KR20160035666A - Vaccine against influenza virus based on hemagglutinin displayed-baculovirus and method for producing the same - Google Patents

Abstract

The present invention relates to a versatile influenza virus vaccine composition and a manufacturing method of the vaccine composition. The vaccine composition comprises recombinant baculovirus expressed in the form of hemagglutinin protein included in the outer skin of the baculovirus, or a virus particle thereof as an active ingredient. The vaccine composition has protective effects with respect to the same type and the different type of influenza virus infection by inducing a humoral immune response and a stock-specific antibody, and can be used as a versatile vaccine.

Description

[0001] The present invention relates to a baculovirus-based influenza virus vaccine expressing hemagglutinin on the surface thereof and a method for producing the vaccine against influenza virus vaccine,

The present invention relates to a general-purpose influenza virus vaccine composition and a method for producing the vaccine composition, which comprise, as an active ingredient, a recombinant baculovirus or a viral particle thereof expressed in the form that the hemagglutinin protein is contained in the envelope of baculovirus will be.

In April 2009, the swine flu H1N1 influenza A virus (pH1N1), the first pandemic of the 21st century in North America, spread rapidly and threatened public health. According to the World Health Organization (WHO), the virus has been reported in more than 214 countries and has reportedly resulted in more than 18449 deaths by August 1, 2010. Influenza viruses occur every year and are a major respiratory pathogen causing epidemics. Influenza viruses belong to Orthomyxoviridae and have single-stranded segmental RNA genomes. Influenza viruses are classified as types A, B, and C based on the sequence of core proteins, while type B and C viruses are predominantly infected to humans, whereas type A viruses infect various mammalian animals and birds. Only types A and B cause human illness. Influenza A virus is classified based on the antigenicity of its hemagglutinin (HA) and neuraminidase (NA) molecules, which are classified into 18 different HA subtypes and 11 different NA subtypes (WHO, 2013; CDC, 2014).

Vaccination against influenza viruses is an effective way to prevent the spread of pathogens. However, most recently licensed influenza vaccines were based on hatching eggs. Using eggs to produce influenza vaccines can lead to potential problems, such as the need for large quantities of fertilized eggs and the risk of egg-related allergic reactions (McPherson, CE, 2008). There is also the problem that it usually takes 6 to 8 months to produce a vaccine based on a hatching egg.

To prevent the spread of influenza viruses from accelerating, new strategies have been proposed to overcome the problem of egg-based influenza vaccines. For example, the production of an influenza vaccine based on cell culture can be mentioned (Drugs, 2008, 68, 1483-1491). In addition, recombinant vaccine technology using a variety of vector systems and major viral structural proteins such as HA, NA, nuclear protein (NP) and membrane protein (M) (PloS one, 2013, 8, e75460; Microbes and infection / Institut Pasteur, 2010, 12, 280-28; Vaccine, 2010, 28, 4771-4776).

Recently, the baculovirus expression system has attracted attention as a platform for vaccine delivery. Baculovirus Autographa Carly poniah nuclear polyhedral virus (Autographa California nucleopolyhedrosis virus, AcNPV) is a double-stranded DNA virus and is a well-studied baculovirus for the mass production of recombinant proteins. As a vaccine delivery tool, baculoviruses have the following advantages: (i) baculovirus and its replicas in mammalian cells do not all induce cytotoxicity, thus minimizing side effects; (ii) large, flexible viral genomes allow insertion of other large genes; (iii) Baculovirus expression systems enable folding, phosphorylation, glycosylation and proteolytic cleavage, which are posttranslational protein modification processes occurring in mammalian cells over prokaryotic systems; (iv) There is no antibody present in the human against existing vectors. Due to these advantages, baculovirus is widely used as a promising vaccine delivery tool.

Influenza hemagglutinin (HA), a glycoprotein of the viral envelope that functions as an antigen, binds to host cell receptors and initiates viral infection. Therefore, HA is a major target for antibody neutralization and is now considered one of the most important components of the influenza vaccine. HA is a homotrimeric molecule and each monomer consists of subunits HA1 and HA2 with two disulfide linkages, which subunits are generated by proteolytic cleavage using host serine proteases. The spherical head domain HA1 binds to the terminal of the sialic acid residue at the host cell receptor and plays an important role in the initiation of viral introduction. After the virus enters the cell, the endosomal acidic environment promotes the conformational change of HA2, a stalk domain that allows fusion of virus and cell membrane. The function of HA1 and HA2 causes the virus to enter the host cell and release the viral genome into the cytoplasm, leading to progression of the infection.

It is well known that a large number of neutralizing antibodies against specific antigenic sites located in the spherical head domain of HA are produced. In addition, many recent studies have shown that stock-specific antibodies can effectively neutralize subtypes of various influenza viruses because stock domains are conserved in a variety of influenza viruses. In particular, the HA stock domain is well conserved between group 1, between group 2 and influenza B virus. Thus, while stock-specific antibodies are promising therapeutics for influenza virus infection, there is a problem that it is difficult to induce the antibody to recognize the stock portion because the stock portion is masked by the membrane.

In addition, there have been many attempts to selectively increase stock-specific antibody production, and there have been attempts to induce stock-specific antibodies using chimera HA expressing the spherical head of the HA and the stock portion of another subtype Journal of virology, 2013 87, 6542-6550). However, in order to induce stock-specific antibodies using the chimeric HA, three types of chimeric HA have to be produced and the chimeric HA has to be inoculated three times.

Under this background, the inventors have developed a recombinant baculovirus (rBac-HA) vaccine that displays a hemagglutinin full-length protein in the envelope of baculovirus, Specific antibody and induce the stock-specific antibody to have an excellent protective effect against the influenza virus infection of the homozygous and subtypes through the induction of the stock-specific antibody, thus confirming that the vaccine can be used as a general avian influenza virus vaccine.

