caagcagatt agtccttgcg actgggctca gaaatagccc tcaaagagag 1020 aaaagaaaaa agagaggact atttggagct atagcaggtt ttatagaggg aggatggcag 1080 ggaatggtag atggttggta tgggtaccac catagcaatg agcaggggag tgggtacgct 1140 gcagacaaag aatccactca aaaggcaata gatggagtca ccaataaggt caactc gatc 1200 attgacaaaa tgaacactca gtttgaggcc gttggaaggg aatttaataa cttagaaagg 1260 agaatagaaa atttaaacaa gaagatggaa gacggattcc tagatgtctg gacttataat 1320 gctgaacttc tggttctcat ggaaaatgag agaactctag actttcatga ctcaaatgtc 1380 aagaaccttt acgacaaggt ccgactacag cttagggata atgcaaagga gctgggtaac 1440 ggttgtttcg agttctatca cagatgtgat aatgaatgta tagaaagtgt aagaaacgga 1500 acgtatggct acccgcagta ttcagaagaa gcaagattaa aaagagagga aataagtgga 1560 gtaaaattgg aatcaatagg aacttaccaa atactgtcaa tttattcaac agtggcaagt 1620 tccctagcac tggcaatcat ggtggctggt ctatctttat ggatgtgctc caatggatcg 1680 ttacaatgca gaatttgcat ttaa 1704 <210> 4 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> M2e400 gene of A/California/04/2009(H1N1) > 4 atgagtcttc taaccgaggt cgaaacgcct accagaagcg aatgggagtg cagatgcagc 60 gattcaagtg atcctctcgt cattgcagca aatatcattg ggatcttgca cctgatattg 120 tggattactg atcgtctttt tttcaaatgt atttatcgtc gctttaaata cggtttgaaa 180 agagggcctt ctacggaagg agtgcctgag tccatgaggg aagaatatca acaggaacag 24 0 cagagtgctg tggatgttga cgatggtcat tttgtcaaca tagagctaga gtaa 294 <210> 5 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> M2e gene of A/canine/Korea/01/2007(H3N2) <400> 5 atgagtcttc taaccgaggt cgaaacgcct accagaaacg gatgggagtg cagatgcagc 60 gatttaagtg atcctctcgt tattgtcgca agtatcattg ggatcttgca cttgatattg 120 tggatttttg atcgtctttt cttcaaatgt atttatcgtc gccttaaata cggtttgaaa 180 agagggcctt ctacggaagg agtgcctgag tctatgaggg aagagtatcg gcaggaacag 240 cagagtgctg tggatgttga cgatggtcat tttgtcaaca tagagctgga gtaa 294 <210> 6 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> M2e gene of A/chiken/korea/es/2003(H5N1) <400> 6 atgagtcttc taaccgaggt cgaaacgcct accagaaacg aatgggagtg cagatgcagc 60 gattcaagtg atcctcttgt tgttgccgca agtatcattg ggatcttgca cttgatattg 120 tggattcttg atcgtctttt cttcaaatgc atttatcgtc gccttaaata cggtttgaaa 180 agagggcctt ctacggcagg ggtacctgag tctatgaggg aagagtaccg gcaggaacag 240 cagagtgctg tggatgttga cgatggtcat tttgtcaaca tagaattgga gtaa 294 <210> 7 <211> 32 < 212> PRT <213> Artificial Sequence <220> <223> HA2SH of A/California/04/2009(H1N1) <400> 7 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 1 5 10 15 Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 20 25 30 <210> 8 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> HA2SH of A/canine/Korea/ 01/2007(H3N2) <400> 8 Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala 1 5 10 15 Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn 20 25 30 < 210> 9 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> HA2SH of A/chiken/korea/es/2003(H5N1) <400> 9 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala 1 5 10 15 Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn 20 25 30 <210> 10 <211> 96 <212> DNA <213> Artificial Sequence <220> < 223> HA2SH gene of A/California/04/2009(H1N1) <400> 10 gagcaggggt caggatatgc agccgacctg aagagcacac agaatgccat tgacgagatt 60 actaacaaag taaattct gt tattgaaaag atgaat 96 <210> 11 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> HA2SH gene of A/canine/Korea/01/2007(H3N2) <400> 11 tccgaaggta caggacaagc agcagacctt aaaagcactc aggcagccat tgaccagatt 60 aatgggaaat tgaacagagt gattgaaaaa acgaat 96 <210> 12 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> HA2SH gene of A/chiken/korea/es/2003(H5N1) <400> 12 gagcagggga gtgggtacgc tgcagacaaa gaatccactc aaaaggcaat agatggagtc 60 accaataagg tcaactcgat cattgacaaa atgaac 96 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> M2400e of A/California/04/2009 13 Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg Ser 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> M2e of A/canine/Korea /01/2007(H3N2) <400> 14 Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg Ser 1 5 10 15 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> M2e of A/chiken/korea/es/2003(H5N1) <400> 15 Thr Glu Val