KR20100058141A - Vaccine composition againt equine herpesvirus for targeting fc receptor of dendritic cells - Google Patents

Abstract

PURPOSE: A horse herpesvirus vaccine formulation targeting Fc receptor of dendritic cells is provided to effectively enhance horse immunity. CONSTITUTION: An EHV-1(equine Herpesvirus-1) vaccine formulation contains a fusion protein as an active ingredient, in which inactivated EHV-1 and immunoglobulin heavy chain constant region are conjugated by covalent bond. The inactivated EHV-1 is obtained using ethylene imine, derivative of ethylene imine, beta-propiolactone, aldehyde compound, formalin, phenol, hypochlorite, or detergent. The EHV-1 vaccine formulation additionally contains an adjuvant. The vaccine formulation is used for injection.

Description

Vaccine Composition Againt Equine Herpesvirus for Targeting Fc Receptor of Dendritic Cells}

The present invention relates to equine herpes virus vaccine preparations targeting Fc receptors of dendritic cells.

Dendritic cells are present in very small numbers in vivo, but are superior to other antigen presenting cells such as B cells and macrophages in terms of obtaining, processing and presenting antigens.

The function of dendritic cells depends on their maturity, which is the degree of differentiation of the cells, and immature dendritic cells are expressed by cluster determinants (CDs) such as CD40, CD54, and CD86, which play an important role in the reaction between T lymphocyte receptors and antigen-MHC conjugates. Although it does not activate T cells, receptors such as the mannose receptor, CD205 (also known as LY75, DEC-205), DC-SIGN, and the immunoglobulin Fc receptor are well expressed on the cell membrane, and thus, antigens with very low concentration of antigen It possesses the ability to capture and process, produce a major histocompatibility, and generate a major histocompatibility-external peptide bond.

Immature dendritic cells are matured by bacteria, inflammatory products, and bacterial cell wall components such as LPS (lipopolysaccharide), IL-4, GM-CSF (CSF2), and TNF-α. Mature dendritic cells co-stimulate with cell surface antigens. It is possible to induce an immune response of primary T cells by the expression or increase in expression of factors (costimulatory molecules). Stimulated by dendritic cells, CD4 helper T cells secrete cytokines to activate cytotoxic T cells, natural killer cells, antigen presenting cells, and other inflammatory cells.

Currently, there are two strategies for using dendritic cells as an immunotherapeutic agent. One is an anticancer immunotherapy strategy in which dendritic cells separated and differentiated from a patient are exposed to tumor antigens and then put back into the patient. It is a strategy to promote antigen recognition by targeting mannose receptor, CD205, DC-SIGN, Fc receptor expressed on the surface of dendritic cells.

Equine herpesvirus (EHV), on the other hand, is a causative agent of equine rhinopneumonitis in equine herpesvirus type 1 (hereinafter referred to as "EHV-1"; also referred to as the first subtype of EHV-1) and equine herpesvirus fourth The type (hereinafter “EHV-4”; also known as the second subtype of EHV-1) is known. EHV-1 is associated with respiratory disease, miscarriage, mortality in newborns, neurological disorders, and EHV-4 is associated with respiratory disease and miscarriage.

The present invention utilizes the targeting technology of Fc receptors in dendritic cells in the development of EHV-1 and EHV-4 vaccine formulations.

It is an object of the present invention to provide an EHV-1 vaccine formulation and an EHV-4 vaccine formulation capable of targeting Fc receptors among dendritic cell receptors.

Other objects of the present invention will be presented below.

In the present invention, when fusion of EHV-1 or EHV-4 and Fc receptor of Equine immunoglobulin is administered to experimental animals, humoral and cellular immunity of experimental animals compared to the case of administration of EHV-1 or EHV-4 alone. It is provided based on the fact that it enhances.

In one aspect, the invention can be understood as EHV-1 vaccine formulation.

EHV-1 vaccine formulation of the present invention comprises a fusion protein (hereinafter referred to as "EHV-1-Fc fusion protein") to which the constant region (Fc) of the inactivated EHV-1 and immunoglobulin heavy chains is combined as an active ingredient. It is done.

The EHV-1-Fc fusion protein included as an active ingredient in the EHV-1 vaccine preparation of the present invention specifically binds to the Fc receptor of dendritic cells and thus more potent biological immunity than when the antigen EHV-1 is administered alone. Can induce a reaction.