It is an object of the present invention to provide a universal influenza virus vaccine composition comprising, as an active ingredient, a recombinant baculovirus or a viral particle thereof expressed in the form that the hemagglutinin protein is contained in the envelope of baculovirus.

Another object of the present invention is to provide a method for producing a recombinant baculovirus, comprising the steps of: (a) culturing a cell transfected with recombinant baculovirus expressed in the form that the hemagglutinin protein is contained in the envelope of baculovirus; And (b) obtaining a recombinant baculovirus from the culture of step (a).

In order to achieve the above object, one aspect of the present invention provides a recombinant baculovirus or viral particle thereof transformed such that the hemagglutinin protein is expressed in the envelope of baculovirus, To provide a universal influenza virus vaccine composition.

The term " hemagglutinin (HA) "of the present invention means a substance which causes aggregation of erythrocytes. For the purpose of the present invention, the hemagglutinin is expressed in baculovirus and has immunity against influenza virus Lt; RTI ID = 0.0 > and / or < / RTI > Specifically, the hemagglutinin may be a hemagglutinin full-length protein of H1N1 virus, a fragment thereof, an amino acid sequence variant thereof, or a multimer thereof in which the hemagglutinin is linked in plural. Specifically, the hemagglutinin may be a full-length protein, A polypeptide comprising the amino acid sequence of SEQ ID NO: 2, but is not particularly limited thereto.

In addition, the hemagglutinin has not only the amino acid sequence shown in SEQ ID NO: 2 but also the amino acid sequence having 70% or more, specifically 80% or more, more specifically 90% or more, still more specifically 95% ≪ / RTI > may comprise an amino acid sequence that exhibits at least 98% similarity. Also, as the sequence having such similarity, peptide mutants having an amino acid sequence in which a part of the sequences are deleted, modified, substituted or added are also included in the scope of the present invention if it is an amino acid sequence having substantially the same or corresponding biological activity as the hemagglutinin It is self-evident.

The term "amino acid sequence variant " of the present invention means a protein having a sequence which differs from the native amino acid sequence of the protein by deletion, insertion, non-conservative or conservative substitution or a combination thereof. For purposes of the present invention, the amino acid sequence variant refers to an amino acid sequence variant of a hemagglutinin protein, which enhances the glycosylated, lipidated, antigen presentation of the hemagglutinin protein And a form derivatized to contain a molecule that enhances the target of the antigen to antigen-presenting cells, but the present invention is not limited thereto.

The term " baculovirus "of the present invention means a virus comprising a circular double-stranded genomic DNA of 130-200 kbp in a rod-like nucleocapsid and causing a fatal disease mainly by infecting an insect cell, Is used as a gene vector for carrying out a series of experiments for introducing a polynucleotide encoding a eukaryotic cell-derived protein into a host eukaryotic cell (for example, an insect cell) to express the protein in the host cell.

In the present invention, the recombinant baculovirus refers to baculovirus inserted into the genomic DNA of baculovirus so that a polynucleotide encoding the hemagglutinin protein is operable. The recombinant baculovirus is characterized in that the hemagglutinin protein is expressed on the envelope of baculovirus. Generally, baculovirus is produced by introducing a polynucleotide encoding a protein derived from a eukaryotic cell into a host eukaryotic cell (for example, an insect cell) by a gene recombination method and carrying out a series of experiments for expressing the protein in the host cell However, in the present invention, the hemagglutinin protein is expressed on the outer surface thereof and used as an active ingredient of the vaccine composition. The recombinant baculovirus of the present invention or itself can be used as an active ingredient of a vaccine for the prevention or treatment of infection of influenza virus.

The term " universal influenza virus vaccine " as used herein means a vaccine having an immunological effect against not only influenza viruses having the same hemagglutinin protein expressed on the recombinant baculovirus envelope but also different influenza viruses. This is because, in the case of a conventional hemagglutinin-based vaccine, an antibody specific to the head portion of hemaglutinin is induced and has only an immune effect against the homologous virus. In the present invention, the hemagglutinin is a baculovirus Because the antibody specific for the stock portion of hemagglutinin, which is present in the form expressed on the envelope of hemagglutinin, which is known to be preserved in various influenza strains, can be induced. Therefore, the vaccine composition of the present invention has an advantage that it can be used as a general-purpose vaccine because it induces a stock-specific antibody that can show immunity against various influenza virus subtypes. According to one embodiment of the present invention, when the hemagglutinin protein is derived from H1N1 virus, the vaccine composition may have an immunological effect against H1N1 virus or H5N1 virus, but is not limited thereto.

The recombinant baculovirus or viral particle thereof of the present invention may be administered as a vaccine to induce immunity against a disease caused by an influenza virus infection, wherein the recombinant baculovirus or virus particle thereof is administered together with a pharmaceutically acceptable carrier ≪ / RTI > The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a pigment and a flavoring agent in the case of oral administration. In the case of an injection, A non-aqueous solvent such as an aqueous solvent such as water and a ring gel liquid, a vegetable oil, a higher fatty acid ester (e.g., oleic acid), and an alcohol (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) A buffering agent, a preservative, an anhydrous agent, a solubilizing agent, an isotonic agent and a stabilizer may be mixed and used. In the case of a propellant, it can be conveniently delivered in the form of an aerosol sprayer body from a pressurized pack or sprayer using a suitable propellant, such as compressed air, nitrogen, carbon dioxide, or a hydrocarbon-based low boiling point solvent.

The formulation of the vaccine composition of the present invention may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like in the case of oral administration, and in the case of injections, unit dosage ampoules or a plurality of dosage forms.

The route of administration of the vaccine composition can be administered via any conventional route so long as it can reach the target tissue.

The term "administration" of the present invention means introduction of a predetermined substance into a patient by any suitable method, and is formulated into human or veterinary and administered by various routes. The vaccine composition of the present invention may be administered by parenteral routes such as intravenous, intravenous, intraarterial, intramuscular or subcutaneous routes and administered by the inhalation route via oral, nasal, rectal, transdermal or aerosol And may be administered as bolus or slowly, but specifically administered sublingually, intramuscularly, or nasally, and more specifically, but not exclusively, the nasal passages.

