VACCINE CONTAINING VIRUS INACTIVATED BY GREEN TEA EXTRACT, AND PREPARATION METHOD THEREFOR

Abstract

The present invention relates to a vaccine composition containing a virus inactivated by a green tea extract, and a preparation method therefor. According to the present invention, when a virus is treated with a green tea extract, there are simultaneous effects of virus inactivation and excellent immunogenicity maintenance, and thus an inactivated vaccine can be prepared by mixing the green tea extract of the present invention and a virus with a proliferative capacity, and infectious diseases caused by the corresponding virus can be effectively prevented since an immune reaction to the corresponding virus is induced when a vaccine composition prepared by the preparation method of the present invention is administered to a subject. In addition, there are advantages of enabling the preparation of a safe virus vaccine since the green tea extract of the present invention is nontoxic, and a preparation process is economical since, unlike a chemical material-based preparation process, a dialysis process is unnecessary.

Claims

1.-9. (canceled)

10. A method for preparing an inactivated virus vaccine, the method comprising: (a) adding a green tea extract to a replicative virus, followed by mixing; and (b) incubating a mixture of the virus and the green tea extract.

11. The method of claim 10, further comprising (c) adding an excipient.

12. The method of claim 11, further comprising (d) performing filtration, sterilization, and dilution.

13. The method of claim 10, wherein in step (a), the virus is an influenza virus and the virus and the green tea extract are mixed at a ratio of 51010 to 5103 PFU:0.1-100 mg.

14. The method of claim 10, wherein in step (a), the virus is coronavirus and the virus and the green tea extract are mixed at a ratio of 1010 to 103 EID50:0.1-100 mg.

15. The method of claim 10, wherein the incubation in step (b) is carried out at a temperature of 15-50 C.

16. The method of claim 10, wherein the incubation in step (b) is carried out for 1 hour or longer.

17. A method for preventing a viral infectious disease, the method comprising administering, to a subject, the vaccine composition comprising a virus inactivated by a green tea extract.

18. The method of claim 18, wherein the viral infectious disease is caused by an infection with an influenza virus, coronavirus, human papillomavirus, or norovirus.

19. The method of claim 17, wherein the green tea extract comprises ()-epigallocatechin gallate (EGCG).

20. The method of claim 17, wherein the virus is influenza A, B, or C virus.

21. The method of claim 20, wherein the influenza A virus is influenza A/H1N1, A/H3N2, A/H5N2, or A/H9N2 virus.

22. The method of claim 17, wherein the virus is coronavirus, human papillomavirus, or norovirus.

23. The method of claim 22, wherein the coronavirus is infectious bronchitis virus strain M41.

24. The method of claim 17, wherein the green tea extract binds to a protein of the virus.

25. The method of claim 24, wherein the virus is an influenza virus and the green tea extract binds to a nucleoprotein or hemagglutinin of the influenza virus.

26. The method of claim 25, wherein the hemagglutinin is a globular domain or a stalk region of the hemagglutinin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1a shows the results of SDS-PAGE analysis after nucleoprotein of A/Puerto Rico/8/34(H1N1) virus was reacted with a green tea extract.

[0051] FIGS. 1b, 1c, and 1d show the results of SDS-PAGE analysis after Lysyl-tRNA synthetase (LysRS)-HA fusion proteins of A/Korea/01/2009(H1N1) virus were reacted with a green tea extract.

[0052] FIG. 1e shows the results of SDS-PAGE analysis after hemagglutinin protein of A/Puerto Rico/8/34(H1N1) virus was reacted with EGCG.

[0053] FIG. 1f shows the results of LCMS/MS analysis of hemagglutinin protein reacted with EGCG.

[0054] FIG. 2a shows virus replication activity and hemagglutination activity when a mixture of equal amounts of virus (510.sup.7 PFU/ml) and a green tea extract (1 mg/ml) was incubated according to the temperature.

[0055] FIG. 2b shows virus replication activity and hemagglutination activity when various concentrations of virus (510.sup.7, 110.sup.8, and 510.sup.8 PFU/ml) was mixed with a green tea extract (1 mg/ml) in equal amounts and the mixture was incubated.