In the vaccine formulation of the present invention, inactivated EHV-1 can be obtained by using various chemical inerts known in the art, and as such inactivating agents, ethyleneimine and derivatives of ethyleneimine (for example, binary ethylene imine (binary) ethyleneimine, acetylimine, etc.), beta-propiolactone, aldehyde compounds (e.g. formaldehyde, glutadialdehyde, etc.), formalin, phenol, hypochlorite, Detergents and the like.

The amount of the inactivating agent used, the reaction temperature, half the time, etc. in order to inactivate the virus will depend on the type of inactivating agent used, and those skilled in the art can determine this experimentally within the range of their usual capabilities. For example, when formalin is used as an inactivating agent, the amount of formalin added to the virus suspension will range from about 0.005% to about 0.1% (v / v), the reaction temperature will range from about 4 ° C to about 38 ° C. The time will be about 20 hours to 50 days when the reaction temperature is about 4 ° C.

In the EHV-1 vaccine formulation of the present invention, the EHV-1-Fc fusion protein can be obtained by genetic engineering method or by chemical method.

Genetic engineering method is a method using a recombinant vector designed to express the protein of the fused form, including the glycoprotein D (gD) gene and Fc gene that determines the EHV-1 antigenicity. Here, the base sequence of the gD gene is known as shown in Table 1 below, and the method of expressing the known base sequence in the form of a fusion protein by binding to the horse Fc gene is disclosed in US Pat. No. 5,795,578, US Pat. No. 7,067,110, US Pat. No. 5,726,044, and the like.

TABLE 1

Genebank gD gene sequence of EHV-1

Chemical methods include glutaaldehyde, p, p'-difluoro-m, m'-dinitrodiphenyl sulphon, cyanide, used in the examples below. Cyanuric chloride, Carbodi-imides, Toluene di-isocyanate, Sodium periodate, p-Benzoquinone, N-succinimidyl 3- (2-pydyldithio) propionate (N-Succinimidyl 3- (2-pydyldithio) propionate) and the like is a method for binding a fused protein.

Both the bond by the genetic engineering method and the bond by the chemical method will be covalent bonds.

The EHV-1 vaccine formulation of the present invention may further comprise an adjuvant (immune adjuvant) in addition to the EHV-1-Fc fusion protein to enhance the immune response to EHV-1. Adjuvants can be used as long as they are non-toxic to the body without interfering with the antigenicity of the vaccine components. Examples of adjuvants that can be used include aluminum gel (Glenny, AT et al. J. Pathol. Bacteriol., 29, 38 (1926)), Freund's Complete or Incomplete Adjuvant (FCA; FIA) (Freund, J. Adv. Tubercul Res., 7, 130 (1956)), saponins (Newman, MJ et al., 5th Annual Meeting of the National Coorperative Vaccine Development Groups for AIDS., August 30-September 3, 1992 (Abstract)), muramyl peptides (Telzak, E. et al., J. Infect. Dis., 153, 628-633 (1986)), liposomes (Reddy, R. et al., Sem. Immunol., 4, 91-96 (1992)) , ISCOMS (Claassen, I. et al., Res. Immunol., 143, 531-541 (1992)), proteosomes (Miller, MD, J. Exp. Med., 176, 1739-1744 (1992)) , SAF (Byars, NE et al., Vaccine, 9, 309-318 (1991)), IL-2, IL-8, IL-12, β-interferon, λ-interferon, γ-interferon, GCSF, GM- CSF etc. can be mentioned.

In the vaccine formulation of the present invention, the Fc region of the EHV-1-Fc fusion protein refers to an immunoglobulin heavy chain constant region or a portion of its constant region that retains the ability to bind Fc receptors. As is known, the heavy chain constant region of an immunoglobulin consists of 4 or 5 domains (CH1 domain-hinge region-CH2 domain-CH3 domain (-CH4 domain)). CH4 is present in IgM that does not have a hint region. The Fc region of the EHV-1-Fc fusion protein of the present invention comprises an immunoglobulin hinge region and may optionally comprise a CH2 domain, a CH3 domain, a CH4 domain or a combination of these domains.