The vaccine composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means an amount sufficient to exhibit a vaccine effect and an amount not causing side effects or serious or excessive immune response, The severity of the particular compound, the activity of the particular compound, the route of administration, the rate of removal of the recombinant baculovirus or viral particles thereof, the duration of the treatment, the drug in combination with or used concurrently with the recombinant baculovirus or its viral particles, , Dietary habits, general health status, and factors known in the pharmaceutical and medical arts. Various general considerations to be considered in determining the "therapeutically effective amount" are known to those skilled in the art and are described, for example, in Gilman et al., Eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990 , And Remington ' s Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990. In the case of administration of the vaccine composition of the present invention, the number administered per dose is usually about 1 × 10 ⁷ to 1 × 10 ¹¹ , more specifically 1 × 10 ⁸ to 5 × 10 ¹⁰ , Is in the range of 5 × 10 ⁸ to 2 × 10 ¹⁰ .

The vaccine 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.

In one embodiment of the present invention, the expression of HA protein in the envelope of recombinant baculovirus (rBac-HA) was analyzed by western blot analysis. As a result, Sf9-II cells transformed with rBac-HA were found to express wild-type baculovirus Bac-ctrl) or non-transformed cells (Fig. 1c). Thus, rBac-HA was expressed and HA was expressed on the surface of rBac-HA.

In addition, in one embodiment of the present invention, rBac-HA was added to female BALB / c mice (5 weeks) and wild-type baculovirus (Bac-ctrl) as a control for immunization analysis of recombinant baculovirus (rBac- Or PBS in the nasal cavity. Then, to promote immunization, the same amount of baculovirus was inoculated two weeks after the first immunization (Example 4-1). As a result, after the second vaccination (stimulation of immunization), the mean level of HA-specific IgG antibody was significantly increased only in the group immunized with rBac-HA, and the rBac-HA vaccine of the present invention induced HA-specific IgG antibody The vaccination of two doses of the vaccine can produce a stronger antibody than one vaccination. Inoculation of 1 × 10 ⁶ PFU vaccine can result in a concentration of 3 × 10 ⁶ PFU vaccine Antibody was produced (Example 5-1).

Further, in one embodiment of the present invention, in order to confirm whether rBac-HA can induce a stock specific antibody, the serum obtained from the vaccinated mice and the purified HA stock protein (A / Korea / 1/09 (H1N1)) and the recombinant chimeric H9 / 1 protein were measured by ELISA (Fig. 2c and 2d). As a result, it was confirmed that the stock-specific antibody reaction strongly occurred after the second inoculation (stimulation of immunization) in the group of rBac-HA immunized mice. In addition, IgG titer to the HA stock domain could not be confirmed as a positive control (FIG. 2d) using the serum of a mouse inoculated with an adenovirus-based vaccine expressing HA on the surface with a strong HA stock antibody reaction. Thus, it was found that the baculovirus-based rBac-HA vaccine of the present invention was more effective than the adenovirus-based vaccine in inducing antibodies against the HA stock domain.

In conclusion, the inoculation of the rBac-HA vaccine of the present invention produces an antibody against the HA stock domain and the antibody has cross protection ability against various influenza viruses. Therefore, the rBac-HA vaccine of the present invention can be used as a general- And it was also found that there was a remarkable effect on the induction of antibodies against the HA stock domain over the adenovirus-based vaccine.

In addition, in one embodiment of the present invention, bronchoalveolar lavage (BAL) fluid was collected on the fifth day of virus infection to measure the efficacy of rBac-HA with respect to the induction of IgA, which plays an important role in the mucosal immune response, Respectively. As a result, the rBac-HA immunization group of the mice induced the HA-specific IgA response of the mucosa, whereas the IgA of the Bac-ctrl and PBS groups was not measured (Fig. 2E). This suggests that HA-specific humoral immune response is effectively induced by intranasal administration of rBac-HA.

In addition, in one embodiment of the present invention, when the activity of a hemagglutination-inhibiting (HAI) antibody is high, influenza virus infection can be inhibited. As a result of the HAI antibody assay, (1 × 10 ⁶ PFU) and 1 × 160 (1 × 10 ⁶ PFU) HAI antibody titers, respectively. The sera of rBac-HA inoculated mice showed an HAI antibody titer of 1: 640 The potency was not measured (Figure 3). As a result, the rBac-HA vaccine of the present invention has high defense against the homozygous virus infection due to the HAI antibody produced after the second inoculation.

Also, in one embodiment of the present invention, in order to confirm that the rBac-HA vaccine can protect mice from influenza virus infection, a vaccine was inoculated with 10 LD50 of A / California / 04/09 virus 3 weeks after the last inoculation Mice and control mice. Five days later, virus titers were measured in the lungs of mice and the survival rate of the infected mice was measured. As a result, mice inoculated with rBac-HA did not measure virus in the lung (<10 PFU / ml). On the other hand, mice inoculated with Bac-ctrl or PBS had higher virus titers in the lung (~1 × 10 ⁶ PFU / ml) (FIG. 4a). In addition, mice inoculated with Bac-ctrl or PBS survived 0% and 20%, respectively, but mice inoculated with rBac-HA survived without weight loss (FIGS. 4b and 4c). This demonstrates that the intranasal inoculated rBac-HA vaccine has a complete protective effect against a fatal isogenic influenza virus infection.

In addition, in one embodiment of the present invention, immunized mice were immunized with 5 (H5N1 (A / Vietnam / 1203/04) influenza virus LD50. As a result, mice inoculated with rBac-HA showed a weight loss of less than 15% until day 6 after the virus infection, but the weight loss rapidly recovered to the pre-infection level. The survival rate of rBac-HA-inoculated mice was 80%. However, control mice treated with Bac-ctrl or PBS showed severe weight loss and none survived the virus infection (Figures 5a and 5b). Thus, it was found that the nBAC-inoculated rBac-HA vaccine has protective effects against not only homozygous virus infection but also subtype influenza virus infection, and that the HA stock-specific antibody induced by the vaccine has cross-protective ability.

