Recombinant vaccinia virus

Abstract

Provided are a recombinant vaccinia virus, which is a clinically usable preventive vaccine for COVID-19 (a vaccine for SARS-CoV-2), etc. The recombinant vaccinia virus according to the present invention is characterized by comprising all or part of a cDNA, said cDNA encoding a non-structural protein or a structural protein derived from SARS-CoV-2, and an expression promoter.

Description

Technical Field

[0001] The present invention relates to recombinant vaccinia virus and the like as a vaccine for SARS-CoV-2.

Background Art

[0002] COVID-19, an infectious disease caused by the highly pathogenic novel coronavirus (SARS-CoV-2), first identified in Wuhan, China in 2019, has spread rapidly across countries worldwide throughout 2020. As of July 2020, the confirmed number of infected people exceeds 16 million worldwide and over 30,000 in Japan, with the death toll exceeding 650,000 worldwide and reaching approximately 1,000 in Japan. It has been reported that about 30% of patients recovered from COVID-19 have insufficient immune induction even after virus elimination (Non-Patent Document 1), which raises concerns about the risk of reinfection with SARS-CoV-2. On the other hand, the immunity induced by common cold coronavirus infection decreases and disappears within a relatively short period of one to two years after infection, and infection occurs repeatedly in periodic cycles. Therefore, considering that currently most people do not have immunity against SARS-CoV-2, that even patients who have already been infected can experience a decrease in immunity within a short period of time, and that SARS-CoV-2 may continue to spread worldwide in the future, the development of a COVID-19 preventive vaccine (vaccine for SARS-CoV-2) that can strongly induce immunity and maintain it for a long period of time is essential.

[0003] In addition, since continuous spread can lead to genetic mutations in SARS-CoV-2, there is a need for vaccines with broad cross-reactivity that can respond to antigenic changes accompanying mutations. Furthermore, since the emergence of SARS coronavirus (CoV) in 2002, novel human-infecting CoVs such as MERS-CoV and the current SARS-CoV-2 have emerged in a short period of time, and there is a possibility that unknown CoVs will emerge in the future. Therefore, the development of a pan-coronavirus infectious disease preventive vaccine that exhibits protective effects against multiple types of CoV is also an important issue.

[0004] Prior art documents Non-patent document 1: MedRxiv, 2020 (Fan Wu et al., Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications., medRxiv preprint doi: (https: / / doi.org / 10.1101 / 2020.03.30.20047365.) [Overview of the Initiative]

[0005] Under these circumstances, the development of a COVID-19 preventive vaccine (SARS-CoV-2 vaccine) that could be used clinically was highly desired.

[0006] This invention has been made in consideration of the above circumstances and provides recombinant vaccinia virus, pharmaceutical compositions, etc., as shown below. (1) Recombinant vaccinia virus comprising all or part of a cDNA encoding a non-structural or structural protein derived from SARS-CoV-2, and an expression promoter. (2) Recombinant vaccinia virus as described in (1) above, wherein the vaccinia virus is the DIs strain. (3) The recombinant vaccinia virus according to (1) or (2) above, wherein the cDNA encoding the non-structural protein includes a cDNA encoding the ORF1b region (a non-structural protein gene region that encodes a protein essential for viral replication) in a non-structural protein derived from SARS-CoV-2.

[0007] (4) A recombinant vaccinia virus according to any one of (1) to (3) above, wherein the cDNA encoding the non-structural protein is one of the DNAs (a) to (d) below. (a) DNA consisting of the nucleotide sequence shown in Sequence ID No. 1 (b) DNA having 80% or more identity with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1, and encoding a non-structural protein derived from SARS-CoV-2. (c) DNA consisting of the base sequence shown in Sequence ID No. 3 (d) DNA having more than 80% identity with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 3, and encoding a non-structural protein derived from SARS-CoV-2. (5) The recombinant vaccinia virus according to (1) or (2) above, wherein the cDNA encoding the structural protein includes the cDNA encoding the spike protein in the structural protein derived from SARS-CoV-2.

[0008] (6) The recombinant vaccinia virus described in (1), (2), or (5) above, wherein the cDNA encoding the structural protein is any of the following DNAs (a) to (d). (a) DNA consisting of the base sequence shown in Sequence ID No. 5 (b) DNA having 80% or more identity with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 5, and encoding a structural protein derived from SARS-CoV-2. (c) DNA consisting of the base sequence shown in Sequence ID No. 7 (d) DNA having more than 80% identity with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 7, and encoding a structural protein derived from SARS-CoV-2. (7) Recombinant vaccinia virus as described in any one of (1) to (6) above, which can maintain stability even at room temperature. (8) A pharmaceutical composition comprising any one of the recombinant vaccinia virus described in (1) to (7) above.