The Fc region of the invention can be obtained from any of the immunoglobulins namely IgA, IgD, IgE, IgG and IgM. Preferably, it is obtained from IgG, and when obtained from IgG, it may be obtained from any subclass of IgG (ie, IgG1, IgG2, IgG3 and IgG4).

The Fc region of the present invention can be obtained by genetically preparing a gene encoding the Fc region by using a recombinant vector or by cleaving with a suitable protein cleavage enzyme such as papain or pepsin from purified polyclonal or monoclonal antibodies. .

Meanwhile, the formulation of the vaccine formulation of the present invention can be made according to standardized techniques known in the art. Typically the vaccine formulation of the present invention will comprise an EHV-1-Fc fusion protein with a pharmaceutically acceptable carrier.

When the vaccine formulation of the present invention is prepared in an injectable formulation, it is usually prepared in the form of a sterile aqueous solution or dispersion. The solvent or dispersion medium may be a polyol such as water, ethanol, glycerol, or a mixture thereof. In the case of injectable formulations, the fluidity must be maintained to be suitable for injection, in which case the fluidity can be maintained by the use of a coating agent such as lecithin or a surfactant.

Vaccine formulations of the present invention may further include antimicrobial agents, stabilizers and the like to prevent contamination by microorganisms. Suitable antimicrobial agents include parabens, chlorobutanol, phenol, sorbic acid, gentamycin, and the like, and stabilizers include carbohydrates such as glycerol / EDTA and sorbitol, proteins such as albumin and casein, gelatin, and the like. have.

Vaccine formulations of the present invention can be prepared in the form of buffered solutions by methods known in the art using sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate and the like.

The vaccine formulation of the present invention may be administered orally or parenterally, and when administered parenterally, it may be administered by a method suitable for subcutaneous, intramuscular, intravenous, intraarterial, and the like.

Meanwhile, the dosage of the vaccine formulation of the present invention will be determined according to the administration schedule, the unit dose of the fusion antibody included in the formulation, the state of health of the patient, and the like.

In another aspect, the present invention relates to a method for enhancing the immunity to EHV-1 in horses comprising parenterally administering the vaccine formulation of the present invention as described above.

In another aspect, the present invention relates to an EHV-4 vaccine preparation comprising a fusion protein in which the constant region (Fc) of an inactivated EHV-4 and an immunoglobulin heavy chain is bound.

In another aspect, the present invention relates to a method for enhancing the immunity to EHV-4 of a horse comprising parenterally administering the vaccine formulation.

The method of enhancing the immunity to EHV-1 in horses, the EHV-4 vaccine preparation and the method of enhancing the immunity to EHV-4 in horses, as described in connection with the EHV-1 vaccine preparation of the present invention is effective as it is. Do.

On the other hand, when preparing a fusion protein in which EHV-4 inactivated by genetic engineering method and the constant region (Fc) of an immunoglobulin heavy chain are bound, a sequence of genes involved in determining antigenicity known on the Genebank. (Accession no. S65633.1) may be used.

As described above, the present invention can provide an EHV-1 vaccine formulation and an EHV-4 vaccine formulation capable of targeting Fc receptors among dendritic cell receptors. These vaccine formulations can more effectively enhance horse's immunity than when administered alone with antigen.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

<Example 1> Preparation of EHV-Fc Fusion Vaccine

Example 1-1 Preparation of Virus Seeds

EHV-1 seed was aborted in February and March 2005 by two horses (formal and storsam) of Daeyoung Ranch in Jocheon-eup, Bukjeju-gun, Jeju Island, respectively. A pleural effusion was found. The cause of abortion was identified as EHV-1 and swabs of organs (thoracic, blood, placenta, lung, and liver) were swabed with a cotton swab. Each organ except for pleural effusion and blood was added with 5 ml of DMEM to about 2.4 g of the organ, and then mixed appropriately. Supernatant and pleural blood of each organ were sterile filtered (0.45 μm). EHV-4 seeds were imported from the United States (American Type Culture Collection.ATCC number: VR-2230TM).