Another aspect of the present invention is a method for producing a recombinant baculovirus, comprising: (a) culturing a cell transfected with recombinant baculovirus expressed in the form that a hemagglutinin protein is contained in a baculovirus envelope; And (b) obtaining a recombinant baculovirus from the culture of step (a). &Lt; IMAGE &gt;

The above-mentioned "hemagglutinin", "baculovirus", "recombinant baculovirus" and "universal influenza virus vaccine" are as described above.

In the present invention, the term "transfection" refers to a phenomenon in which a gene is introduced as a host and a gene is replicable as a chromosome factor or by integration of chromosome integration, thereby introducing an external gene into a cell, it means. The transfection method of the present invention can be carried out using any transformation method and can be easily carried out according to a conventional method in the art. Specifically, the recombinant baculovirus can be transfected into Sf9 cells by a known method, but is not limited thereto.

In one embodiment of the present invention, to prepare recombinant baculovirus (rBac-HA) expressing HA on the surface, hemagglutinin of A / California / 04/09 (pH1N1) HA) was amplified by PCR and inserted into the T easy vector. The autographa California nucleopolyhedrosis virus (AcNPV) polyhedrin promoter (pPH), which expresses the strong expression of the protein, HA were introduced into the Sal I and Not I sites of the pFast Bac dual vector to ligate the nucleotide sequences coding for HA (Fig. 1A). Following the protocol of the Bac-to-Bac system (Invitrogen), specific transposition sites by the expression cassette was inserted into the viral genome into baculovirus in DH10Bac ^TM (Invitrogen, USA). Recombinant bacmid was transfected into Sf9 cells.

The term "culture" of the present invention means a product obtained by culturing the transfected cells according to a known microbial culture method. Such cultures include both culture supernatant and cell lysate, and may or may not contain cells.

The term "obtaining" of the present invention means separating the recombinant baculovirus from the culture, and the recombinant baculovirus can be isolated from the culture by methods known in the art. Specifically, the recombinant baculovirus may be separated from the culture by centrifugation using a sugar concentration gradient, but the present invention is not limited thereto.

In one embodiment of the present invention, recombinant baculovirus-transformed Sf9 cells were cultured at 28 DEG C for about 96 hours and harvested at a viability of 70-80% or more. The final supernatant was collected and concentrated via centrifugation (25% w / w sucrose in 5 mM NaCl, 10 mM EDTA in PBS) (24000 rpm for 75 min at 4 ° C) using a centrifuge. After concentration, the cells were resuspended in phosphate buffered saline (PBS, pH 6.2) to wash the virus and then centrifuged again (27000 rpm for 150 minutes at 4 ° C). The pellet was then resuspended in 0.25M sucrose and the virus stock was stored in the LN ₂ tank. The purified recombinant baculovirus was titrated according to the protocol through the plaque method with Sf9 cells (Invitrogen, No. 10359).

Another aspect of the present invention is a method for preventing an infection caused by an influenza virus infection comprising the step of administering the influenza virus vaccine composition to an individual in which an infection of the influenza virus is expected or an illness caused by an influenza virus infection is caused Or &lt; / RTI &gt;

The term "individual" of the present invention means a living organism capable of infecting influenza virus and capable of causing respiratory disease due to infected influenza virus. Specifically, respiratory diseases may develop, , More specifically a mammal, and more specifically a primate, but is not limited thereto.

The term "a disease caused by an influenza virus infection" as used herein means a respiratory disease caused by an influenza virus infection, including, but not limited to, cough, sneezing, high fever, wheezing, bronchitis, bronchiolitis, , Asthma, croup and respiratory failure.

The term "prevention" of the present invention means any action that inhibits or delays the onset of a disease caused by an influenza virus infection by the administration of the vaccine composition of the present invention.

The term "treatment" of the present invention means any action that alleviates or alleviates symptoms of a disease already caused by an influenza virus infection due to the administration of the vaccine composition of the present invention.

The vaccine composition comprising the recombinant baculovirus or the viral particle thereof transformed so that the hemagglutinin protein of the present invention is expressed in the envelope of the baculovirus as an active ingredient is useful for the humoral immune response and the stock specific antibody To protect against influenza virus infection of the same type and subtype, and thus can be used as a general vaccine.

