WO2006038742A1 - 組み換えウイルスおよびその用途 - Google Patents
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- WO2006038742A1 WO2006038742A1 PCT/JP2005/019151 JP2005019151W WO2006038742A1 WO 2006038742 A1 WO2006038742 A1 WO 2006038742A1 JP 2005019151 W JP2005019151 W JP 2005019151W WO 2006038742 A1 WO2006038742 A1 WO 2006038742A1
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Definitions
- the present invention relates to a recombinant virus capable of expressing a SARS coronavirus gene and use of the virus. More specifically, the present invention relates to a recombinant virus found by preventive medical research and development for SARS infection, which has become the most important and urgent issue in current medical administration, and a vaccine for SARS coronavirus using the same. Background art
- SARS severe Acute Respiratory Syndrome
- Coronawinores SARS-CoV
- SARS-CoV severe Acute Respiratory Syndrome Coronawinores
- live vaccines are one of the most effective, but generally it takes a very long time to develop an attenuated vaccine for an emerging virus. This is also known to be the case with SARS-CoV.
- a genetic engineering technique for producing a “recombinant vaccinia virus” as a live vaccine is well known.
- the recombinant vaccinia virus for rabies virus and Linda paste developed by the present inventors is known (for example, Tsukiyama K, Yosikawa Y, Kamata H et al., Arch. Virol., 1989, Vol.107, p.225-235), these have already been demonstrated to have excellent infection prevention effects in field trials.
- vaccinia virus LCl6m8 strain for example, Sugimoto, Yasuda A, iki K et ai., Microbiol Immunol "1985, vol.29, p.421-428.
- the LC16m8 strain was isolated from the Lister strain and has actually been used as a preventive vaccine. It is the only vaccine strain currently in general production that has been confirmed to be safe and effective.
- the present inventor has succeeded in developing a gene expression promoter that can greatly enhance the ability to produce antibodies and induce cellular immunity in the course of research and development of recombinant vaccinia virus against Linda paste and HIV.
- Specific examples include plasmid vectors pSFJl-10 and pSFJ2_16 (see, for example, Jin N-Y, Funahashi S and Shida H, Arch. Virol., 1994, vol.138, p.315-330). Disclosure of the invention
- the problem to be solved by the present invention is to provide a recombinant virus that is effective in preventing the onset of SARS infection and is highly safe, and a vaccine for SARS coronavirus containing the same.
- the inventor has gained from research on viral infections such as poliovirus, Linda pastilles and hepatitis C virus, which have been involved for many years. Based on our experience, we made extensive studies to solve the above problems by making use of our track record in developing live vaccines for Linda plague. As a result, the SARS coronavirus protein was successfully expressed from the vaccinia virus genome, and the present invention was completed.
- the present invention is as follows.
- a recombinant virus capable of expressing the SARS coronavirus gene (1) A recombinant virus capable of expressing the SARS coronavirus gene.
- the SARS coronavirus gene may include, for example, at least the structural protein gene of the virus.
- the structural protein gene includes, for example, at least a spike of the virus. It may contain a protein gene.
- the recombinant virus of (1) above may be, for example, one capable of producing pseudo SARS coronavirus particles, may be a vaccinia virus transformant, or the vaccinia virus may be Even if it is the LC16m8 stock.
- the recombinant virus of (1) above may be one in which the SARS coronavirus gene is inserted into, for example, the HA gene region in the genome of the vaccinia virus, or a hybrid program motor. It may be inserted into the genome of the vaccinia virus so as to be located downstream of the vaccinia virus.
- FIG. 1 is a schematic diagram of the plasmid vector “pSFJl-10-SARS-S” obtained in Example 1 (construction diagram of SARS-CoVS protein expression vector).
- vvHA Vaccinia virus Hemagglutinin gene TRS: Transcription Regulating Sequence
- Figure 2 is a photograph of agarose gel electrophoresis showing the results of S protein gene transfer confirmation by PCR.
- FIG. 3A is a schematic diagram showing the binding positions of the S probe (Spike probe) and the HA probe used for plaque hybridization.
- Fig. 3B is a photograph of the culture plate showing the results of confirmation of S protein gene transfer by plaque hybridization. As a result, only sample 1 was positive.
- Figure 4 is a photograph of a PVDF membrane showing the results of S protein expression confirmation by Western plotting (24 hours after infection).