Example 1-2 Incubation of EHV-1

MDBK cells (Korea Cell Line Bank, 2005) were incubated in DMEM (10% FBS, 0.1% SPK, pH 7.16) for 20 hours to form approximately 90-100% of monolayers, followed by EHV-1 The multiplicity of infection (MOI) was inoculated at about 1 and reacted at 37 ° C. for 4 hours. The inoculum was removed, and DMEM medium containing 2% of FBS was added thereto, followed by incubation at 37 ° C. for about 40 hours to confirm that 60% of CPE was generated. The recovered medium was centrifuged at 500 g for 15 minutes and the supernatant was collected and stored at -70 ° C and used for the experiment.

Example 1-3 Incubation of EHV-4

MDBK cells (Korea Cell Line Bank, 2005) were cultured in DMEM (10% FBS, 0.1% SPK, pH 7.16) for 18 hours to form approximately 90% of monolayers, and then EHV-4 multiplicity of The infection (MOI) was inoculated to about 0.5 and reacted at 37 ° C. for 4 hours. The inoculum was removed, and DMEM medium containing 2% of FBS was added thereto, followed by incubation at 37 ° C. for about 60 hours to confirm that 10% of CPE was generated. The recovered medium was centrifuged at 160g for 5 minutes and the supernatant was collected and stored at 4 ° C. 20 hours after the medium was recovered and the remaining EHV-4 infected MDBK was subcultured, the purified EHV-4 supernatant stored at 4 ° C was inoculated at 37 ° C for 4 hours. Without removing the inoculum, the medium was recovered by adding DMEM medium containing 2% of FBS and incubating at 37 ° C. for about 40 hours to generate 70% of CPE. The recovered medium was centrifuged at 160 g for 5 minutes and the supernatant was collected and stored at -70 ° C.

Example 1-4 Virus Inactivation

<1> Inactivation condition experiment in formalin treatment

Dispense 20 μl of purified EHV-1 (0.16 mg / ml) and EHV-4 (0.11 mg / ml), and add 20 μl of 0.05%, 0.1%, 0.2%, and 0.5% formalin, respectively. After 4 hours, 12 hours, 24 hours, 48 hours, and 72 hours of reaction at 4 ° C., 40 μl of 0.2% BSA (Bovine serum albumin) was added and quenched. The microcon Ym-100 (millipore. 42413) was centrifuged at 12000 g for 3 minutes at 4 ℃ to remove formalin and BSA and centrifuged at 1000 g to recover the virus. Inactivation of EHV-1 and EHV-4 was inoculated into 6 μl of 96-well cell culture flasks in which MDBK (90% cell seat) was grown to determine the degree of inactivation.

In FIG. 1 and FIG. 2, the inactivation conditions for EHV-1 and EHV-4 are shown graphically. As can be seen in FIGS. 1 and 2, EHV-1 completely fluorinated when reacted for 48 hours at 0.5% formalin and 72 hours at 0.1% formalin treatment, 0.1% for EHV-4. The reaction was completely inactivated when reacted for 24 hours with 0.2% and 0.5% formalin and 48 hours with 0.05% formalin.

<2> inactivation of EHV-1

  Inactivated EHV-1 antigen was prepared by applying the formalin treatment conditions established in the above experiment. 500 μl of 0.2% formalin was added to 500 μl (0.16 mg / ml) of EHV-1, followed by stirring at 4 ° C. for 24 hours, followed by centrifugation at 12,000 g for 3 minutes at 4 ° C. using microcon Ym-100 (millipore. 42413). Formalin was removed, purified inactivated EHV-1 antigen was obtained, aliquoted and stored at -70 ° C.

<3> Inactivation of EHV-4

  Inactivated EHV-4 antigen was prepared by applying the formalin treatment conditions established in the experiment. 600 μl of 0.2% formalin was added to 600 μl of EHV-4 (0.11 mg / ml), followed by stirring at 4 ° C. for 24 hours, followed by centrifugation at 12,000 g for 3 minutes at 4 ° C. using microcon Ym-100 (millipore. 42413). Formalin was removed, purified inactivated EHV-1 antigen was obtained, aliquoted and stored at -70 ° C.