Glu T hr Pro Thr Arg Asn Glu Trp Glu Ser Arg Ser 1 5 10 15 <210> 16 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> M2e gene of A/California/04/2009(H1N1 ) <400> 16 accgaggtcg aaacgcctac cagaagcgaa tgggagagca gaagc 45 <210> 17 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> M2e gene of A/canine/Korea/01/2007(H3N2) < 400> 17 accgaggtcg aaacgcctac cagaaacgga tgggagagca gaagc 45 <210> 18 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> M2e gene of A/chiken/korea/es/2003(H5N1) <400> 18 accgaggtcg aaacgcctac cagaaacgaa tgggagagca gaagc 45 <210> 19 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> HA2SH-M2e of A/California/04/2009(H1N1) <400> 19 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 1 5 10 15 Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg Ser 35 40 45 <210> 20 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> HA2SH-M2e of A/ca nine/Korea/01/2007(H3N2) <400> 20 Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala 1 5 10 15 Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg Ser 35 40 45 <210> 21 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> HA2SH-M2e of A/ chiken/korea/es/2003(H5N1) <400> 21 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala 1 5 10 15 Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Ser Arg Ser 35 40 45 <210> 22 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> HA2SH-M2e gene of A /California/04/2009 (H1N1) <400> 22 gagcaggggt caggatatgc agccgacctg aagagcacac agaatgccat tgacgagatt 60 actaacaaag taaattctgt tattgaaaag DNA Sequence atgaataccg aggagatcgaaac <211> 141 <220 a > <223> HA2SH-M2e gene of A/canine/Korea/0 1/2007(H3N2) <400> 23 tccgaaggta caggacaagc agcagacctt aaaagcactc aggcagccat tgaccagatt 60 aatgggaaat tgaacagagt gattgaaaaa acgaataccg aggtcgaaac gcctaccaga 120 223 <211 DNA < 141 Sequence 141 220 <211 aacggatgg Artificial < 212 aacggatg > HA2SH-M2e gene of A/chiken/korea/es/2003(H5N1) <400> 24 gagcagggga gtgggtacgc tgcagacaaa gaatccactc aaaaggcaat agatggagtc 60 accaataagg tcaactc 25gat cattgaca 212 211 <400> > PRT <213> Artificial Sequence <220> <223> 3XH1SH-M2e <400> 25 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 1 5 10 15 Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg Ser Glu 35 40 45 Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile 50 55 60 Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr 65 70 75 80 Glu Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg Ser Glu Gln 85 90 95 Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp 100 105 110 Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Glu 115 120 125 Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg Ser 130 135 140 <210> 26 <211> 141 <212> PRT <213> Artificial Sequence <220> <223> 3XH3SH-M2e <400> 26 Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala 1 5 10 15 Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg Ser Ser 35 40 45 Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile 50 55 60 Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Thr 65 70 75 80 Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg Ser Ser Glu 85 90 95 Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp 100 105 110 Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Thr Glu 115 120 125 Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg Ser 130 135 140 <210> 27 <211> 141 <212> PRT <213> Artificial Sequence <220 > <223> 3XH5SH-M2e <400> 27 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala 1 5 10 15 Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Ser Arg Ser Glu 35 40 45 Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile 50 55 60 Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr 65 70 75 80 Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Ser Arg Ser Glu Gln 85 90 95 Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp 100 105 110 Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr Glu 115 120 125 Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Ser Arg Ser 130 135 140 <210> 28 <211> 141 <212> PRT <213> Artificial Sequence <220> <223> H135SH-M2e <400> 28 