[0056] FIG. 2c shows virus replication activity and hemagglutination activity when virus (510.sup.7 PFU/ml) was mixed with various concentrations of a green tea extract (0.1, 0.5, 1 mg/ml) in equal amounts, and incubated. The dotted lines represent the detection limit. The detection limit for virus replication activity assay was 5 PFU/ml and the detection limit for hemagglutination activity assay was 2 HAU/ml.

[0057] FIG. 3a shows the results of plaque assay performed to investigate whether virus was completely inactivated.

[0058] FIG. 3b shows the results of confirming that GT-V lost its ability to infect chicken embryos.

[0059] FIG. 4a shows toxicity test results of GT-V. FIG. 4b shows toxicity test results of a green tea extract.

[0060] FIG. 5a shows hemagglutination inhibition (HI) test results of GT-V. FIG. 5b shows virus neutralization test (VNT) results of GT-V. The dotted lines represent the detection limit. The detection limit for HI assay was 8 (HI titer) and the detection limit for neutralization ability assay was 20 (NT titer).

[0061] FIG. 6 shows the results of analysis of protective effect of GT-V against virus challenge.

[0062] FIG. 7 shows the results of confirming the inhibition of infectious virus replication in the lung of mice immunized with GT-V. The dotted lines represent a detection limit of 50 PFU/ml.

[0063] FIG. 8 shows toxicity test results of dialyzed or non-dialyzed GT-V.

[0064] FIG. 9 shows the results of SDS-PAGE assay and Western blotting assay after infectious bronchitis virus (IBV) strain M41 was reacted with a green tea extract.

[0065] FIG. 10 shows the result of dot-immunoblot assay (DIB) to confirm that IBV was inactivated by GT.

[0066] FIG. 11 shows the results of analysis of antibody titer of IgG in the serum of mice immunized with GT-IBV.

[0067] FIG. 12a shows the results of dot-immunoblot assay (DIB) of neutralization antibody of mouse serum collected at 2 weeks after GT-IBV inoculation.

[0068] FIG. 12b shows the results of DIB detection of neutralizing antibody of mouse serum collected at the 6th week after GT-IBV inoculation.

[0069] FIG. 13 shows the results of SDS-PAGE analysis after hRBD-L1 fusion protein of human papillomavirus was reacted with a green tea extract.

[0070] FIG. 14 shows the result of SDS-PAGE analysis after hRBD-NoV VP1 fusion protein of norovirus was reacted with a green tea extract.

SUMMARY

[0071] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

[0072] Materials

[0073] Cell Lines

[0074] Madin-Darby canine kidney (MDCK) and Vero cells were obtained from American Type Culture Collection (ATCC), and the cells were incubated using 10% fetal bovine serum (FBS, HyClone, US) and minimal essential medium (MEM, HyClone, US) under the conditions of 5% CO.sub.2 and 37 C.

[0075] Virus and Green Tea Extract

[0076] A/Puerto Rico/8/34 (H1N1) virus was inoculated into 11-day-old specific pathogen free (SPF) chicken embryos, and incubated for 2 days in a 37 C. incubator. Then, an allantoic fluid was collected, followed by impurity removal therefrom, and stored in 80 C. refrigeration equipment.

[0077] Infectious bronchitis virus (IBV) strain M41 was inoculated into 11-day-old specific pathogen free (SPF) chicken embryos, and incubated for 2 days in a 37 C. incubator. Then, an allantoic fluid was collected, followed by impurity removal therefrom, and stored in 80 C. refrigeration equipment.

[0078] L1 protein (HPV 16L1), which is type 16 virus-like particle (VLP)-derived enveloped protein, was used for human papillomavirus (HPV).

[0079] VP1 (NoV VP1), which is a structural protein of Hu/GII.4/Hiroshima/55/2005/JPN strain, was used for norovirus (NoV).

[0080] For a green tea extract, powdered green tea (100% green tea, AmorePacific, Korea) was dissolved in tertiary distilled water, and then purified using a 0.2 m syringe filter.

[0081] EGCG (EGCG 98%, Changsha Sunfull Bio-tech, China) was dissolved in tertiary distilled water, and then purified using a 0.2 m syringe filter.