Figures 1A-1C illustrate the structure and characteristics of recombinant baculovirus (rBac-HA).
Figure 1a shows a pFastBac dual vector designed to encode the pH1N1 HA gene from A / California / 04/09 under the control of a polyhedrin (PH) promoter.
Figure 1B shows the results of Western blot analysis of the presence of recombinant HA protein in purified virus particles. 3 × 10 ⁶ PFU of virus particles were loaded into each lane.
FIG. 1C shows the results of flow cytometry analysis of expression levels of HA protein expressed on baculovirus surface. FIG. Sf9 cells were transformed with 10 MOI of rBac-HA or Bac-ctrl. Forty-eight hours after transformation, the cells were analyzed for HA-specific pAb to measure HA expression on the surface. Untransfected cells were used as a negative control (dashed line: untransformed cells, dotted line: Bac-ctrl, solid line: rBac-HA).
Figures 2a-2e illustrate the humoral immune response induced by intranasally inoculated rBac-HA. Mice were immunized twice at 0 and 16 days with 3 × 10 ⁶ PFU or 1 × 10 ⁶ PFU of recombinant baculovirus (rBac-HA) inoculated intranasally with Balb / c mice (n = 4 / group) . Serum was obtained from all mice three weeks after the second vaccination. Control mice were inoculated with 3x10 ⁶ PFU of wild-type baculovirus (Bac-ctrl, bacimid) intranasally and mice inoculated with PBS were used as negative control.
Figure 2a is a diagram showing the duration of vaccination and virus infection.
FIG. 2B shows the titer of HA-specific IgG antibody measured by ELISA in the sera from the first vaccination and the second vaccination (immunostimulated) mice.
FIG. 2c is a diagram showing the HA-specific IgG antibody titer of a second vaccine (immunostimulated) mouse serum measured by ELISA using A / Korea / 1/09 (H1N1) stock protein.
FIG. 2d shows the HA-specific IgG antibody titer of a second vaccine (immunostimulated) mouse serum measured by ELISA using recombinant chimeric H9 / 1 protein. Serum from mice inoculated with adenovirus-based vaccine expressing HA on the surface was used as a positive control.
Figure 2e shows HA-specific IgA present in mucosa measured by ELISA in BAL fluid 5 days after infection with influenza A / California / 04/09. The result is expressed as Log ₂ (last point value).
FIG. 3 shows hemagglutination-inhibiting (HAI) inhibition of erythrocyte aggregation induced by rBac-HA. Antibody (Negative control: mice inoculated with Bac-ctrl (3 × 10 ⁶ PFU) or phosphate buffered saline (PBS)). The single values measured in the second-inoculated mouse serum at day 38 were plotted on a graph.
Figures 4a-4c illustrate protection against homozygous A / California / 04/09 viral infection through intranasal vaccination of rBac-HA. Balb / c mice (n = 4 / group) were inoculated with rBac-HA through the nasal passages at days 0 and 16. Control mice were immunized with Bac-control. Each group of mice was infected with 10 LD50 A / California / 04/09 virus through the nasal passages.
FIG. 4A is a graph showing virus titers measured by standard plaque assay on MDCK (Madin-Darby canine kidney) cells in the lungs of mice on the 5th day of infection. log ₁₀ (4 mice per group).
4B is a graph showing a result of measuring weight loss in mice infected for 14 days. At the beginning of the experiment, weight was expressed as a percentage value.
4c is a graph showing the results of measurement of survival of mice for 14 days.
Figures 5a-5b illustrate the protective effect against subtype H5N1 (A / Vietnam / 1203/04) viral infection via intranasal vaccination with rBac-HA. Mice (n = 5 / group) were vaccinated twice through the nasal passages on days 0 and 16, and mice in each group were challenged with 5 LD50 of H5N1 (A / Vietnam / 1203/04) .
FIG. 5A is a graph showing the results of measurement of weight loss in mice infected for 14 days. FIG. At the beginning of the experiment, weight was expressed as a percentage value.
FIG. 5B is a graph showing the results of measurement of survival of mice for 14 days.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1. Materials

Example  1-1. Cells and viruses

Spodoptera frugiperda, Sf9), the SF-900II serum was cultured in a medium (Gibco) with 28 ℃, the human embryonic kidney 293 cells (HEK 293) Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Gaithersburg, MD cell) 10% Bovine fetal serum was added and cultured. In addition, Madin-Darby dog kidney cells (MDCK) were cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum.

The virus (H1N1; mouse adapted) of influenza A / California / 04/09 virus (pH1N1; mouse adapted) and A / Vietnam / 1203/04-PR8 / CDC- RG was obtained from the International Vaccine Institute . The weakened virus of A / Vietnam / 1203/04-PR8 / CDC-RG was isolated from A / Vietnam / Haemophilus influenzae using the genetic information of A / Puerto Rico / 8/34 (H1N1) It is the only recombinant virus from 1203/04 (H5N1).

Example  1-2. mouse

Five-week-old female BALB / c mice were purchased from Charles River Laboratories Inc. (Yokohama, Japan) and raised in Ehwa Women's University experimental facility under disease-free conditions. All mouse experiments were performed according to the guidelines approved by IACUC (Approval No. 2010-9-4).

2. Recombination Baculovirus ( rBac - HA ) Produce

Example  2-1. Recombination Of baculovirus (rBac-HA)  Design and production

To determine the efficacy of immunization with recombinant baculovirus, a full-length hemagglutinin (HA) protein of A / California / 04/09 (SEQ ID NO: (RBac-HA), which encodes a recombinant baculovirus (SEQ ID NO: 2).

First, the hemagglutinin (HA) whole gene (SEQ ID NO: 1) of A / California / 04/09 (pH1N1) was prepared by the International Vaccine Institute using pUC57 vector, and the HA gene was subjected to PCR using the following primer set (Underlined is restriction enzyme cleavage site).

HA Forward Sal I: 5 'GG G TCG AC G CCA CCA TGA AGG CAA TAC TA 3 '(SEQ ID NO: 3)

HA Reverse NotI: 5 'GG G CGG CCG C TT AAA TAC ATA TTC TA 3 '(SEQ ID NO: 4)

After the amplified HA gene was inserted into the T easy vector, the nucleotide sequence encoding the HA was amplified using Autographa California nucleopolyhedrosis virus (AcNPV) polyhedrin promoter (pPH ) Into the Sal I and Not I sites of the pFast Bac dual vector (Fig. 1a). According to the protocol of the Bac-to-Bac system (Invitrogen ) via a site-specific potential (transposition sites) was inserted into the expression cassette into the viral genome into baculovirus in DH10Bac ^TM (Invitrogen, USA).

Recombinant bacmid was transfected into Sf9 cells and recombinant baculovirus (rBac-HA) expressing HA on surface was harvested after 96 hours of transfection. Recombinant viruses were selected by plaque isolation and amplified according to the usual protocol.