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 1 5 10 15 Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg Ser Ser 35 40 45 Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile 50 55 60 Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Thr 65 70 75 80 Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg Ser Glu Gln 85 90 95 Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp 100 105 110 Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn Thr Glu 115 120 125 Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Ser Arg Ser 130 135 140 <210> 29 <211> 423 <212> DNA <213> Artificial Sequence <220> <223> 3XH1SH-M2e gene <400> 29 gagcagggg t caggatatgc agccgacctg aagagcacac agaatgccat tgacgagatt 60 actaacaaag taaattctgt tattgaaaag atgaataccg aggtcgaaac gcctaccaga 120 agcgaatggg agagcagaag cgagcagggg tcaggatatg cagccgacct gaagagcaca 180 cagaatgcca ttgacgagat tactaacaaa gtaaattctg ttattgaaaa gatgaatacc 240 gaggtcgaaa cgcctaccag aagcgaatgg gagagcagaa gcgagcaggg gtcaggatat 300 gcagccgacc tgaagagcac acagaatgcc attgacgaga ttactaacaa agtaaattct 360 gttattgaaa agatgaatac cgaggtcgaa acgcctacca gaagcgaatg ggagagcaga 420 agc 423 <210> 30 <211> 423 <212> DNA <213> Artificial Sequence <220> <223> 3XH3SH-M2e gene <400> 30 tccgaaggta caggacaagc agcagacctt aaaagcactc aggcagccat tgaccagatt 60 aatgggaaat tgaacagagt gattgaaaaa acgaataccg aggtcgaaac gcctaccaga 120 aacggatggg agagcagaag ctccgaaggt acaggacaag cagcagacct taaaagcact 180 caggcagcca ttgaccagat taatgggaaa ttgaacagag tgattgaaaa aacgaatacc 240 gaggtcgaaa cgcctaccag aaacggatgg gagagcagaa gctccgaagg tacaggacaa 300 gcagcagacc ttaaaagcac tcaggcagcc attgaccaga ttaatgggaa attgaacaga 360 gtgattgaaa aaacgaatac cgaggtcgaa acgcctacca gaaacggatg ggagagcaga 420 agc 423 <210> 31 <211> 423 <212> DNA <213> Artificial Sequence <220> <223> 3XH5SH-M2e gene <400> 31 gagcagggga gtgggtacgc tgcagacaaa gaatccactc aaaagcaacta agat catggga atgaacaccg aggtcgaaac gcctaccaga 120 aacgaatggg agagcagaag cgagcagggg agtgggtacg ctgcagacaa agaatccact 180 caaaaggcaa tagatggagt caccaataag gtcaactcga tcattgacaa aatgaacacc 240 gaggtcgaaa cgcctaccag aaacgaatgg gagagcagaa gcgagcaggg gagtgggtac 300 gctgcagaca aagaatccac tcaaaaggca atagatggag tcaccaataa ggtcaactcg 360 atcattgaca aaatgaacac cgaggtcgaa acgcctacca gaaacgaatg ggagagcaga 420 agc 423 <210> 32 <211> 423 < 212> DNA <213> Artificial Sequence <220> <223> H135SH-M2e gene <400> 32 gagcaggggt caggatatgc agccgacctg aagagcacac agaatgccat tgacgagatt 60 actaacaaag taaattctgt tattgaaaag atgaataccg aggtcgaaac gcctaccaga 120 agcgaatggg agagcagaag ctccgaaggt acaggacaag cagcagacct taaaagcact 180 caggcagcca ttgaccagat taatgggaaa ttgaacagag tgattgaaaa aacgaatacc 240 gaggtcgaaa cgcctaccag aaacggatgg gagagcagaa gcgagcaggg gagtgggtac 300 gctgcagaca aagaatccac tcaaaaggca atagatggag tcaccaataa ggtcaactcg 23 gaaaccaca aaatgaacca ga 420 g ggtacca 33 145 <212> PRT <213> Artificial Sequence <220> <223> 3XH1SH-M2e <400> 33 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 1 5 10 15 Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg Ser Gly 35 40 45 Ser Glu Gly Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn 50 55 60 Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met 65 70 75 80 Asn Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg Ser 85 90 95 Lys Leu Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln 100 105 110 Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys 115 120 125 Met Asn Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg 130 135 140 Ser 145 <210 > 34 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> 3XH3SH-M2e <400> 34 Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr G ln Ala Ala 1 5 10 15 Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg Ser Gly 35 40 45 Ser Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala 50 55 60 Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr 65 70 75 80 Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg Ser 85 90 95 Lys Leu Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln 100 105 110 Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys 115 120 