[0082] Methods and Results

[0083] Analysis of Influenza Protein Treated with Green Tea Extract

[0084] To investigate the effect of the green tea extract according to the present invention on an influenza protein, nucleoprotein (NP) of A/Puerto Rico/8/34 (H1N1) virus was reacted with the green tea extract, and then analyzed through SDS-PAGE.

[0085] First, a nucleic acid sequence encoding the nucleoprotein was inserted into pGE-LysRS(3) vector, expressed in E. coli, and then separated and purified using nickel chromatography. Next, 1 g/10 l purified nucleoprotein was reacted with 10, 100, and 1000 g/10 l green tea extract at room temperature for 6 hours. Thereafter, the nucleoprotein treated with the green tea extract was loaded on 10% PAGE gel to perform electrophoresis, and the gel was stained with Coomassie-blue to identify stained protein bands.

[0086] As a result, it was confirmed that, in a group treated with 1000 g of green tea extract, the molecular weight of the nucleoprotein was increased due to the binding of the protein and the green tea extract, leading to an increase in band size, but there was no significant difference at low concentrations (FIG. 1a).

[0087] Then, Lysyl-tRNA synthetase (LysRS)-HA fusion protein of A/Korea/01/2009(H1N1) virus was reacted with a green tea extract, and then analyzed through SDS-PAGE.

[0088] A nucleic acid sequence encoding LysRS-HA fusion protein was inserted into pGE-LysRS(3) vector, expressed in E. coli, and then separated and purified using nickel chromatography. The LysRS-HA fusion protein may have three different structures, HA globular domain (LysRS-HA GD) and HA stalk region (LysRS-HA Stalk), which correspond to a head part of hemagglutinin, and HA full (LysRS-HA full) of HA globular domain plus HA stalk region. The respective LysRS-HA Full, LysRS-HA GD, and LysRS-HA Stalk fusion proteins were treated with TEV protease (Invitrogen, US) to digest LysRS proteins, and then 1 g/10 l LysRS-HA Full, LysRS-HA GD, and LysRS-HA Stalk were reacted with 10, 100, and 1000 g/10 l green tea extract at room temperature for 6 hours. Thereafter, the reaction products were loaded on 10% PAGE gel to perform electrophoresis, and the gel was stained with Coomassie-blue to identify stained protein bands.

[0089] As a result, it was confirmed that, in a group treated with green tea extract (1000 g), the molecular weights of all of the LysRS-HA full protein, HA full protein, and HA globular and HA stalk were increased, leading to an increase in band size, but there was no significant difference at low concentrations. Therefore, it was confirmed that the green tea extract according to the present invention bound to virus full proteins (FIGS. 1b to 1d).

[0090] Assay of Influenza Protein Treated with EGCG

[0091] Hemagglutinin (HA) of A/Puerto Rico/8/34(H1N1) virus was reacted with EGCG, followed by analysis through SDS-PAGE

[0092] Hemagglutinin was expressed in human cells. 2 g/10 l hemagglutinin was reacted with 100 g/10 l EGCG at room temperature for 2 hours. Thereafter, hemagglutinin treated with EGCG was loaded on 10% PAGE gel to perform electrophoresis, and the gel was stained with Coomassie-blue to identify stained protein bands.

[0093] As a result, it was confirmed that the reaction with EGCG increased the molecular weight of the protein and thus the band size was increased. Therefore, it was confirmed that EGCG according to the present invention bound to the hemagglutinin protein of the influenza virus (FIG. 1e).

[0094] In addition, the hemagglutinin protein reacted with EGCG was analyzed through liquid chromatography mass spectrometry (LCMS/MS). The protein bands stained with Coomassie blue were separated by in-gel digestion, subjected to alkylation and de-staining processes, and then prepared into peptide fragments using trypsin. The prepared peptide fragments were analyzed using LCMS/MS [(Q-Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled with an Easy-nLC system (Thermo Fisher Scientific, Odense, Denmark)].

[0095] As a result, it was confirmed that EGCG is modified in the form of dihydro epigallocatechin (C.sub.15H.sub.11O.sub.6) and bound to the cysteine residue, which is the 152nd amino acid of the influenza hemagglutinin protein (FIG. 1f).