Example  2-2. Recombination Of baculovirus (rBac-HA)  refine

Recombinant baculovirus was produced by transfecting Sf9 cells with 0.1 MOI. Transfected cells were cultured at 28 ° C for about 96 hours and harvested when viability was 70-80% or more. The final supernatant was collected and concentrated via centrifugation (25% w / w sucrose in 5 mM NaCl, 10 mM EDTA in PBS) (24000 rpm for 75 min at 4 ° C) using a centrifuge. After concentration, the cells were resuspended in phosphate buffered saline (PBS, pH 6.2) to wash the virus and then centrifuged again (27000 rpm for 150 minutes at 4 ° C). The pellet was then resuspended in 0.25M sucrose and the virus stock was stored in the LN ₂ tank. The purified recombinant baculovirus was titrated according to the protocol through the plaque method with Sf9 cells (Invitrogen, No. 10359).

Example  3. Recombination Of baculovirus (rBac-HA)  On the envelope HA  Confirmation of protein expression

Example  3-1. Western Blot

Expression of recombinant baculovirus expressing HA on the surface was confirmed by western blotting. The purified virus contained in loading buffer (50 mM Tris-HCl, pH 6.8), 100 mM dithiothreitol, 2% sodium dodecyl sulfate, 0.1% Bromophenol blue, 10% glycerol) was boiled at 100 ° C for 5 minutes, And separated into 10% sodium dodecyl sulfate-polyacrylamide gel. The gel was transferred to PVDF membrane (Pall, Ann Arbor, MI, USA) at 300 mA for 1 hour 30 minutes in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). Membranes were blocked with TBST buffer containing 5% defatted milk at 37 ° C for 1 hour and incubated overnight at 4 ° C in anti-HA mouse serum diluted 1: 1000. After washing, the membranes were incubated in HRP-conjugated goat anti-mouse IgG diluted 1: 1000 for 1 hour at 37 ° C. After the last wash, the protein was detected with a FUJIFILM luminescent image analyzer LAS-3000.

Example  3-2. Flow cell  analysis

To analyze the expression of the HA protein, Sf9 cells were transfected with 10 MOI of rBac-HA or Bac-ctrl in 6 well plates. Forty-eight hours after transfection, cells were harvested and washed twice with FACS buffer (PBS containing 0.5% FBS and 0.09% NaN ₃ ) and blocked with Fc block for 5 minutes at room temperature. The cells were then incubated with polyclonal antibodies against HA protein (1: 100 dilution) for 40 min at 4 &lt; 0 &gt; C and then washed with FACS buffer. Cells were stained with FITC conjugated anti-mouse IgG (1: 100 dilution) (eBioscience, San Diego, Calif.). After staining, the cells were washed with FACS buffer. Finally, flow cytometry was performed by detecting fluorescence signals using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA) and Flowjo software (TreeStar Inc., Ashalend, OR, USA).

Example  3-3. Recombination Of baculovirus (rBac-HA)  On the envelope HA  Confirmation of protein expression

HA protein expression was measured by Western blot analysis using virus particles purified using a sucrose gradient. Using a polyclonal antibody against the HA protein, an HA0 specific band with a molecular weight of approximately 70 kDa was found in the purified rBac-HA particles. HA0 was not cleaved into HA1 and HA2 subunits because insect cells lacked purines (Yang, DG et al., 2007). As a positive control, the influenza virus (A / California / 04/09) was used and the molecular weight was found to be 70 kDa. No specific bands were found in control wells loaded with wild-type baculovirus (Bac-ctrl, bacimid) (Fig. 1b). The expression level of HA protein on baculovirus surface was stained with anti - HA antibody and measured by flow cytometer. After 48 hours of infection, Sf9-II cells transfected with rBac-HA showed increased fluorescence intensity compared to cells transfected with wild-type baculovirus (Bac-ctrl) or non-transfected cells (Fig. Thus, rBac-HA was expressed and HA was expressed on the surface of rBac-HA.

Example  4. Recombination Of baculovirus (rBac-HA)  Immunization analysis

Example  4-1. Immunization and virus infection

Female BALB / c mice (5 weeks) were purchased from Charles River Laboratories (Yokohama, Japan). For immunization, mice were anesthetized by inhalation of an isoflurane (Ifran; Hana Pharm, Gyeonggi-do, Korea), rBac-HA of 3 x 10 ⁶ plaque forming units (PFU) and wild-type baculovirus ) Or PBS in the nasal cavity. Then, to promote immunization, the same amount of baculovirus was inoculated two weeks after the first immunization. For the virus infection experiment, mice were nasally inoculated with 10 LD50 of influenza virus A / California / 04/09 (pH1N1) or 5 LD50 of A / Vietnam / 1203/04 (H5N1) three weeks after the stimulation. All animal experiments were performed in accordance with the guidelines of the Committee on Animal Experiments at Ewha Womans University.

Example  4-2. Bronchoalveolar lavage ( BAL )

Five days after infection, mice were sacrificed and BAL was obtained by flushing the lung airway with 1 ml of PBS. The secretory IgA level was measured with the supernatant obtained by centrifuging the BAL liquid.

Example  4-3. ELISA

All groups of mice were anesthetized and blood drawn using a heparin-treated capillary tube in a retro-orbital plexus. The titer of the HA-specific antibody of the immunized mice was measured by an enzyme-linked immunosorbent assay (ELISA). Purified recombinant HA protein (50 ng / well) (A / California / 07/09 (H1N1), Immune Technology, USA) diluted in PBS; Purified HA stock protein (500 ng / well) (A / Korea / 1/09 (H1N1), Professor Sung Baeklin, Yonsei University); (F96 Polysorp NUNC immunoplate) with protein (200 ng / well) (Dr. Huan H. Nguyen, International Vaccine Institute) or recombinant chimeric H9 / 1 (H9 HA head portion connected to the top of H1 Respectively. The 96-well plate was then blocked with PBS containing 1% defatted milk for 2 hours at room temperature and washed 4 times with PBS (PBST) containing 0.05% Tween-20. After washing, diluted serum or BAL samples were added to 96-well plates and incubated at room temperature for 2 hours. The plate was washed 4 times with PBS (PBST) containing 0.05% Tween-20. Next, HRP-conjugated rabbit anti-mouse IgG (Abcam, Cambridge, UK) (1: 2000 dilution); Or HRP-conjugated goat anti-mouse IgA (Zymed Laboratories, San Francisco, CA) (1: 3000 dilution) was added as a secondary antibody and incubated at room temperature for 1 hour. After the final wash, the ELISA plate was treated with 3,3 ', 5,5'-tetramethylbenzidine peroxidase (KPL, Gaithersburg, Md.) To develop color, and the color reaction was stopped with 1M H ₃ PO ₄ . Colors were analyzed at 450 nm using a Thermo ELISA plate reader.