Example 1-5 Purification of Equine IgG Fc Fraction

<1> Purification of Equine IgG

Equine IgG was isolated by affinity chromatography using Protein G Sepharose 4 Fast Flow (Amersham Pharmacia Biotech, sweden). Sepharose gel was charged into a syringe column (16 × 80 mm) and 40 ml of horse serum diluted twice with 200 mM phosphate buffered saline (PBS, pH 7.2) was added (flow rate: 1 ml / min). Other components of serum not bound with Protein G were removed by washing with about 50 ml of PBS. Bound Equine IgG was eluted by addition of 3 M NaSCN (pH 5.0). Fractions were recovered in 2 ml portions, and the fractions with high protein content were combined, dialyzed in 20 mM PBS for 72 hours, and filtered and stored in the refrigerator.

<2> Preparation of Fc Fraction

Fc fractions were prepared by treating purified IgG with proteases. Treating IgG with papain can cut several parts of the IgG molecule complex and thus form multiple fractions. Cystein-HCl is used to obtain Fc fractions (dimer-type fractions) that retain Di-sulfide bonds. Digestion buffer (75 mM NaCl, 2 mM EDTA, 75 mM Sodium Phosphate, 10 mM Cystein-HCl, pH 7.1) was prepared, and papain (Sigma, CAT: p-4792) was added to the buffer immediately before digestion experiment. Melting for 5 minutes to prepare a final digestion solution. IgG was digested by adjusting the amount of papain to 10 ug at 1 mg for 4 hours at 37 ° C. to digest the IgG. 20 mM iodoacetate (Signa, CAT: I4386) was added to stop the reaction.

<3> Purification of Fc Fraction

In order to purify the Fc fraction, IgG treated with papain was reacted with protein G sepharose at 4 ° C for 2 hours, and then filled in a column to remove unbound sup and washed twice with PBS. Bound Fc was eluted with 3 M NaSCN (pH 5.0), dialyzed in PBS for 72 hours and sterile filtered to purify the final Fc fraction. Confirmation of purified Fc was carried out through SDS-PAGE analysis.

<4> Preparation and Purification of EHV-Fc Protein Fusion

To prepare the EHV-Fc protein fusion, conjugation using two-step glutaraldehyde was used. 2 ml of purified EHV (5 mg / ml) was slowly mixed with 1 ml of PBS containing 2% glutaraldehyde, followed by reaction at room temperature for 8 hours while stirring continued. The reaction solution was passed through the PD-10 column (Pharmacia-LKB, Sweden) to receive the fraction and the absorbance at 280 nm to confirm the activated EHV fraction. After combining the identified fractions (about 1 ml), slowly mix 1 ml of purified Fc (1 mg / ml) and add 0.1 ml of 1 M carbonate-bicarbonate buffer (pH 9.6) at about 12 ° C at 4 ° C with gentle stirring. The reaction was time. 0.1 ml of Lysine solution (0.2M) was added again and reacted at room temperature for 2 hours, and then dialyzed at 4 ° C. for 10 times in PBS solution (pH 7.2). The dialysis material was purified through a protein G column, and the final eluted material was stored at -70 ° C. Preparation of the fusion protein was confirmed by SDS-PAGE analysis. The analysis results of SDS-PAGE can be seen in FIG. 3.

<Example 2> Humoral and Cellular Immune Activation Experiments Using EHV-Fc Vaccine

Example 2-1 Experimental Method

<1> Use and care of the mouse

The mice used in this experiment were Balb / c or C57BL / 6 strain and 7-12 weeks old females were used in all experiments. Animal testing was conducted in accordance with the regulations on animal use of the Animal Experiment Committee of Jeju National University.

<2> Inoculation and Blood Collection of Vaccine Candidates Including EHV-Fc

The vaccine candidates shown in Table 2 below, which were treated with formalin, which inactivated the infectivity of the virus, were inoculated with 1 μg, 0.2 μg or 0.04 μg each mouse. It was inoculated subcutaneously and the site was inoculated by diluting the vaccine candidates of Table 2 in 100 μl of phosphate buffer using a 1 ml syringe on the dorsal part of the mouse. Vaccine candidates were stored at −80 ° C. after preparation and immediately melted when needed for the experiment. As a control group, mice with similar strain and age and no antigen sensitization of EHV were used.