[0096] Inactivation Effect of Green Tea Extract on Influenza Virus

[0097] In order to investigate the inactivation effect when influenza was directly treated with a green tea extract, virus replication activity, hemagglutination activity, and growth kinetic tests were carried out under various conditions.

[0098] First, in order to investigate the degree of inactivation depending on the temperature, virus (510.sup.7 PFU/ml) was mixed with a green tea extract (1 mg/ml) in equal amounts, followed by incubation in a constant-temperature water bath at 20, 25, 30, and 35 C. The mixed solution was inoculated on a 12-well plate in which MDCK cells have been cultured, and the virus titer was examined by plaque assay. As a result, it was confirmed that the virus replication activity was decreased by about 3 log.sub.10 PFU/ml with increasing temperature, and virus replication was all inhibited at 35 C. It was confirmed that the hemagglutination activity was also decreased depending on the temperature and the hemagglutination activity was all inhibited at 35 C. (FIG. 2a).

[0099] Then, in order to investigate the degree of inactivation according to the virus titer, virus with various titers (510.sup.7, 110.sup.8, and 510.sup.8 PFU/ml) and a green tea extract (1 mg/ml) were mixed in equal amounts, followed by incubation in a constant-temperature water bath at 35 C. at which the virus has been effectively inhibited in the previous test. The mixed solution was inoculated on a 12-well plate in which MDCK cells have been cultured, and the virus titer was examined by plaque assay. As a result, it was confirmed that the virus replication activity was increased as the titer of virus was higher, and the virus replication activity was inhibited at both of the titers of 110.sup.8 PFU/ml and 510.sup.7 PFU/ml. It was confirmed that hemagglutination activity was also decreased depending on the titers and hemagglutination activity was all inhibited at the titers of 510.sup.7 PFU/ml (FIG. 2b).

[0100] On the basis of the above test results, 510.sup.7 PFU/ml virus and the green tea extract with various concentrations (0.1, 0.5, and 1 mg/ml) were mixed in equal amounts, and then the growth kinetic of virus depending on the time was examined while the mixture was incubated at 35 C. for 24 hours. The mixed solution was inoculated on a 12-well plate in which MDCK cells were cultured, and the virus titer was examined by plaque assay. As a result, it was confirmed that virus replication activity was decreased according to the concentration and time, and for 1 mg/ml green tea extract, virus replication activity was all inhibited within 6 hours. The hemagglutination activity was also decreased as the concentration of the green tea extract was increased, and the treatment time was longer, and for 1 mg/ml green tea extract, the hemagglutination activity was all inhibited 24 hours after the treatment (FIG. 2c).

[0101] Preparation of Inactivated Influenza Virus Vaccine (GT-V)

[0102] On the basis of the above test results, 510.sup.7 PFU/ml A/Puerto Rico/8/34(H1N1) and 1 mg/ml green tea extract were mixed in equal amounts, followed by incubation at 35 C. for 24 hours, thereby preparing an influenza inactivated vaccine (GT-V) after the treatment with the green tea extract. In order to investigate whether the virus was completely inactivated, the vaccine was inoculated into MDCK cells, followed by plaque assay. As a result, it was confirmed that no plaque was generated, indicating that viral activity was abolished (FIG. 3a). For more accurate validation, the prepared GT-V stock solution was inoculated into 11-day-old embryos and cultured at 37 C. for 2 days, and then an allantoic fluid was collected to examine hemagglutination ability. As a test result, hemagglutination ability was not observed, confirming that the influenza inactivated vaccine (GT-V) of the present invention lost its ability to infect chicken embryos (FIG. 3b).

[0103] Investigation of Toxicity of Inactivated Influenza Virus Vaccine

[0104] In order to investigate toxicity of the GT-V prepared above, mice were intraperitoneally administered with GT-V (200 l/mice) with various concentrations (GT (Green tea) 12.5 g-V (virus) 6.2510.sup.5 PFU, GT 25.0 g-V 1.2510.sup.6 PFU, and GT 50.0 g-V 2.5010.sup.6 PFU) and PBS together with alum (100 l) as an adjuvant, and the body weight change was monitored for 14 days. Although a slight weight loss was observed until 2 days after the inoculation, the weight loss was about 5% compared with a control group, indicating no significant difference, and then the body weight was continuously recovered, and returned to the normal weight after day 5. Therefore, it was confirmed that GT-V of the present invention showed no toxicity in animal test results (FIG. 4a).