Example  4-4. Red Cell Coagulation Reaction Inhibition Assay Hemagglutination inhibition assay )

To inactivate nonspecific inhibitors, serum samples were diluted 1: 4 with receptor-destroying enzyme (RDE, Denka-Seiken, Tokyo, Japan) and treated at 37 ° C for 18-20 hours. RDE-treated serum was incubated at 56 ° C for 1 hour to inactivate RDE, and 6-fold PBS was added. Serum was diluted 1: 10 to 2-fold in 96-well V-bottom plates and the same volume of influenza virus (8 HA units) was added to each well. The microplate was incubated for 1 hour at room temperature and Alsever's solution (Sigma Aldrich, St. Louis, Mo.) containing 0.5% chicken erythrocyte was added. Plates were mixed gently and incubated at room temperature for 40 minutes. The HI titer was measured through the reciprocal of the last dilution point containing the non-aggregation reaction. Serum HAI antibody titers above 1:40 are associated with a protective effect against infections of at least 50% (Human vaccines & immunotherapeutics, 2013, 9 (2), 405-408).

Example  4-5. Lung virus Potency

Five days after infection with influenza A virus, lung tissue was recovered at the expense of each group of mice. To separate single cells, the tissue was homogenized in 3 ml serum-free MEM via a 70 [mu] m cell strainer (BD Labware, Franklin Lakes, NJ). After centrifugation, the supernatant was collected and viral titers were measured in Madin-Darby dog kidney (MDCK) cells via plaque assay. Data are expressed as PFU per gram of lung tissue.

Example  4-6. Statistical method

Differential comparisons were performed using an unpaired two-tailored Student's t-test. When P value ≤0.05, the deviation is statistically significant.

Example  5. rBac - HA  Inoculation induced antibody analysis

Example  5-1. rBac - HA Induced by HA  Specific IgG  Antibody

To determine the immunogenicity of rBac-HA, groups of mice were inoculated with rBac-HA, wild-type baculovirus (Bac-ctrl, bacmid) or PBS via nasal administration. The immunization reaction was promoted at the same concentration 2 weeks after the first inoculation, and sera were obtained at 2 and 5 weeks.

As a result, as shown in FIG. 2B, low-level HA-specific IgG antibodies were confirmed only in the group immunized with rBac-HA 14 days after the first inoculation. In addition, after the second vaccination (stimulation of immunization), the mean level of HA-specific IgG antibody was significantly increased only in the group immunized with rBac-HA.

However, there was no difference in HA-specific IgG antibody titer between mice immunized with rBac-HA (3 × 10 ⁶ ) and rBac-HA (1 × 10 ⁶ ), and PBS or Bac-ctrl was inoculated No HA - specific IgG antibody titers were detected in one mouse.

Thus, it was found that the rBac-HA vaccine of the present invention can induce a HA-specific IgG antibody, and inoculation of the vaccine twice can produce a stronger antibody than 1 inoculation, When inoculated with 10 ⁶ PFU vaccine was found that 3 × 10 ⁶ PFU produce antibodies at a concentration similar to the vaccine.

Example  5-2. rBac - HA Induced by stock  Specific antibody

(A / Korea / 1/09 (H1N1)) and purified recombinant chimera H9 / 1 (H1N1) were used to confirm that rBac-HA could induce stock specific antibodies The reactivity of the proteins was measured by ELISA (Fig. 2c and 2d).

As a result, it was confirmed that the stock-specific antibody reaction strongly occurred after the second inoculation (stimulation of immunization) in the group of rBac-HA immunized mice. However, IgG titers to the HA stock domain could not be detected in the sera from rBac-HA mice inoculated for the first time. Also, IgG titer against the HA stock domain could not be confirmed in serum of the group immunized with PBS or Bac-ctrl.

In addition, IgG titer to the HA stock domain could not be confirmed as a positive control (FIG. 2d) using the serum of a mouse inoculated with an adenovirus-based vaccine expressing HA on the surface with a strong HA stock antibody reaction. This suggests that the baculovirus-based rBac-HA vaccine of the present invention has a remarkable immune effect than the adenovirus-based vaccine for antibody induction to the HA stock domain.

In conclusion, the inoculation of the rBac-HA vaccine of the present invention produces an antibody against the stock domain of HA, which is conserved in various influenza viruses. Therefore, the antibody has cross-protective ability against various influenza viruses, HA vaccine could be used as a general avian influenza virus vaccine. It was also found that the rBac-HA vaccine of the present invention had a remarkable effect on the induction of antibodies against the HA stock domain over the adenovirus-based vaccine.

Example  5-3. rBac - HA Induced by HA  Specific IgA  Antibody

Bronchoalveolar lavage (BAL) fluid was collected on day 5 of viral infection and ELISA was performed to determine the efficacy of rBac-HA in connection with the induction of IgA, which plays an important role in the mucosal immune response.

As a result, the rBac-HA immunization group of the mice induced the HA-specific IgA response of the mucosa, whereas the IgA of the Bac-ctrl and PBS groups was not measured (Fig. 2E). This suggests that HA-specific humoral immune response is effectively induced by intranasal administration of rBac-HA.