125 Thr Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg 130 135 140 Ser 145 <210> 35 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> 3XH5SH-M2e <400> 35 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala 1 5 10 15 Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met Asn 20 25 30 Thr Glu Val Glu T hr Pro Thr Arg Asn Glu Trp Glu Ser Arg Ser Gly 35 40 45 Ser Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys 50 55 60 Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys Met 65 70 75 80 Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Ser Arg Ser 85 90 95 Lys Leu Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln 100 105 110 Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys 115 120 125 Met Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Ser Arg 130 135 140 Ser 145 <210> 36 <211> 145 <212> PRT <213> Artificial Sequence < 220> <223> H135SH-M2e <400> 36 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 1 5 10 15 Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 20 25 30 Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp Glu Ser Arg Ser Gly 35 40 45 Ser Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala 50 55 60 Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr 65 70 75 80 Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Ser Arg Ser 85 90 95 Lys Leu Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln 100 105 110 Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp Lys 115 120 125 Met Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Ser Arg 130 135 140 Ser 145 < 210> 37 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> 3XH1SH-M2e <400> 37 gagcaggggt caggatatgc agccgacctg aagagcacac agaatgccat tgacgagatt 60 actaacaaag taaattctgt tattgaaaag atgaataccg aggtcgaaac gcctaccaga 120 agcgaatggg agagcagaag cggatccgag caggggtcag gatatgcagc cgacctgaag 180 agcacacaga atgccattga cgagattact aacaaagtaa attctgttat tgaaaagatg 240 aataccgagg tcgaaacgcc taccagaagc gaatgggaga gcagaagcaa gcttgagcag 300 ccattgacgaggat atgcagccga gaagtaagaataacg a acattagaagat 360 t ctgttattga aaagatgaat accgaggtcg aaacgcctac cagaagcgaa 420 tgggagagca gaagc 435 <210> 38 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> 3XH3SH-M2e <400> 38 tcca gaatta agcagacct aggaatta acagagta gaatta 60 acgaataccg aggtcgaaac gcctaccaga 120 aacggatggg agagcagaag cggatcctcc gaaggtacag gacaagcagc agaccttaaa 180 agcactcagg cagccattga ccagattaat gggaaattga acagagtgat tgaaaaaacg 240 aataccgagg tcgaaacgcc taccagaaac ggatgggaga gcagaagcaa gctttccgaa 300 ggtacaggac aagcagcaga ccttaaaagc actcaggcag ccattgacca gattaatggg 360 aaattgaaca gagtgattga aaaaacgaat accgaggtcg aaacgcctac cagaaacgga 420 tgggagagca gaagc 435 <210> 39 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> 3XH5SH-M2e <400> 39 gagcagggga gtgggtacgc tgcagacaaa gaatccactc aaaaggcaat agatggagtc 60 accaataagg tcaactcgat cattgacaaa atgaacaccg aggtcgaaac gcctaccaga 120 aacgaatggg agagcagaag cggatccgag caggggagtg ggtacgctgc agacaaagaa 180 tccactcaaa aggcaataga tgg agtcacc aataaggtca actcgatcat tgacaaaatg 240 aacaccgagg tcgaaacgcc taccagaaac gaatgggaga gcagaagcaa gcttgagcag 300 gggagtgggt acgctgcaga caaagaatcc actcaaaagg caatagatgg agtcaccaat 360 aaggtcaact cgatcattga caaaatgaac accgaggtcg aaacgcctac cagaaacgaa 420 tgggagagca gaagc 435 <210> 40 <211> 507 <212> DNA <213> Artificial Sequence <220> < 223> H135SH-M2e <400> 40 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggagcagg ggtcaggata tgcagccgac ctgaagagca cacagaatgc cattgacgag 120 attactaaca aagtaaattc tgttattgaa aagatgaata ccgaggtcga aacgcctacc 180 agaagcgaat gggagagcag aagcggatcc tccgaaggta caggacaagc agcagacctt 240 aaaagcactc aggcagccat tgaccagatt aatgggaaat tgaacagagt gattgaaaaa 300 acgaataccg aggtcgaaac gcctaccaga aacggatggg agagcagaag caagcttgag 360 caggggagtg ggtacgctgc agacaaagaa tccactcaaa aggcaataga tggagtcacc 420 aataaggtca actcgatcat tgacaaaatg aacaccgagg tcgaaacgcc taccagaaac 480 gaatgggaga gca 213gaagccia <gt cga l Sequence <220> <223> 3XH1SH-M2e <400> 41 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys 20 25 30 Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val 35 40 45 Ile Glu Lys Met Asn Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp 50 55 60 Glu Ser Arg Ser Gly Ser Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu 65 70 75 80 Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser 85 90 95 Val Ile Glu Lys Met Asn Thr Glu Val Glu Thr Pro Thr Arg Ser Glu 100 105 110 Trp Glu Ser Arg Ser Lys Leu Glu Gln Gly Ser Gly Tyr Ala Ala Asp 115 120 125 Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn 130 135 140 Ser Val Ile Glu Lys Met Asn Thr Glu Val Glu Thr Pro Thr Arg Ser 145 150 155 160 Glu Trp Glu Ser Arg Ser Leu Glu 165 <210> 42 <211 > 168 <212> PRT <213> Artificial Sequence <220> <223> 3XH3SH-M2e <400> 42 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys 20 25 30 Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val 35 40 45 Ile Glu Lys Thr Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp 50 55 60 Glu Ser Arg Ser Gly Ser Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu 65 70 75 80 Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg 85 90 95 Val Ile Glu Lys Thr Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Gly 100 105 110 Trp Glu Ser Arg Ser Lys Leu Ser Glu Gly Thr Gly Gln Ala Ala Asp 115 120 125 Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn 130 135 140 Arg Val Ile Glu Lys Thr Asn Thr Glu Val Glu Thr Pro Thr Arg Asn 145 150 155 160 Gly Trp Glu Ser Ar g Ser Leu Glu 165 <210> 43 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> 3XH5SH-M2e <400> 43 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu 20 25 30 Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile 35 40 45 Ile Asp Lys Met Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp 50 55 60 Glu Ser Arg Ser Gly Ser Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys 65 70 75 80 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser 85 90 95 Ile Ile Asp Lys Met Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Glu 100 105 110 Trp Glu Ser Arg Ser Lys Leu Glu Gln Gly Ser Gly Tyr Ala Ala Asp 115 120 125 Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn 130 135 140 Ser Ile Ile Asp Lys Met Asn Thr Glu Val Glu Thr Pro Thr Arg Asn 145 150 155 160 Glu Trp Glu Ser Arg Ser Leu Glu 165 <210> 44 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> H135SH-M2e <400> 44 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys 20 25 30 Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val 35 40 45 Ile Glu Lys Met Asn Thr Glu Val Glu Thr Pro Thr Arg Ser Glu Trp 50 55 60 Glu Ser Arg Ser Gly Ser Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu 65 70 75 80 Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg 85 90 95 Val Ile Glu Lys Thr Asn Thr Glu Val Glu Thr Pro Thr Arg Asn Gly 100 105 110 Trp Glu Ser Arg Ser Lys Leu Glu Gln Gly Ser Gly Tyr Ala Ala Asp 115 120 125 Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn 130 135 140 Ser Ile Ile Asp Lys Met Asn Thr Glu Val Glu Thr Pro Thr Arg Asn 145 150 155 160 Glu Trp Glu Ser Arg Ser Leu Glu 165 <210> 45 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> 3XH1SH-M2e gene <400> 45 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggagcagg ggtcaggata tgcagccgac ctgaagagca cacagaatgc cattgacgag 120 attactaaca aagtaaattc tgttattgaa aagatgaata ccgaggtcga aacgcctacc 180 agaagcgaat gggagagcag aagcggatcc gagcaggggt caggatatgc agccgacctg 240 aagagcacac agaatgccat tgacgagatt actaacaaag taaattctgt tattgaaaag 300 atgaataccg aggtcgaaac gcctaccaga agcgaatggg agagcagaag caagcttgag 360 caggggtcag gatatgcagc cgacctgaag agcacacaga atgccattga cgagattact 420 aacaaagtaa attctgttat tgaaaagatg aataccgagg tcgaaacgcc taccagaagc 480 gaatgggaga gcagaagcct cgagtga 507 <210> 46 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> 3XH3SH-M2e gene <400> 46 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgtgccgacgcg cggcagccat ctacagcg cggcagca taccagct 60 atttagacagca taccagt atggga aattgaacag agtgattgaa aaaacgaata ccgaggtcga aacgcctacc 180 agaaacggat gggagagcag aagcggatcc tccgaaggta caggacaagc agcagacctt 240 aaaagcactc aggcagccat tgaccagatt aatgggaaat tgaacagagt gattgaaaaa 300 