[0105] In order to examine toxicity of only the green tea extract, four mice per group were intraperitoneally injected (100 l) with a green tea extract (0.05, 0.1, 1 mg) and PBS. The mice were observed for the weight loss change and survival rate for 14 days. As a result, compared with a mouse group (control group) administered with PBS, all mouse groups administered with green tea extract showed no significant body weight loss at all doses, and showed 100% survival rates. In the GT-V animal test, the highest dose of the green tea extract was 0.05 mg, and it was confirmed that toxicity was not observed even when mice were administered with a green tea extract of 1 mg, which is 20-fold higher than 0.05 mg (FIG. 4b).

[0106] Investigation of Immunogenicity of GT-V

[0107] GT-V Inoculation and Blood Collection

[0108] In order to investigate immunogenicity and protective effect of GT-V, five mice per group were intraperitoneally administered with 100 l of GT-V (100 l/mice) with various concentrations (GT 12.5 g-V 6.2510.sup.5 PFU, GT 25.0 g-V 1.2510.sup.6 PFU, GT 50.0 g-V 2.5010.sup.6 PFU) together with 100 l of alum as an adjuvant, and additionally inoculated at the same concentrations after 2 weeks. The mouse body weight change was observed daily for 2 weeks after the inoculation, and after 2, 4, and 6 weeks of the first inoculation, blood was collected, and subjected to centrifugation to collect only serum, which was then used for immunogenicity analysis.

[0109] All the test procedures were carried out according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Yonsei Laboratory Animal Research Center.

[0110] Hemagglutination Inhibition Assay

[0111] In order to analyze hemagglutination inhibition characteristics of GT-V, hemagglutination inhibition (HI) analysis was performed. First, the serum was treated with a receptor destroying enzyme, which was then inactivated by heating at 56 C. for 1 hour. Then, 25 l of the serum was diluted 2-fold serially with PBS in a 96-well plate. Then 4 HAU/25 l of the same wild type of A/Puerto Rico/8/34 (H1N1) virus was added to the diluted serum, followed by incubation at 37 C. for 1 hour. Thereafter, 50 l of 1% chicken red blood cells (cRBC, chicken RBC) was added, followed by incubation at 4 C. for 1 hour, and then the highest dilution rate for inhibiting hemagglutination activity was calculated.

[0112] As a result, it was confirmed that the HI titer was not shown at the lowest inoculation concentration at the 2nd week, but after the additional inoculation, the HI titer was significantly increased, and was highly induced by the concentration at each week. It was confirmed that the HI titer showed the highest value at the 6th week, confirming that the immune-induced response by GT-V of the present invention was maintained for 6 weeks or longer (FIG. 5a).

[0113] Virus Neutralization Assay

[0114] In order to investigate virus neutralization ability of the serum of mice inoculated with GT-V, virus neutralization test (VNT) was carried out. First, the serum of a mouse inoculated with GT-V, the serum being collected in the above example, was inactivated by heating at 56 C. for 1 hour. Then, 25 l of each serum was diluted 2-fold serially with PBS in a 96-well plate. Next, 100 PFU/100 l of virus was added to the diluted serum, followed by a neutralization reaction at 37 C. for 1 hour. Thereafter, the virus and serum, which had been subjected to the neutralization reaction, were inoculated on a 12-well plate in which MDCK cells were cultured, and then plaque assay was performed. The dilution ratio showing a 50% plaque reduction compared with a control group was calculated.

[0115] As a result, it was confirmed that the neutralization titer (NT titer) was hardly increased at the 2nd week after the first inoculation, but the neutralization titer was greatly increased after the additional inoculation, and further increased at the 6th week to maintain the immune response (FIG. 5b).