- FIG. 5 is a graph showing the evaluation results of neutralizing ability against SARS coronavirus.
- the X-axis shows the period since virus inoculation, and the Y-axis shows NT 50 measurements.
- the recombinant virus according to the present invention is a virus capable of expressing the SARS coronavirus gene.
- the recombinant virus of the present invention is, but not limited to, a so-called recombinant vaccinia virus (maternal vaccinia virus) in which the recombinant mother is a vaccinia virus and the SARS coronavirus gene is incorporated into the genome so that the protein can be expressed.
- ⁇ Cinnia virus is generally an “attenuated strain” that is capable of growing in an animal individual (animal cell) and has a very low growth potential in nerve cells.
- the recombinant mother vaccinia virus is not limited, but includes, for example, vaccinia virus LC16m8 strain, Wyeth strain and Lister strain.
- vaccinia virus strain LC16m8 which has extremely low proliferation ability in nerve cells, is preferable.
- the LC16m8 strain has been approved as a pressure ulcer vaccine in Japan, and no serious side effects have occurred as a result of vaccination in approximately 100,000 children.
- Virus, vol.3, No.3, p269 (1975)) and the immunity-inducing ability has been reported to be the same as the parent Lister strain (Morita M, Suzuki K, Yasuda A, et al. al., Vaccine, 1987, vol.5, p.65-70), because it is a safe and effective vaccine strain. Therefore, the recombinant vaccinia virus of the present invention is preferably a transformant of LC16m8 strain.
- SARS coronavirus is an RNA virus having a 29751base ss-RNA genome, and the entire DNA base sequence corresponding to the ss-RNA is shown in "GenBank No. NC-004718". At the same time, the sequence portions encoding various proteins of SARS coronavirus are also shown.
- the SARS coronavirus gene integrated into the genome is a DNA gene.
- the DNA gene for example, a DNA fragment obtained by isolating the above ss-RNA and obtaining a full-length cDNA using reverse transcriptase, amplifying the desired protein gene portion by PCR, and collecting it can be used.
- the base sequence of the DNA fragment may contain a base sequence that is recognized as a transcription termination signal in the recombinant vaccinia virus (for example, "TTTTTNT”) and is incorporated into the virus genome as it is. And protein expression decreases.
- a mutation (silent, mutation) into a part of the base sequence serving as a transcription termination signal before incorporation into the genome.
- the mutation can be introduced using a known site-directed mutagenesis method (for example, Quick-change kit (Strategene, model number: 200523)).
- Specific examples of SARS coronavirus genes that can be expressed in the recombinant virus of the present invention include structural protein genes of the virus. Although there are nonstructural protein genes, it is preferable that they contain at least a structural protein gene.
- a structural protein gene is a gene that has undergone transcription / translation and regulates the amino acid sequence of the structural protein, and the structural protein forms / holds a structure, form, etc.
- a nonstructural protein gene is a gene that has undergone transcription / translation and defines the amino acid sequence of the nonstructural protein.
- the nonstructural protein is a function other than the function of the structural gene in vivo. It means a protein that can perform its function.
- SARS coronavirus structural protein genes include, for example, spike protein (S protein) gene, membrane protein (M protein) gene, envelope protein (E protein) gene, nucleocapsid protein (N protein) gene, etc.
- S protein spike protein
- M protein membrane protein
- E protein envelope protein
- N protein nucleocapsid protein
- those containing at least the S protein gene are more preferable. This is because, when an S protein expressed from at least an S protein gene is used as an antigen, it becomes a recombinant virus that is extremely excellent in immunity induction.
- the structural protein gene contains at least the S protein gene, whether it contains only the S protein gene or a combination of the S protein gene and another structural protein gene, the same applies.
- the same applies for example, the N protein gene, the M protein gene, and the E protein gene are preferred as other structural protein genes.
- combinations that can produce pseudo SARS coronavirus particles are also possible.
- One of the preferred forms are also possible.
- SARS coronavirus nonstructural protein genes include helicase protease.
- the recombinant virus of the present invention is preferably, but not limited to, one capable of producing pseudo SARS coronal virus particles.
- the pseudovirus particle By using the pseudovirus particle as an antigen, it can be expected that a recombinant virus with excellent immunity induction can be obtained.