After inoculation of vaccine candidates, we confirmed whether humoral and cellular immunity were increased to verify vaccine efficacy. Efficacy experiments were conducted after some modifications were made to suit the laboratory's circumstances based on the established experimental methods of other research teams. Each vaccine candidate was inoculated once in mice and blood was collected from the tail vein three weeks later. Blood was collected from each mouse and 10 µl of each mouse was mixed with 100 µl of phosphate buffer solution, and then only red blood cells were centrifuged using a microcentrifuge to separate plasma. EHV-specific antibodies were quantified by applying plasma isolated from mice in the control group and the vaccine candidate group to the EHV-specific enzyme-linked immunosorbent assay (ELISA) optimized in the laboratory.

TABLE 2

Vaccine candidate

EHV type formula EHV 1 formalin treatment + Fc conjugation EHV 4 formalin treatment + Fc conjugation EHV 1 formalin treatment EHV 4 formalin treatment

<3> ELISA (Measuring Humoral Immunity) for Quantification of EHV-Specific Antibodies

EHV antigen with a concentration of 0.625 μg / ml was coated onto 8-well strips for ELISA overnight (approximately 16 hours) at 100 μl / well. The next day, the antigen solution of each well was removed using a wash solution and blocked for 1 hour at room temperature using an assay buffer (hereinafter referred to as "analysis buffer"). Plasma obtained from the tail vein of the mouse was gradually diluted and put in each well and reacted at 37 ° C. for 2 hours. After that, horseradish peroxidase (HRP) labeled goat anti-mouse IgG, a secondary antibody capable of recognizing mouse antibodies, was added and reacted at 37 ° C. for 2 hours. As a chromophoric agent, a TMB solution used in an ELISA using HRP was used. The color development time was optimized until the difference between samples was clear within 10 minutes or the absorbance of high concentration samples was about 1.5. Absorbance was measured at 490 nm using a microplate reader.

<4> Flow Cytometry for the Measurement of EHV-Specific Cellular Immune

 <4-1> Mouse Spleen Cell Collection

Splenocytes were harvested after euthanizing mice inoculated with vaccine candidates (when treated with 1 μg above). Briefly, mouse spleens were taken, placed in a culture dish containing phosphate buffer, and mechanically pressed using a syringe to obtain splenocytes. Hemolysed erythrocytes contained in splenocytes using ammonium chloride potassium (ACK) lysis buffer, a hypotonic solution, and placed in RPMI-1640 medium (Sigma) containing 5% fetal bovine serum (FBS) for 1 hour. Were incubated in a T75 tissue culture flask. Splenocytes floating in the cell culture were passed through the cell strainer to obtain a single cell was used in the experiment.

<4-2> Syngeneic Mouse Dendritic Cell Culture

The femur and tibia of C57BL / 6 mice were harvested to obtain bone marrow cells by flowing HBSS solution into the bone marrow cavity, and then erythrocytes were hemolyzed by reacting for 10 minutes at room temperature using 0.83% ammonium chloride potassium lysis buffer. Bone marrow cells were suspended in RPMI-1640 (5% FBS) medium (Sigma) containing mouse GM-CSF (granulocyte macrophage-colony stimulating factor), and placed in a 6-well plate and incubated in a 37 ° C incubator supplied with 5% CO _2. It was. Every two days, fresh medium with GM-CSF was added. On the second and fourth days of culture, the whole medium was replaced to remove granulocytes and lymphocytes from the culture medium. On days 6-10, more than 60% of the medium was replaced with new ones. Floating dendritic cells were used for the experiment.

<4-3> Activated Lymphocyte Production by Coculture of Dendritic and Spleen Cells

Dendritic cells were treated with EHV-1 for 4 hours and then incubated with splenocytes. Splenocytes were 2 × 10 ⁵ / well and dendritic cells were 1 × 10 ⁴ / well and placed in 100 μl / well in 96-well plates, respectively, and cultured together for 5 days, followed by immunofluorescence staining.