[0116] Analysis of Protective Effect of GT-V Against Virus Challenge

[0117] Mice inoculated with GT-V (GT 12.5 g-V 6.2510.sup.5 PFU, GT 25.0 g-V 1.2510.sup.6 PFU, and GT 50.0 g-V 2.5010.sup.6 PFU) were additionally inoculated in equal amounts after two weeks. At the 4th week after the additional inoculation, A/Puerto Rico/8/34 (H1N1) virus was intranasally challenged in 10.sup.4 PFU/50 l, which was a concentration of 10 times the 50% mortality rate (10 MLD.sub.50), and then the body weight change and survival rate were monitored for 2 weeks after the challenge.

[0118] As a result, the mice inoculated with GT-V showed a body weight loss of about 10% until the 6th day after the challenge, but thereafter, the body weight was recovered. A control group not inoculated with GT-V showed a rapid body weight loss, and then all mice were dead on the 6th day after the challenge. Regardless of the inoculation concentration of GT-V, survival rate was 100% even in the group inoculated with the lowest concentration of GT-V (FIG. 6).

[0119] Investigation of Inhibition of Virus Replication in Lung

[0120] In order to further investigate protective effect of GT-V against fatal influenza virus infection, mice were inoculated twice, and 4 weeks later, intranasally challenged with 10 MLD.sub.50 (10.sup.4 PFU/50 l) of A/Puerto Rico/8/34 (H1N1) virus as in the above example, and 2, 4 and 6 days later, the mice were sacrificed to collect lungs thereof. The collected lungs were put into 500 ml of PBS, followed by disruption, and then centrifuged to separate only supernatant. The separated supernatant was inoculated into MDCK cells, and plaque analysis was performed to check the titer of virus present in the lungs of mice.

[0121] As a result, the infectious virus identified in the lungs of mice inoculated with GT-V showed a virus titer, which was approximately 10.sup.3 times lower than that in the mice not inoculated with GT-V. This value was observed on even day 2 and day 4 as well as day 6 after the inoculation, and the viral replication was not completely inhibited, but a sufficiently low inhibition value was confirmed (FIG. 7).

[0122] Investigation of Need of Dialysis

[0123] In order to investigate whether dialysis was needed when virus was inactivated by the green tea extract according to the present invention like in a case where the virus was inactivated by formaldehyde, the toxicity of GT-V subjected to a dialysis procedure for removing the green tea extract and GT-V not subjected to a dialysis procedure was tested by the same method as in the foregoing GT-V toxicity test, and the results were compared. The mixed solution of the green tea extract and the virus was dialyzed with PBS buffer (pH 7.4) at 4 C. for 24 hours. As a result, there was no significant difference in body weight between a mouse group inoculated with GT-V subjected to a dialysis procedure and a mouse group inoculated with GT-V not subjected to a dialysis procedure (FIG. 8).

[0124] Therefore, GT-V of the present invention does not require a dialysis process, indicating that GT-V of the present invention is highly economical in manufacturing vaccines.

[0125] Analysis of Coronavirus Treated with Green Tea Extract

[0126] In order to investigate the effect of the green tea extract according to the present invention on coronavirus, infectious bronchitis virus (IBV) strain M41 was reacted with the green tea extract, followed by analysis through SDS-PAGE. 100 l of virus (10.sup.65 EID.sub.50/ml) was reacted with 100 l of the green tea extract (10 mg/ml) at room temperature for 2 hours. Thereafter, the reaction product was loaded on 8% PAGE gel, followed by electrophoresis. Thereafter, the gel was stained with Coomassie-blue to identify stained protein bands, and, at the same time, western blotting was performed. The protein bands on the gel were transferred to polyvinylidene fluoride (PVDF) membrane, and for the reduction of non-specific reactions, the membrane was blocked with 5% skim milk, and then washed with TBST. The serum of mice inoculated with IBV was diluted to 1:1000, and used as primary antibody with respect to the membrane. The membrane was washed with TBST, and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (HRP-conjugated anti-mouse IgG) was diluted to 1:10000, and thus, the membrane was treated with secondary antibody. The membrane was washed with TBST, and then treated with WEST-ZOL plus Western Blot Detection System (iNtRON, Korea), and developed on X-ray film.

[0127] As a result, it was confirmed that the reaction with the green tea extract increased the molecular weight of the protein, and thus the band size was increased. Therefore, it was confirmed that the green tea extract according to the present invention bound to the coronavirus protein (FIG. 9).