Example  5-4. rBac - HA Lt; RTI ID = 0.0 &gt; (&lt; / RTI &gt; hemagglutination - inhibiting , HAI ) Antibody

3 × 10 ⁶ PFU or 1 × 10 ⁶ PFU rBac-HA to measure the HAI antibody can be used to inhibit influenza virus infection if the activity of a hemagglutination-inhibiting (HAI) antibody is high. And a negative control Bac-ctrl (3 x ¹⁰⁶ PFU) or PBS.

As a result, after 2 weeks of the first inoculation, the titers of HAI antibody to homozygous A / California / 04/09 virus in the obtained sera were unmeasured or low (<1:20). However, at 2 weeks after the second inoculation, the rBac-HA-inoculated mouse serum showed an HAI antibody titer of 1: 640 (3 × 10 ⁶ PFU) and 1: 160 (1 × 10 ⁶ PFU) And the HAI antibody titers of the PBS group were not measured (Figure 3). In addition, HAI antibody titers above 1:40 are generally known to provide at least 50% protection against infection (Cox, RJ et al., 2013).

As a result, the rBac-HA vaccine of the present invention has high defense against the homozygous virus infection due to the HAI antibody produced after the second inoculation.

Example  6. rBac - HA  Protection effect against influenza virus infection by inoculation

Example  6-1. rBac - HA  Protective effect against influenza virus infection by inoculation

Three weeks after the last inoculation, mice infected with the vaccine with A / California / 04/09 virus 10 LD50 and control mice were infected with rBac-HA to determine if they could protect the mice from influenza virus infection. Five days later, virus titers were measured in the lungs of mice and the survival rate of the infected mice was measured.

As a result, mice inoculated with rBac-HA did not measure virus in the lung (<10 PFU / ml). On the other hand, mice inoculated with Bac-ctrl or PBS had higher virus titers in the lung (~1 × 10 ⁶ PFU / ml) (FIG. 4a).

 In addition, mice inoculated with Bac-ctrl or PBS survived 0% and 20%, respectively, but mice inoculated with rBac-HA survived without weight loss (FIGS. 4b and 4c).

This demonstrates that the intranasal inoculated rBac-HA vaccine has a complete protective effect against a fatal isogenic influenza virus infection.

Example 6-2. Protective effect against subtype influenza virus infection by rBac-HA inoculation

Immunized mice were infected with 5 LD50 of H5N1 (A / Vietnam / 1203/04) influenza virus to confirm that cross protection against the group 1 influenza virus infection was possible by rBac-HA inoculation.

As a result, mice inoculated with rBac-HA showed a weight loss of less than 15% until day 6 after the virus infection, but the weight loss rapidly recovered to the pre-infection level. The survival rate of rBac-HA-inoculated mice was 80%. However, control mice treated with Bac-ctrl or PBS showed severe weight loss and none survived the virus infection (Figures 5a and 5b).

Thus, it was found that the nBAC-inoculated rBac-HA vaccine has protective effects against not only homozygous virus infection but also subtype influenza virus infection, and that the HA stock-specific antibody induced by the vaccine has cross-protective ability.

In conclusion, the inoculation of the rBac-HA vaccine of the present invention produces an antibody against the HA stock domain and the antibody has cross protection ability against various influenza viruses. Therefore, the rBac-HA vaccine of the present invention can be used as a general- .

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> Ewha University - Industry Collaboration Foundation <120> Vaccine against influenza virus based on hemagglutinin          displayed-baculovirus and method for producing same <130> KPA140781-KR <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1701 <212> DNA <213> A / California / 04/09 virus <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 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 <210> 2 <211> 566 <212> PRT <213> A / California / 04/09 virus <400> 2 Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn  12 16 21 26 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr              31 36 41 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn          46 51 56 Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val      61 66 71 Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly  76 81 86 91 Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile                  96 101 106 Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe             111 116 121 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe         126 131 136 Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp     141 146 151 Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 156 161 166 171 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro                 176 181 186 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val             191 196 201 Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu         206 211 216 Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser     221 226 231 Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 236 241 246 251 Glu Gly Arg Met Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys                 256 261 266 Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe             271 276 281 Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro         286 291 296 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn     301 306 311 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys 316 321 326 331 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg                 336 341 346 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly             351 356 361 Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr         366 371 376 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser     381 386 391 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 396 401 406 411 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His                 416 421 426 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe             431 436 441 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn         446 451 456 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu     461 466 471 Lys Val Arg Ser Glu Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 476 481 486 491 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val                 496 501 506 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu             511 516 521 Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr         526 531 536 Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val     541 546 551 Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 556 561 566 571 Gln Cys Arg Ile Cys Ile                 576 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HA forward primer <400> 3 gggtcgacgc caccatgaag gcaatacta 29 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> HA reverse primer <400> 4 gggcggccgc ttaaatacat attcta 26

Claims (10)

A universal influenza virus vaccine composition comprising, as an active ingredient, a recombinant baculovirus or a viral particle thereof expressed in the form that the hemagglutinin protein is contained in the envelope of baculovirus.
The method according to claim 1,
Wherein the hemagglutinin protein is derived from a H1N1 virus.
The method according to claim 1,
Wherein the hemagglutinin protein is defined by the amino acid sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein said vaccine composition induces an antibody to the stalk domain of a hemagglutinin protein.
The method according to claim 1,
Wherein said influenza virus is a H1N1 virus or a H5N1 virus.
The method according to claim 1,
Wherein said vaccine composition is a formulation suitable for sublingual, nasal, or intramuscular administration.
The method according to claim 1,
Wherein said vaccine composition further comprises at least one immunostimulant.
(a) culturing a recombinant baculovirus-transfected cell expressed in a form wherein the hemagglutinin protein is contained in the envelope of baculovirus; And
(b) obtaining a recombinant baculovirus from the culture of step (a). &lt; Desc / Clms Page number 20 &gt;
9. The method of claim 8,
Wherein said vaccine composition induces an antibody to the stalk domain of a hemagglutinin protein.
9. The method of claim 8,
Wherein said influenza virus is H1N1 virus or H5N1 virus.

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