<4-4> Immunofluorescence staining of cell surface molecules and intracellular cytokines (BD Cytofix / Cytoperm ^TM kit)

In order to stimulate cytokine secreting cells to induce cytokine production, 10 ng / ml PMA (phorbol 12-myristate 13-acetate) and 1 ng / ml ionomycin were added together and incubated for 5 hours and intracellular protein transport inhibitors at the last 2 hours. BD GolgiStop ^™ solution containing phosphorus monensin was added. After cell recovery, the anti-mouse CD16 / 32 antibody was reacted for 15 minutes at 4 ° C to block non-specific reactions. PBS solution containing 5% FBS and 0.1% sodium azide was used as staining buffer and washing buffer for cell surface staining, and cells were washed twice after each antibody reaction. FITC-conjugated anti-mouse CD4 antibody was reacted at 4 ° C. for 30 minutes for cell surface staining. Fixation / Permeabilization solution was added for intracellular cytokine staining and cell fixation and permeability increased for 20 minutes at 4 ° C. In order to maintain the permeability of the cells was washed twice using BD Perm / Wash ^TM buffer included in the kit. PE-conjuated anti-mosue interferon (IFN) -gamma antibody was added and reacted for 30 minutes at 4 ° C to stain intracellular cytokine, IFN-gamma. The cells were fixed in staining buffer containing 1% paraformaldehyde and analyzed using BD CellQuest software and FACS Calibur.

<Example 2-2> Experiment result

<1> experimental results of humoral immunity

As suggested in the above experimental methods, EHV antigen-coated plasma from blood obtained from the vein after 3 weeks of inoculating mice with PBS (control) and EHV vaccine candidates once at 1, 0.2 or 0.04 μg per mouse. The result of ELISA after the treatment on the bottom and the secondary antibody, the substrate in turn is shown in the graph below.

Referring to FIG. 4, both classes of vaccine candidates showed higher antibody titers with higher antigen amounts inoculated into mice. In addition, it can be seen that the fusion vaccination group of EHV-1-Fc produced more antibodies specific to EHV-1 than the EHV-1 vaccination group as a whole. It is expected that the EHV-Fc fusion vaccine will induce more potent humoral immunity than the general inactivated vaccine.

<2> Experimental Results of Cellular Immunity

Along with humoral immunity, which produces antigen-specific antibodies in the body, the production of cellular immunity that can attack and eliminate cells already infected with the virus is a major part of the vaccine effect.

Cellular immunity is primarily due to the production and maintenance of antigen-specific cytotoxic T lymphocytes (CTLs). To verify CTL, this study identified the percentage of Th1 (T helper 1) cells capable of producing interferon-gamma in cells.

Peripheral blood of the mouse has a very small amount of blood cells that can be obtained per horse, so splenocytes were used in the experiment. The results are shown in FIG. 5, where the ratio of IFN-γ ⁺ positive cells in the splenic CD4 ⁺ cells was 2.1% in the control group (PBS administration group), 3.3% in the EHV-1-Fc fusion vaccination group, and EHV-1. 2.3% in the vaccinated group. In addition, the ratio of IFN-γ ⁺ cell / CD4 ⁺ cells was 0.18 in the control group, 0.27 in the EHV-1-Fc fusion vaccine group, and 0.19 in the EHV-1 vaccine group.

Therefore, it can be seen that the EHV-Fc fusion vaccine increases the ratio of IFN-γ producing CD4 ⁺ T cells, that is, Th1 cells, relative to the general inactivated vaccine. Increasing Th1 cells may be directly linked to an increase in cellular immunity, which may maximize the effect of the vaccine along with humoral immunity.

1 and 2 are graphs showing inactivation conditions for EHV-1 and EHV-4, respectively, during formalin treatment.

Figure 3 is the result confirmed by the SDS-PAGE analysis whether the production of inactivated EHV-1 or EHV-4 Fc fusion protein. Lanes 1, 2, 3 and 4 in FIG. 3 are protein markers, EHV-1-Fc conjugates, EHV-4-Fc conjugates and Fc used for conjugation, respectively.

4 is a graph showing the results of enhanced humoral immunity following administration of a fusion protein of Fc of inactivated EHV-1 or EHV-4.

5 is a graph showing the results of enhanced cellular immunity following administration of a fusion protein of Fc of inactivated EHV-1 or EHV-4.