- pseudo-SARS coronavirus particles are not virus particles that have the pathogenicity unique to SARS coronavirus, but those that do not have the pathogenicity but that can be mimicked from the virus particles from a structural point of view. means.
- the SARS coronavirus gene has at least N protein gene, M protein gene and E protein among its structural protein genes. It must contain genes, and these genes must be incorporated into the viral genome so that the protein corresponding to each gene can be expressed.
- the production of the recombinant vaccinia virus of the present invention is not limited, and can be performed using a conventional homologous recombination method.
- a foreign gene SARS coronavirus gene
- its expression can be expressed in the genome of a vaccinia virus as a recombinant mother (preferably in a sequence of a gene that is not essential for the propagation of the virus (referred to as gene a)).
- gene a a sequence of a gene that is not essential for the propagation of the virus.
- a plasmid vector containing a DNA sequence having a promoter inserted therein is constructed.
- homologous recombination between the plasmid vector and the maternal vaccinia virus resulted in the recombination in which the above promoter and SARS coronavirus gene were inserted into the maternal vaccinia virus genome (preferably in the sequence of gene a).
- Vaccinia virus can be produced.
- the homologous recombination can be performed by a conventional transfection method.
- Maternal vaccinia virus is infected with pre-cultured animal cells (for example, monkey kidney cell CV-1 and rabbit rabbit cell RK13), and then the plasmid vector is transfected into the infected cells by the calcium phosphate method or the like. And obtain a candidate recombinant virus.
- the candidate recombinant virus is then subjected to various selection methods, confirmations and tests to obtain the desired recombinant vaccinia virus.
- hemagglutinin (HA) gene is preferably selected as the gene a in the genome of the maternal vaccinia virus such as the LC16m8 strain.
- HA hemagglutinin
- TK thymidine kinase
- HA protein expression deficiency has almost no effect on growth, the usefulness inherent in the LC16m8 strain, which is the maternal vaccinia virus, can be fully utilized.
- the HA gene region in the genome of the LC16m8 strain, etc. changes to a region into which a foreign gene has been inserted due to homologous recombination, the HA protein will not be expressed and the hemagglutination characteristic of the HA protein will not occur .
- erythrocyte eg, ⁇ chicken erythrocytes
- HA- white plaque
- the plasmid vector used for homologous recombination is not limited as long as it contains a DNA sequence having the characteristics described above.
- a known plasmid vector is used as a parent and a DNA sequence having the above-mentioned characteristics is inserted, or the DNA sequence is finally inserted.
- the plasmid vector that serves as a matrix include pSFJl-10 (Arc. Virol., 1994, vol. 138, p. 315-330, JP-A-6-237773 (Examples 1 to 3)), pSFJ2-16 etc. are mentioned. Of these, pSFJl-10 is preferred.
- the plasmid vector pSFJl-10 contains a “box virus type A inclusion body (ATI) promoter” and “multiple repeat vaccinia virus LCl6m8 strain 7.5 kDa in the hemagglutinin (HA) gene region of the LC16m8 strain corresponding to the gene a.
- ATI hemagglutinin
- a DNA sequence having a hybrid promoter composed of a protein (p7.5) expression mutant promoter "and a multiple cloning site" This is a plasmid vector comprising
- a recombinant vector can be constructed in which the desired SARS coronavirus gene is inserted into the desired restriction enzyme site in the cloning site located downstream of the hybrid promoter (Fig. 1). See).
- homologous recombination using this recombination vector is performed, and the SARS coronavirus gene is located downstream of the hybrid promoter (specifically, together with the hybrid promoter) in the genome of the maternal vaccinia virus (HA gene)
- the protein corresponding to the inserted SARS coronal virus gene is expressed continuously and in large quantities from the early stage to the late stage with complete glycosylation. Can be made.
- the vaccine for SARS coronavirus according to the present invention contains the above-described recombinant virus of the present invention. Since the recombinant virus of the present invention is safe, the vaccine for SARS coronavirus is not only a preventive agent intended to prevent SARS infection in advance, but also a therapeutic agent aimed at reducing symptoms after SARS infection. Can also be used.
- SARS coronavirus vaccines generally contain other components in addition to the recombinant virus of the present invention so that they can be used as live vaccines.