[0009] (9) The pharmaceutical composition described in (8) above, which is a preventive drug for SARS-CoV-2 infection. (10) The pharmaceutical composition described in (8) above, which is a treatment for SARS-CoV-2 infection. (11) A vaccine for SARS-CoV-2 containing any one of the recombinant vaccinia virus described in (1) to (7) above. Effects of the invention

[0010] The present invention provides recombinant vaccinia virus as a COVID-19 preventive vaccine (SARS-CoV-2 vaccine) usable in clinical settings, and pharmaceutical compositions using the same. The recombinant vaccinia virus of the present invention is extremely useful as a SARS-CoV-2 vaccine that strongly induces immunity and can maintain immunity for a long period of time, making it ideal for the prevention or treatment of COVID-19 infection. Furthermore, the recombinant vaccinia virus of the present invention maintains its stability as a SARS-CoV-2 vaccine even at room temperature, and its storage and transportation temperatures can be refrigerated or at room temperature, making it highly practical. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the effectiveness of SARS-CoV-S recombinant vaccines (recombinant vaccinia virus) in preventing the onset of the disease (based on the results of SARS vaccines). [Figure 2] This figure shows the gene sequence homology in the coronavirus genome. [Figure 3] This figure shows the results of a vaccination trial of SARS-CoV-S recombinant vaccine (rVV-S) in rabbits (vaccinia virus LC16m8 pre-immunized group and unvaccinated group).

[0012] [Figure 4] This diagram shows the genetic makeup of a SARS-CoV-2 recombinant vaccinia virus. [Figure 5] This is a diagram illustrating the SARS-CoV-2 recombinant vaccine. [Figure 6] This diagram shows the configuration of the pSMART-DIs-L3-mnCoV-Japan-1b GND-GPTF vector.

[0013] [Figure 7] FIG. 1 is a diagram showing the construction of the pSMART-DIs-L3-mnCoV-Japan-S-GPTF vector. [Figure 8] FIG. 2 is a diagram showing the identification results of SARS-CoV-2 mRNA expression in cells infected with SARS-CoV-2 recombinant vaccinia virus (rDIs-1b-GND) by real-time detection polymerase chain reaction. [Figure 9] FIG. 3 is a diagram showing the identification results of SARS-CoV-2 spike protein expression in cells infected with SARS-CoV-2 recombinant vaccinia virus (rDIs-S) by western blotting.

[0014] [Figure 10] FIG. 4 is a diagram showing the protective mechanism of a novel coronavirus (SARS-CoV-2) preventive vaccine (SARS-CoV-2 recombinant vaccinia virus). [Figure 11] FIG. 5 is a diagram showing the results of confirming the expression of introduced antigens in the prepared recombinant vaccines (rDIs-S and rDIs-1b-GND) by western blotting. A (left panel): Detection results of SARS-CoV-2 spike protein (S protein) using a specific antibody against the S protein. B (right panel): Detection results of 1b protein using a specific antibody against SARS-CoV-2 ORF1b constituent protein (non-structural protein encoded by the ORF1b region). [Figure 12] FIG. 6 is a diagram showing the results of analysis of SARS-CoV-2-specific T cell responses in rDIs-1b-GND-inoculated human ACE2-expressing transgenic mice (hACE2-expressing Tg mice).

[0015] [Figure 13] FIG. 7 is a diagram showing the method for evaluating the efficacy and safety of recombinant vaccine (rDIs-S) using cynomolgus monkeys. [Figure 14] FIG. 8 is a diagram showing the measurement results of body temperature changes after SARS-CoV-2 infection in rDIs-S-inoculated cynomolgus monkeys. [Figure 15] This figure shows the time course of infectious virus levels in nasal swabs and respiratory swabs in rDIs-S-inoculated cynomolgus monkeys. A (left): Virus level in nasal swabs; B (right): Virus level in respiratory swabs

[0016] [Figure 16] This figure shows the results of measuring the amount of viral RNA in the lungs of cynomolgus monkeys vaccinated with rDIs-S, 7 days after SARS-CoV-2 infection. [Figure 17] This figure shows the pulmonary pathology findings (left panel in Figure 17) and pathology score (right panel in Figure 17) 7 days after SARS-CoV-2 infection in rDIs-S-vaccinated cynomolgus monkeys. [Figure 18] This figure shows the neutralizing antibody induction effect in rDIs-S-vaccinated cynomolgus monkeys. [Figure 19] This figure shows the results of a SARS-CoV-2 infection protection test in rDIs-S inoculated hACE2-expressing transgenic mice. [Figure 20] This figure shows the results of evaluating the in vivo activity of antigen-specific cytotoxic T cells in rDIs-S-inoculated C57BL / 6 mice. [Figure 21] This figure shows the effect of inducing S protein-specific binding antibodies in rDIs-S-vaccinated C57BL / 6J mice. [Figure 22] This figure shows the neutralizing antibody induction effect in rDIs-S-vaccinated C57BL / 6J mice. [Figure 23]This figure shows the results of neutralizing antibody titer measurements against early SARS-CoV-2 strains (TY / WK-521) and variant strains (SUMS2: Shiga University of Medical Science isolate, QHN001: UK variant, TY7-501: Brazilian variant) using rDIs-S inoculated cynomolgus monkey plasma. In the figure, #1 to #4 represent four plasma samples from different rDIs-S inoculated cynomolgus monkeys. SUMS2 (Shiga University of Medical Science isolate) has an amino acid substitution mutation of "D614G, Q675H" in the amino acid sequence (SEQ ID NO: 6) of the SARS-CoV-2 spike protein (S protein), QHN001 (UK type mutant) has an amino acid substitution mutation of "N501Y, D614G" in the amino acid sequence (SEQ ID NO: 6) of the SARS-CoV-2 S protein, and TY7-501 (Brazil type mutant) has an amino acid substitution mutation of "K414T, E484K, N501Y, D614G" in the amino acid sequence (SEQ ID NO: 6) of the SARS-CoV-2 S protein. [Figure 24] This figure shows the results of the protective effect against the SARS-CoV-2 mutant (TY8-612: South African mutant) using rDIs-S inoculated hACE2 Tg mice. TY8-612 (South African mutant) has an amino acid substitution mutation in the amino acid sequence (SEQ ID NO: 6) of the SARS-CoV-2 S protein, specifically in the RBD region: K417N, E484K, N501Y; N-terminal region: D80A, D215G; Subdomain 1 region: D614G; S2 region: A701V, and also lacks amino acids 242-244 (Leu-Ala-Leu). [Modes for carrying out the invention]