acgaataccg aggtcgaaac gcctaccaga aacggatggg agagcagaag caagctttcc 360 gaaggtacag gacaagcagc agaccttaaa agcactcagg cagccattga ccagattaat 420 gggaaattga acagagtgat tgaaaaaacg aataccgagg tcgaaacgcc taccagaaac 480 ggatgggaga gcagaagcct cgagtga 507 <210> 47 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> 3XH5SH-M2e gene <400> 47 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggagcagg ggagtgggta cgctgcagac aaagaatcca ctcaaaaggc aatagatgga 120 gtcaccaata aggtcaactc gatcattgac aaaatgaaca ccgaggtcga aacgcctacc 180 agaaacgaat gggagagcag aagcggatcc gagcagggga gtgggtacgc tgcagacaaa 240 gaatccactc aaaaggcaat agatggagtc accaataagg tcaactcgat cattgacaaa 300 atgaacaccg aggtcgaaac gcctaccaga aacgaatggg tagagc gga caggtaccaga aacgaatggg tagct gga caaa aggcaataga tggagtcacc 420 aataaggtca actcgatcat tgacaaaatg aacaccgagg tcgaaacgcc taccagaaac 480 gaatgggaga gcagaagcct cgagtga 507 <210> 48 <211> 507 <212> DNA <213> gc ca g aggcat c ggcat <213> 507 <212> DNA <213> artificial sequence <220> tggtgccgcg cggcagccat 60 atggagcagg ggtcaggata tgcagccgac ctgaagagca cacagaatgc cattgacgag 120 attactaaca aagtaaattc tgttattgaa aagatgaata ccgaggtcga aacgcctacc 180 agaagcgaat gggagagcag aagcggatcc tccgaaggta caggacaagc agcagacctt 240 aaaagcactc aggcagccat tgaccagatt aatgggaaat tgaacagagt gattgaaaaa 300 acgaataccg aggtcgaaac gcctaccaga aacggatggg agagcagaag caagcttgag 360 caggggagtg ggtacgctgc agacaaagaa tccactcaaa aggcaataga tggagtcacc 420 aataaggtca actcgatcat tgacaaaatg aacaccgagg tcgaaacgcc taccagaaac 480gaatgggaga gcagaagcct cgagtga 507

Claims (22)

a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a vaccine composition for preventing canine influenza virus infection comprising a,
The fusion protein trimer has the following structural formula,
The canine influenza virus is any one subtype selected from the group consisting of H1N1, H3N2, H5N1 and H5N8, the composition:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequence of SEQ ID NO: 7 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequence of SEQ ID NO: 13 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
According to claim 1,
The A1, A2 and A3 are each independently represented by the amino acid sequence of SEQ ID NO: 19, a vaccine composition for preventing canine influenza virus infection.
According to claim 1,
The fusion protein trimer is represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 33 and SEQ ID NO: 41, vaccine composition for preventing canine influenza virus infection.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a vaccine composition for preventing canine influenza virus infection comprising a,
The fusion protein trimer has the following structural formula,
wherein the canine influenza virus is of subtype H3N2 or H5N1, a composition comprising:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequence of SEQ ID NO: 8 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequence of SEQ ID NO: 14 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
5. The method of claim 4,
The A1, A2 and A3 are each independently represented by the amino acid sequence of SEQ ID NO: 20, a vaccine composition for preventing canine influenza virus infection.
5. The method of claim 4,
The fusion protein trimer is represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 34 and SEQ ID NO: 42, vaccine composition for preventing canine influenza virus infection.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a vaccine composition for preventing canine influenza virus infection comprising a,
The fusion protein trimer has the following structural formula,
wherein the canine influenza virus is of subtype H5N1 or H5N8, the composition:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequence of SEQ ID NO: 9 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequence of SEQ ID NO: 15 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
8. The method of claim 7,
The A1, A2 and A3 are each independently represented by the amino acid sequence of SEQ ID NO: 21, a vaccine composition for preventing canine influenza virus infection.