- Other components include, for example, water; an oil phase containing at least one oil (preferably its emulsification system); an ester obtained by condensation of sugar or glycerol with a fatty acid; an emulsion containing a derivative of the ester And the like. These may be used alone or in combination of two or more.
- the content of the recombinant virus of the present invention is not limited. Generally, it is preferably 30% or more when used in any of the SARS preventive agent and the SARS therapeutic agent. More preferably, it is 50% or more, more preferably 80% or more. The content is above Within the range, there are advantages such as efficiently enhancing the immunity of the host and enhancing the neutralizing antibody titer and cytotoxicity.
- the form of vaccination for the SARS coronavirus vaccine is not limited, and in general, it can be used for any of the SARS preventive agents and the SARS therapeutic agents.
- inoculation and nasal inoculation are not limited, for example, oral inoculation may be used.
- SARS coronavirus vaccine is not limited.
- injections subcutaneous injections, etc.
- intramuscular injections intramuscular injections
- oral preparations oral administrations can be used for both SARS preventive agents and SARS therapeutic agents.
- Sprays, nasal sprays, and the like are listed, with injections being preferred.
- the dose when using the SARS coronavirus vaccine as an injection is not limited, but it is preferably 10 2 to 10 10 PFU / body when used for any of the SARS preventive agents and SARS therapeutic agents. .
- the dose when using the SARS coronavirus vaccine as an oral preparation is not limited, but it is preferably 10 4 to 10 12 PFU / body for any of the SARS preventive agents and SARS therapeutic agents. .
- the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. In the following, for convenience, “wt%” is sometimes referred to as “wt%”.
- the S protein gene was isolated and prepared as follows. After SARS coronavirus was propagated in animal cells (Vero cells), full-length RNA (complete genome, 29751base, ss-RN 3 ⁇ 4 extracted and isolated) was synthesized according to a conventional method. , Using a primer of SEQ ID NOs: 1 and 2 specific for the S protein gene (sequence 21482-25259 in the entire base sequence of SARS coronavirus shown in “GenBank No. NC—004718”), PCR was performed using the above cDNA as a saddle type.
- the composition of the reaction solution was DNA polymerase 1U, dNTP0.3mM, F primer 1 ⁇ , R primer ⁇ with respect to 50pL of the buffer solution supplied with the commercially available polymerase, and the cycle conditions were melting at 95 ° C for 0.5 minutes and annealing at 58 ° C.
- the cycle of 0.5 min at ° C and 2 min at 72 ° C was 25 cycles.
- R primer SEQ ID NO: 2:.
- the S protein gene has two sequences called “TTTTTTNT”.
- This sequence serves as a transcription termination signal for the long-term promoter in vaccinia virus (see Virol., 1991, vol. 185, p.432-436), so that the amino acid sequence of the S protein does not change.
- Silent mutation was introduced into the base in the code containing part of the sequence. Specifically, using the Quick-change kit (Strategene), the base sequence “TTTTTTT” of 22569 to 22575 was changed to “TTCTTCT”, and the base sequence “TTTTTGT” of 25580-25586 was changed to “TCTTCGT”.
- pSFJl-10 (Arch. Virol., 1994, vol.138, p.315-330, JP-A-6-237773 (Examples 1 to 3)) was prepared.
- pSFJl-10 is expressed in the hemagglutinin (HA) gene region of the vaccinia virus LC16m8 strain as a “box virus type A inclusion body (ATI) promoter” and “multiple repeat vaccinia virus LC16m8 strain 7.5 kDa protein (p7.5).
- HA hemagglutinin
- ATI inclusion body
- p7.5 multiple repeat vaccinia virus LC16m8 strain 7.5 kDa protein
- It is a plasmid vector having a hybrid promoter composed of a “mutant promoter” and a multicloning site. Proteins expressed by this hybrid promoter are expressed in large quantities in the form of complete sugar modification from the early to late stages of vaccinia virus infection.
- the SARS coronavirus S protein gene was transferred to the Kpnl site on the multicloning site of pSFJl-10 in accordance with conventional methods of gene recombination technology. Incorporation creates a new plasmid vector pSFJl-10-SARS-S in which the S protein gene is inserted downstream of the ATI p7.5 hybrid promoter in the hemagglutinin (HA) gene region of pSFJl-10. (See Figure 1).