[0128] Preparation of Inactivated Coronavirus Vaccine (GT-IBV)

[0129] Infectious bronchitis virus (IBV) strain M41 (10.sup.65 EID.sub.50/ml) and a green tea extract (1 mg/ml) were mixed in equal amounts, followed by incubation at 35 C. for 24 hours, thereby preparing a green tea extract-treated corona inactivated vaccine (GT-IBV). In order to investigate whether the virus was completely inactivated, the GT-IBV stock solution was inoculated onto 11-day-old chicken embryos, followed by incubation at 37 C. for 2 days. Then, an allantoic fluid was collected, and dot-immunoblot assay (DIB) was performed for measuring residual amount of virus. The mixture of the virus and green tea extract was dispensed in 200 l for each nitrocellulose paper (NC paper), followed by vacuum treatment for 10-15 minutes and then washing. The nitrocellulose paper was blocked with 3% bovine serum albumin (BSA) at 37 C. for 2 hours, and then, the serum of mice inoculated with IBV was diluted to 1:1000, followed by incubation at 37 C. for 30 minutes. The reaction product was washed three times with TBST and treated with biotinylated anti-mouse IgG, followed by incubation at 37 C. for 30 minutes. The reaction product was washed three times with TBST and treated with biotin and avidin-conjugated peroxidase complex (ABC) kit, followed by incubation at 37 C. for 30 minutes. The reaction product was washed three times with TBST, treated with diaminobenzidine to perform color development for 1 minute, and washed with flowing water, followed by drying, to investigate staining or non-staining.

[0130] As a result, an allantoic solution of chicken embryos inoculated with GT-IBV of the present invention was not stained with IBV antibody, confirming that IBV activity was lost (FIG. 10).

[0131] Investigation of Immunogenicity of GT-IBV

[0132] GT-IBV Inoculation and Blood Collection

[0133] In order to investigate the immunogenicity of GT-IBV of the present invention, four mice per group were intraperitoneally administered with 100 l of GT-V with various concentrations (GT 12.5 g-IBV 1.2510.sup.4.5 EID.sub.50, GT 25.0 g-IBV 2.5010.sup.4.5 EID.sub.50, and GT 50.0 g-V 5.010.sup.4.5 EID.sub.50) and 100 l of alum as an adjuvant. After 2 weeks, additional inoculation was carried out at the same concentrations. Blood was collected at 2 weeks and 6 weeks after the first inoculation, and centrifuged to collect only serum, which was then used for immunogenicity analysis.

[0134] All the test procedures were carried out according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Yonsei Laboratory Animal Research Center.

[0135] Analysis of IgG Titer

[0136] The serum of mice inoculated with GT-V of the present invention was subjected to ELISA analysis. Wild-type (WT) IBV virus (10.sup.65 EID.sub.50/ml) was dispensed into a 96 well plate at 100 l per each well, followed by coating at 4 C. for one day. The virus-coated plate was washed three times with Tris-HCl (pH 7.4) and blocked with 1 BSA at room temperature for 1 hour. After washing in the same manner, the serum of mice inoculated with GT-V of the present invention was initially diluted to 1:200, then 2-fold serially diluted, and dispensed at 100 l/well in a 96-well plate and treated at room temperature for 1 hour. The reaction product was washed by the same method, and then treated with 1:1000-diluted HRP-conjugated anti-mouse IgG (Mab) at 100 l/well at room temperature for 1 hour. The reaction product was washed by the same method, and then treated with TMB solution at 100 l/well at room temperature for 30 minutes. The reaction was stopped by treatment with 2N H.sub.2SO.sub.4, and analyzed by using a spectrometer at 450 nm.

[0137] As a result, it was confirmed that IgG antibody was hardly produced at the 2nd week after the first inoculation, but the antibody titer was significantly increased at the 6th week after the second inoculation, and thus, the antibody was sufficiently produced at each GT-IBV inoculation concentration (FIG. 11).