Claims (24)

EHV-1 vaccine formulation comprising a fusion protein in which the constant region (Fc) of the inactivated EHV-1 and immunoglobulin heavy chain is covalently bound as an active ingredient. The method of claim 1, The inactivated EHV-1 is EHV-1, ethyleneimine, derivatives of ethyleneimine, beta-propiolactone, β-propiolactone, aldehyde compounds, formalin, phenol, hypochlorite and detergents ( EHV-1 vaccine formulation, characterized in that obtained using a chemical deactivator selected from the group consisting of detergent). The method of claim 1, A fusion protein in which the inactivated EHV-1 and the constant region (Fc) of an immunoglobulin heavy chain are bound is obtained by chemically combining the inactivated EHV-1 and the constant region (Fc) of an immunoglobulin heavy chain. -1 vaccine formulation. The method of claim 1, A fusion protein in which the constant region (Fc) of an inactivated EHV-1 and an immunoglobulin heavy chain is bound is obtained by binding glutaaldehyde to the constant region (Fc) of an inactivated EHV-1 and an immunoglobulin heavy chain. EHV-1 vaccine formulation. The method of claim 1, The EHV-1 vaccine preparation further comprises an adjuvant in addition to the fusion protein. The method of claim 5, The adjuvant is aluminum gel, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, muramyl peptide, liposome, ISCOMS, proteosome, SAF, IL-2, IL-8, IL-12, β-interferon, λ EHV-1 vaccine formulation, characterized in that at least one selected from the group consisting of interferon, γ-interferon, GCSF and GM-CSF. The method of claim 1, The Fc region of the fusion protein is an immunoglobulin heavy chain constant region or part of the constant region possessing the ability to bind to the Fc receptor. The method of claim 1, The Fc region of the fusion protein EHV-1 vaccine preparation, characterized in that it comprises an immunoglobulin hinge region (hinge region). The method of claim 1, The Fc region is an EHV-1 vaccine preparation, characterized in that obtained from IgG. The method of claim 1, The Fc region is an EHV-1 vaccine preparation, characterized in that obtained by cleaving immunoglobulin with protein cleavage enzyme. The method of claim 1, EHV-1 vaccine formulation, characterized in that the vaccine formulation is for injection. A method of enhancing the immunity to EHV-1 in horses, comprising parenterally administering the vaccine formulation of claim 1 to the horse. EHV-4 vaccine formulation comprising a fusion protein in which the constant region (Fc) of the inactivated EHV-4 and the immunoglobulin heavy chain is covalently bound. The method of claim 13, The inactivated EHV-4 is EHV-4, ethyleneimine, derivatives of ethyleneimine, beta-propiolactone, β-propiolactone, aldehyde compound, formalin, phenol, hypochlorite and detergent ( EHV-4 vaccine formulation, characterized in that obtained using a chemical inactivator selected from the group consisting of detergent). The method of claim 13, A fusion protein in which the inactivated EHV-4 and the constant region (Fc) of the immunoglobulin heavy chain are bound is obtained by chemically combining the inactivated EHV-4 and the constant region (Fc) of the immunoglobulin heavy chain. -1 vaccine formulation. The method of claim 13, A fusion protein in which the constant region (Fc) of the inactivated EHV-4 and the immunoglobulin heavy chain is bound is obtained by glutarolehydro linking the constant region (Fc) of the inactivated EHV-4 and the immunoglobulin heavy chain. EHV-4 vaccine formulation. The method of claim 13, The EHV-4 vaccine preparation further comprises an adjuvant in addition to the fusion protein. The method of claim 17, The adjuvant is aluminum gel, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, muramyl peptide, liposome, ISCOMS, proteosome, SAF, IL-2, IL-8, IL-12, β-interferon, λ EHV-4 vaccine formulation, characterized in that at least one selected from the group consisting of interferon, γ-interferon, GCSF and GM-CSF. The method of claim 13, The Fc region of the fusion protein is an immunoglobulin heavy chain constant region or part of its constant region that retains the ability to bind an Fc receptor. The method of claim 13, The Fc region of the fusion protein EHV-4 vaccine preparation, characterized in that it comprises an immunoglobulin hinge region (hinge region). The method of claim 13, The Fc region is an EHV-4 vaccine preparation, characterized in that obtained from IgG. The method of claim 13, The Fc region is an EHV-4 vaccine formulation, characterized in that obtained by cleaving immunoglobulin with protein cleavage enzyme. The method of claim 13, EHV-4 vaccine formulation, characterized in that the vaccine formulation is for injection. 24. A method of enhancing the immunity to EHV-4 in horses comprising parenterally administering the vaccine formulation of any one of claims 13-23.

Images