- RK13 cells in this example, “primary kidney cultured cells” may be used in place of this and the following “RK13 cells” are seeded in a T175 flask and reached a confluent state.
- Plasmid vector pSFJl-10-SARS-S (40 pg) was diluted with HeBS buffer to a total volume of 200 pL, added to the cell suspension and mixed, and allowed to stand on ice for 10 minutes. This cell suspension was transferred to a 0.4 cm cuvette and subjected to electroporation (0.2 kV, 960F) using an electric mouth porator (manufactured by Bio-Rad, product name: Gene-Pulser).
- the cells were peeled off with a scraper to obtain a cell suspension.
- the cell suspension was collected, sonicated in cold water (about 4 ° C) (4 times for 30 seconds), and then centrifuged (2000 rpm, 10 minutes). The supernatant after centrifugation was used as a virus solution.
- This virus solution was diluted with 1.9 mL of 10% FCS / MEM medium, added to RK13 cells previously seeded in a lOOnini dish, and infected at 30 ° C for 1 hour.
- the virus solution was removed by suction, the cells were washed with PBS ( ⁇ ), 10 mL of 10% FCS / 0.5% methylcellulose ZMEM medium was added, and the mixture was cultured at 30 ° C. for 72 hours. After incubation, the supernatant was removed by aspiration, and the cells were washed with PBS (—). 10 mL of a chicken erythrocyte solution (concentration: 0.5%) diluted with PBS (+) was added to a 100 mm dish and incubated at 37 ° C for 30 minutes. Thereafter, the erythrocyte solution was removed by aspiration, and the cells were washed twice with PBS (—).
- plaques not adsorbed with chicken erythrocytes were collected using a Pipetman. For details, see HA gene region 1 of vaccinia virus strain LC16m8. When homologous recombination involving pSFJl-10-SARS-S occurs, the HA protein is not expressed when the S protein gene is inserted. Does not cause hemagglutination. Therefore, after the addition of the chicken erythrocyte solution, the target recombinant vaccine virus into which the S protein gene has been introduced can be efficiently recovered by selecting plaques in which hemagglutination reaction is not observed, ie, white plaques.
- S protein gene introduction was confirmed by PCR and plaque hybridization. The virus purification was repeated 3 times for the virus confirmed to have been introduced.
- the plaque obtained in the third round was suspended in 700 pL of 10% FCS / MEM medium and sonicated in cold water (30 seconds x 4 times). After centrifugation (2000 rpm, 10 minutes), 500 pL of the supernatant was added to RK13 cells previously seeded in a T25 flask and infected at 30 ° C for 2 hours. After infection, the virus solution was removed by suction, and the cells were washed with 2.5 mL of 10% FCSZMEM medium. The medium was removed by aspiration, 2.5 mL of 10% FCSZMEM medium was newly added, and cultured at 30 ° C for 72 hours.
- the cells were removed from the flask using a scraper, and the cell suspension was recovered. This cell suspension was sonicated in cold water (30 seconds x 4 times), then centrifuged, and the supernatant was collected as a virus solution.
- the collected virus solution was serially diluted and separately seeded in advance on a 6-well plate: It was added to K13 cells and infected at 30 ° C for 1 hour. Then, the virus solution is removed by aspiration, and the cells are washed twice with PBS (—) and then 10% FCS. 2 mL of /0.5% methylcellulose / MEM medium was added and cultured at 30 ° C. for 72 hours.
- the titer was calculated by counting the number of plaques formed in the well.
- the stock titer (PFU / mL) is obtained by multiplying the number of plaques in the well by the dilution factor and calculating the PFU contained in the original 1 niL stock solution.
- the moi was adjusted from the number of PFU and the number of cells in the T175 flask, and large-scale culture was performed as described below.
- the cells were removed from the flask using a scraper, and the cell suspension was collected and stored frozen at -80 ° C. After freeze-thawing three times, the cell suspension was sonicated in cold water (30 seconds x 4 times), centrifuged (2000 rpm, 10 minutes), and the supernatant was recovered as a virus solution .
- the collected virus solution was filled into a high-speed centrifuge tube and centrifuged (18000 rpm, 45 minutes) to precipitate the virus. After removing the supernatant by aspiration, the pellet (virus) was resuspended in a small amount of MEM medium. By this operation, it was possible to prepare a virus solution that was 10 times more concentrated than when cultured in a T175 flask.