[0138] Virus Neutralization Assay

[0139] In order to investigate virus neutralization ability of the serum of mice inoculated with GT-IBV, virus neutralization test (VNT) was carried out. First, 100 l of 10.sup.2 EID.sub.50/ml IBV strain was reacted with 100 l of serum (10.sup.3, 10.sup.4, 10.sup.5 dilution) at 37 C., the serum being collected, on the 2nd week and the 6th week, from mice inoculated with GT 12.5 g-IBV 1.2510.sup.4.5 EID.sub.50, GT 25.0 g-IBV 2.5010.sup.4.5 EID.sub.50, and GT 50.0 g-V 5.010.sup.4.5 EID.sub.50. Then, three chicken embryos were inoculated with each of the mixtures, followed by incubation at 37 C. for 3 days. Thereafter, an allantoic solution of each chicken embryo was collected, and dot-immunoblot assay (DIB) was performed by the same method as in the neutralization assay.

[0140] As a result, it was confirmed that the negative viruses accounted for 78% in the GT 12.5 g-IBV 1.2510.sup.4.5 EID.sub.50 group, 67% in the GT 25.0 g-IBV 2.5010.sup.4.5 EID.sub.50 group, and 22% in the GT 50.0 g-V 5.010.sup.4.5 EID.sub.50 group (FIG. 12a). In addition, it was confirmed that negative viruses accounted for 33% in the GT 12.5 g-IBV 1.2510.sup.4.5 EID.sub.50 group, 44% in the GT 25.0 g-IBV 2.5010.sup.4.5 EID.sub.50 group, and 33% in the GT 50.0 g-V 5.010.sup.4.5 EID.sub.50 group (FIG. 12b). Therefore, it was confirmed that the serum of mice inoculated with coronavirus treated with a green tea extract could neutralize coronavirus.

[0141] Analysis of Non-Influenza Protein Treated with Green Tea Extract

[0142] Analysis of HPV Protein Treated with Green Tea Extract

[0143] In order to investigate the effect of the green tea extract according to the present invention on a non-influenza virus, LI protein (HPV 16L1), which is type 16 enveloped protein of human papillomavirus (HPV), was inserted into hRBD vector, expressed in E. coli, purified using nickel affinity chromatography, reacted with a green tea extract, and analyzed through SDS-PAGE. The hRBD-L1 fusion protein of human papillomavirus was treated with TEV protease (Invitrogen, USA) to digest hRBD. The L1 protein (2 g/10 l) was reacted with a green tea extract (10, 100, and 1000 g/10 l) of the present invention at room temperature for 2 hours. Thereafter, the reaction product was loaded on 10% PAGE gel to perform electrophoresis, and the gel was stained with Coomassie-blue to identify stained protein bands.

[0144] As a result, it was confirmed that there was no great difference at low concentrations, but when the L1 protein was reacted with 1000 g/10 l green tea extract, the molecular weight of the L1 protein was increased due to the binding between the protein and the green tea extract, and thus, the band size was increased. Therefore, it was confirmed that the green tea extract according to the present invention bound to the protein of human papillomavirus, which is a non-influenza virus (FIG. 13).

[0145] Analysis of Norovirus Protein Treated with Green Tea Extract

[0146] In order to investigate the effect of the green tea extract according to the present invention on another non-influenza virus, VP1 (NoV VP1), which is a structure protein of norovirus (NoV, Hu/GII.4/Hiroshima/55/2005/JPN), was inserted into hRBD vector, expressed in E. coli, purified using nickel affinity chromatography, reacted with a green tea extract, and analyzed through SDS-PAGE. The hRBD-Nov VP1 fusion protein of norovirus was treated with TEV protease (Invitrogen, USA) to digest hRBD. The VP1 protein (2 g/10 l) was reacted with the green tea extract (10, 100, and 1000 g/10 l) of the present invention at room temperature for 2 hours. Thereafter, the reaction product was loaded on 10% PAGE gel to perform electrophoresis, and the gel was stained with Coomassie-blue to identify stained protein bands.

[0147] As a result, it was confirmed that there was no great difference at low concentrations, but when the L1 protein was reacted with 1000 g/10 l green tea extract, the molecular weight of the VP1 protein was increased due to the binding between the protein and the green tea extract, and thus, the band size was increased. Therefore, it was confirmed that the green tea extract according to the present invention bound to the protein of norovirus, which is a non-influenza virus (FIG. 14).

[0148] Although the present invention has been described in detail with reference to specific features thereof, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.