8. The method of claim 7,
The fusion protein trimer is represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 35 and SEQ ID NO: 43, vaccine composition for preventing canine influenza virus infection.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a vaccine composition for preventing canine influenza virus infection comprising a,
The fusion protein trimer has the following structural formula,
wherein the canine influenza virus is of subtype H3N2 or H5N1, a composition comprising:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
The method of claim 1,
The A1, A2 and A3 are each independently represented by the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, vaccine composition for preventing canine influenza virus infection.
According to claim 1,
The fusion protein trimer is represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 36 and SEQ ID NO: 44, vaccine composition for preventing canine influenza virus infection.
11. The method of any one of claims 1, 4, 7 and 10,
The peptide linker is selected from the group consisting of glycine, serine, alanine, proline, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, threonine, cysteine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid and glutamic acid. A vaccine composition for preventing canine influenza virus infection, comprising one or more amino acids.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a veterinary composition for preventing or treating canine influenza virus infection disease comprising a,
The fusion protein trimer has the following structural formula,
The canine influenza virus is any one subtype selected from the group consisting of H1N1, H3N2, H5N1 and H5N8, the composition:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequence of SEQ ID NO: 7 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequence of SEQ ID NO: 13 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a veterinary composition for preventing or treating canine influenza virus infection disease comprising a,
The fusion protein trimer has the following structural formula,
wherein the canine influenza virus is of subtype H3N2 or H5N1, a composition comprising:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequence of SEQ ID NO: 8 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequence of SEQ ID NO: 14 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a veterinary composition for preventing or treating canine influenza virus infection disease comprising a,
The fusion protein trimer has the following structural formula,
wherein the canine influenza virus is of subtype H5N1 or H5N8, the composition:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequence of SEQ ID NO: 9 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequence of SEQ ID NO: 15 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a veterinary composition for preventing or treating canine influenza virus infection disease comprising a,
The fusion protein trimer has the following structural formula,
wherein the canine influenza virus is of subtype H3N2 or H5N1, a composition comprising:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
18. The method according to any one of claims 14 to 17,
The canine influenza virus infection disease is any one selected from the group consisting of sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea, a veterinary composition for preventing or treating canine influenza virus infection disease.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a pharmaceutical composition for the prevention or treatment of canine influenza virus infection disease comprising a,
The fusion protein trimer has the following structural formula,
The canine influenza virus is any one subtype selected from the group consisting of H1N1, H3N2, H5N1 and H5N8, the composition:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequence of SEQ ID NO: 7 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequence of SEQ ID NO: 13 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a pharmaceutical composition for the prevention or treatment of canine influenza virus infection disease comprising a,
The fusion protein trimer has the following structural formula,
wherein the canine influenza virus is of subtype H3N2 or H5N1, a composition comprising:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequence of SEQ ID NO: 8 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequence of SEQ ID NO: 14 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a pharmaceutical composition for the prevention or treatment of canine influenza virus infection disease comprising a,
The fusion protein trimer has the following structural formula,
wherein the canine influenza virus is of subtype H5N1 or H5N8, the composition:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequence of SEQ ID NO: 9 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequence of SEQ ID NO: 15 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.
a fusion protein trimer comprising the short helical domain of hemagglutinin of influenza virus (HA2SH) or a fragment thereof and the ectodomain of M2 protein (M2e) or a fragment thereof; and
adjuvant;
As a pharmaceutical composition for the prevention or treatment of canine influenza virus infection disease comprising a,
The fusion protein trimer has the following structural formula,
wherein the canine influenza virus is of subtype H3N2 or H5N1, a composition comprising:
A1-(L1)n-A2-(L2)m-A3
Here, A1 is represented by HA2SH1-(l1)k-M2e1, A2 is represented by HA2SH2-(l2)i-M2e2, and A3 is represented by HA2SH3-(l3)j-M2e3,
The HA2SH1, HA2SH2 and HA2SH3 are each independently a short helical domain of hemagglutinin of influenza virus represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or a fragment thereof,
The M2e1, M2e2 and M2e3 are each independently an ectodomain of the M2 protein of influenza virus represented by the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 or a fragment thereof,
Wherein L1, L2, 11, 12 and 13 are each independently a peptide linker consisting of 1 to 8 amino acids linked by a peptide bond,
Wherein n, m, k, i, and j are each independently an integer of 0 or 1.

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