- This concentrated virus solution was serially diluted, and the titer was calculated in the same manner as described above.
- F primer (SEQ ID NO: 3): 5'-GGCTATGGCT GTCTTTCCTG-3 'Primer (SEQ ID NO: 4): 5'-CAAGCGAAAA GGCATCAGAT ATG-3'
- the composition of the reaction solution was DNA polymerase 1U, dNTP0.3mM, F primer 1 ⁇ R, R primer ⁇ with respect to 50pL of the buffer solution attached to the commercially available polymerase, and the cycle conditions were 95 ° C for thawing.
- the cycle for 0.5 minutes at 0.5 ° C, 0.5 minutes at 58 ° C for annealing, and 2 minutes at 72 ° C for elongation was 25 cycles.
- the obtained PCR product was electrophoresed on an agarose gel to confirm the panda. As a result, if a single band of approximately 300 bp assumed in advance by the primer design is recognized, it can be seen that the S protein gene has been introduced into the recombinant virus genome. It will be understood that the gene has not been introduced.
- the probe of SEQ ID NO: 5 (Dig-dUTP addition) specific to the S protein gene and the probe of SEQ ID NO: 6 specific to the HA gene (Dig-dUTP addition) Using the plaque hybridization method, it was confirmed whether the S protein gene was introduced into the genome of the obtained recombinant vaccinia virus (see Fig. 3A).
- GACTTCTAACGCCATCGATGTTTAGATCCATCACACAAATACGAT HA probe (SEQ ID NO: 6): GGTTCTACCATGAACAACAAGTCACAGTCGGTGATTATTATTAAC Specifically, a recombinant virus solution was added to RK13 cells previously seeded on 6-well plates and infected at 30 ° C for 1 hour. After infection, the virus solution was removed by suction, and the cells were washed twice with PBS (—). To each well, 2 mL of lOo / oFCSZO.SQ / o methyl cell ZMEM medium was added and cultured at 30 ° C for 72 hours.
- the developed plaque was transferred to nylon membrane (Hybond N +) (Amersham), treated with denaturing solution (0.5M NaOH, 1.5M NaCl), neutralized solution (1.5M NaCl, 1M (Tris-HCl) and 2XSSC solution.
- the transferred nylon membrane was air-dried for 45 minutes and then UV cross-linked by Auto closslink using UV stratalinker 2400 (Stratagenes). 0.15 mL of rapid hybri buffer (Amersham) per 1 cm 2 of nylon membrane was added, heated at 65 ° C for 30 minutes, and then S probe or HA probe was added at 50 pg / mL (final concentration).
- Lysis buffer (1 ° /. SDS, 0.5% NP-40 0.15M NaCl, lOmM Tris-HCl (pH7.4)
- lOO L is added to lyse the cells.
- the solution was transferred to a 1.5 mL Eppendorf tube.
- the collected solution was sonicated in cold water (4 times for 30 seconds) until the viscosity disappeared.
- the amount of protein in the solution was quantified by the Lowry method.
- electrophoresis was performed using the above solution as a sample. Electrophoresis was performed on 50 pg protein per lane. After completion of electrophoresis, the extracted gel was energized for 60 minutes at 5.5mAZcm2 using a Semi-dry blotter (BIO-RAD, model number: Trans-Blot (registered trademark) SD Cell). Transferred to PVDF membrane. Next, the PVDF membrane was washed with a TBS-T solution and blocked by immersing it in a 5 wt% skim milk-TBS-T solution for 180 minutes. After blocking, the PVDF membrane was washed 3 times with TBS-T solution and reacted with the addition of the primary antibody solution.
- antisera product of SIGMA GENOSYS, manufactured by SIGMA GENOSYS
- SIGMA GENOSYS amino acid sequence of S protein. Name: ST11.68, ST1170
- IgG antibody obtained by purification with Protein A using the Ampure PA kit (manufactured by Amersham, model number: RPN. 1752) was used.
- the purified antibody was quantified by the Lowry method, adjusted to lOpg / mL, and used.
- Peptide A (SEQ ID NO: 7): CTDSVRDPKTSEI
- Peptide B (SEQ ID NO: 8): CKFDEDDSEPVLK
- the PVDF membrane was washed 3 times with TBS-T solution, and the secondary antibody solution was added to react.
- Donkey anti-rabbit IgG-linked HRPO manufactured by Amersham, product name: NA9340
- the PVDF membrane was washed again with TBS-T solution three times.
- the ECL solution was then added to the PVDF membrane, exposed for 3 minutes and developed into a film.
- Recombinant vaccinia virus obtained in Example 1 and vaccinia virus LC16m8 strain were transendothelially inoculated with lxl0 8 PFU to another Usagi (New Zealand White, female), 1, 2, 3, 4, Six weeks later, blood was collected from each ear vein.
- a serum sample used for the evaluation of neutralizing ability a sample obtained by thawing the above cryopreserved serum at 37 ° C and inactivating it at 56 ° C for 30 minutes was used.
- Serum samples were added to SARS coronavirus 200 TCIDso, which was passaged twice in serum-free medium (MEM) in Hanoi 01-03 strain, and kept at 37 ° C for 1 hour and at 4 ° C for 1 hour Later, the cells were infected with Vero E6 cells previously seeded on 96-well plates. Five days after infection, CPE (cytopathic effect) was observed, and Neutralization Titer (NT 50 ), which is an index of neutralization ability of each serum sample, was visually measured and evaluated.
- MEM serum-free medium
- a vaccine capable of simultaneously expressing not only SARS coronavirus S protein but also a plurality of structural proteins or nonstructural proteins is also considered effective.
- the present inventor has confirmed the expression of S protein even in a gene expression system constructed to express four types of structural proteins (S protein, M protein, E protein, N protein).
- S protein, M protein, E protein, N protein The production of pseudo SARS coronavirus particles is also predicted, so a strong vaccine effect can be expected.
- Industrial applicability According to the present invention, it is possible to provide a novel recombinant virus that is effective in preventing the onset of SARS infection and highly safe, and a vaccine for SARS coronavirus containing the same. Sequence listing free text
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Cited By (5)
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WO2009110644A1 (ja) | 2008-03-07 | 2009-09-11 | 財団法人東京都医学研究機構 | C型肝炎ウイルス遺伝子を有する組換えワクシニアウイルス |
WO2012050229A1 (ja) | 2010-10-15 | 2012-04-19 | 財団法人東京都医学総合研究所 | 新型インフルエンザウイルス由来ヘマグルチニンタンパク質遺伝子を有する組換えワクシニアウイルス |
WO2019069561A1 (ja) | 2017-10-03 | 2019-04-11 | 公益財団法人東京都医学総合研究所 | インフルエンザに対する医薬 |
WO2020100990A1 (ja) | 2018-11-14 | 2020-05-22 | 公益財団法人東京都医学総合研究所 | 新型インフルエンザウイルス由来ヘマグルチニンタンパク質遺伝子を有するDIs株由来組換えワクシニアウイルス |
WO2022025298A1 (ja) * | 2020-07-31 | 2022-02-03 | 公益財団法人東京都医学総合研究所 | 組換えワクシニアウイルス |
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JP5944210B2 (ja) * | 2012-04-18 | 2016-07-05 | 一般財団法人化学及血清療法研究所 | 新型インフルエンザウイルス由来ヘマグルチニンタンパク質遺伝子が組み込まれたb5r遺伝子欠損組換えワクシニアウイルス |
TW202144574A (zh) * | 2020-03-30 | 2021-12-01 | 國立大學法人大阪大學 | 冠狀病毒感染或伴隨冠狀病毒感染之症狀的預防或治療疫苗 |
WO2021202893A1 (en) * | 2020-04-03 | 2021-10-07 | Nonigenex, Inc. | Detecting adaptive immunity to coronavirus |
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06237773A (ja) * | 1992-12-22 | 1994-08-30 | Tonen Corp | ポックスウイルスのa型封入体(ati)プロモーター及び前期プロモーターを含んで成る外来遺伝子発現ベクター |
Family Cites Families (1)
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WO2005035556A2 (en) * | 2003-05-06 | 2005-04-21 | Iguazu Biosciences Corp. | Sars-coronavirus virus-like particles and methods of use |
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2004
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06237773A (ja) * | 1992-12-22 | 1994-08-30 | Tonen Corp | ポックスウイルスのa型封入体(ati)プロモーター及び前期プロモーターを含んで成る外来遺伝子発現ベクター |
Non-Patent Citations (6)
Title |
---|
BISHT H. ET AL: "Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice", PROC.NATL.ACAD.SCI. USA, vol. 101, no. 17, April 2004 (2004-04-01), pages 6641 - 6646, XP002332532 * |
JIN N.Y. ET AL: "Constructions of vaccinia virus A-type inclusion body protein, tandemly repeated mutant 7.5 kDa protein, and hemagglutinin gene promoters support high levels of expression", ARCH.VIROL., vol. 138, no. 3-4, 1994, pages 315 - 330, XP002996778 * |
KITAHATA M. ET AL: "Kumikae Vaccinia Virus ni yoru SARS Vaccine no Kaihatsu", DAI 8 KAI THE JAPANESE SOCIETY FOR VACCINOLOGY GAKUJUTSU SHUKAI PROGRAM SHOROKUSHU, September 2004 (2004-09-01), pages 49, XP002996775 * |
OBARA M. ET AL: "Virus no Byogensei Hatsugen Kiko Oyobi Saibo Shi no Bunshi Kiko no Kaiseki", TOKYOTO RINSHO IGAKU SOGO KENKYUSHO KENKYU HOKOKUSHU, HEISEI 15 NENDOBAN, February 2004 (2004-02-01), pages 129 - 143, XP002996776 * |
ODAGIRI T. ET AL: "Jusho Kyusei Kokyuki Shokogun (SARS) no Shindan Oyobi Kensa Shuho nado ni Kansuru Kinkyu Chosa Kenkyu 3.SARS Virus ni Taisuru Vaccine no Kenkyu 3.1. Vaccine Kohokabu no Sentei", JUSHO KYUSEI KOKYUKI SHOKOGUN (SARS) NO SHINDAN OYOBI KENSA SHUHO NADO NI KANSURU KINKYU CHOSA KENKYU HEISEI 15 NENDO SEIKA HOKOKUSHO, August 2004 (2004-08-01), pages 29 - 41, XP002996777 * |
SUGIMOTO M. ET AL: "Gene structures of low-neurovirulent vaccinia virus LC16m0, LC16m8, and their Lister original (LO) strains", MICROBIOL.IMMUNOL., vol. 29, no. 5, 1985, pages 421 - 428, XP002996779 * |
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WO2009110644A1 (ja) | 2008-03-07 | 2009-09-11 | 財団法人東京都医学研究機構 | C型肝炎ウイルス遺伝子を有する組換えワクシニアウイルス |
JP2009232836A (ja) * | 2008-03-07 | 2009-10-15 | Tokyoto Igaku Kenkyu Kiko | C型肝炎ウイルス遺伝子を有する組換えワクシニアウイルス |
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US9000136B2 (en) | 2008-03-07 | 2015-04-07 | Tokyo Metropolitan Institute Of Medical Science | Recombinant vaccinia virus having hepatitis C virus gene |
WO2012050229A1 (ja) | 2010-10-15 | 2012-04-19 | 財団法人東京都医学総合研究所 | 新型インフルエンザウイルス由来ヘマグルチニンタンパク質遺伝子を有する組換えワクシニアウイルス |
JP2016178945A (ja) * | 2010-10-15 | 2016-10-13 | 公益財団法人東京都医学総合研究所 | 新型インフルエンザウイルス由来ヘマグルチニンタンパク質遺伝子を有する組換えワクシニアウイルス |
JP6013185B2 (ja) * | 2010-10-15 | 2016-10-25 | 公益財団法人東京都医学総合研究所 | 新型インフルエンザウイルス由来ヘマグルチニンタンパク質遺伝子を有する組換えワクシニアウイルス |
WO2019069561A1 (ja) | 2017-10-03 | 2019-04-11 | 公益財団法人東京都医学総合研究所 | インフルエンザに対する医薬 |
WO2020100990A1 (ja) | 2018-11-14 | 2020-05-22 | 公益財団法人東京都医学総合研究所 | 新型インフルエンザウイルス由来ヘマグルチニンタンパク質遺伝子を有するDIs株由来組換えワクシニアウイルス |
WO2022025298A1 (ja) * | 2020-07-31 | 2022-02-03 | 公益財団法人東京都医学総合研究所 | 組換えワクシニアウイルス |
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