[0017] The present invention will now be described in detail. The scope of the present invention is not limited to this description, and the invention may be modified and implemented in any way that does not impair the spirit of the invention, in addition to the examples given below. This specification encompasses the entirety of Japanese Patent Application No. 2020-130717 (filed July 31, 2020), Japanese Patent Application No. 2020-215454 (filed December 24, 2020), and Japanese Patent Application No. 2021-078781 (filed May 6, 2021), which form the basis of this application's priority claim. All publications cited herein, such as prior art documents, and published gazettes, patent gazettes, and other patent documents are incorporated herein by reference.

[0018] 1. Outline of the present invention The aim is to develop and quickly put into practical use a genetically modified live vaccine for COVID-19, which involves introducing a SARS-CoV-2-derived gene into a highly attenuated DIs strain of vaccinia virus, a smallpox vaccine. By using vaccinia virus as the parent organism, it is expected that a strong immune response to SARS-CoV-2 can be induced in a short period after vaccination, and that the induced immunity will be long-lasting and exhibit broad cross-reactivity that can cope with antigenic mutations.

[0019] The inventors have established, as candidates for a COVID-19 preventive vaccine (SARS-CoV-2 vaccine), 1) a recombinant vaccine (antibody-inducing and T-cell-inducing type) that expresses the spike protein (S protein), which is expressed on the surface of the virus particle and is important for adsorption to cells and infection, based on the track record of SARS vaccines (Figures 1 and 3), and 2) a recombinant vaccine (T-cell-inducing type) that expresses the non-structural protein gene region (ORF1b), which is the region with the highest gene sequence homology on the coronavirus genome (Zhou et al., Nature. 2020 Mar; 579(7798): 270-273) (Figure 2) and encodes a protein essential for viral replication.

[0020] Recombinant vaccines expressing the SARS-CoV S protein, which have been established and evaluated to date, were able to induce neutralizing antibodies within one week after vaccination and protected mice from attack infection by SARS-CoV (Fumihiko Yasui et al., J Immunol, 181:6337-6348 (2008)) (Figure 1). Furthermore, SARS-CoV-specific immunity was induced in rabbits that had already been vaccinated with smallpox, at a level equivalent to that of unsensitized rabbits (Masahiro Kitabatake et al., Vaccine, 25:630-637 (2007)) (Figure 3). Therefore, recombinant vaccinia virus is considered effective even in the elderly population, who are at risk of severe COVID-19 and have a history of smallpox vaccination. Furthermore, to ensure even greater safety, the inventors have also developed a recombinant vaccine using a highly attenuated vaccinia virus DIs strain (Isamu Tagaya et al., Nature, 192:381-382, 1961), which was adapted by Dr. Isamu Tagaya et al. of the National Institute of Preventive Health to chicken fibroblasts (CEF) and does not proliferate in many normal mammalian cells. The SARS-S vaccine using the DIs strain also protected against SARS-CoV infection (Koji Ishii et al., Virology, 351(2):368-380, 2006). Furthermore, the inventors have also been developing recombinant avian influenza vaccines expressing H5 subtype and H7 type hemagglutinin (HA) protein genes from DIs parent organisms (rDIs-H5 HA and rDIs-H7 HA). They have demonstrated that these vaccines exhibit disease-preventing effects within a short period of one week after vaccination, and that the immunity conferred once provides long-term immunity that can be described as lifelong immunity in mouse models. Based on these findings, they have created recombinant vaccinia viruses (vaccinia vaccines) capable of expressing SARS-CoV-2 viral structural protein genes and non-structural protein genes (Figures 4 and 5).

[0021] 2. Production of recombinant vaccinia virus Specifically, information on all genes encoding SARS-CoV-2 proteins, genes encoding the outer shell protein region, and genes encoding non-structural protein regions involved in replication is available in the form of the gene sequence information (SEQ ID NO: 9) for the SARS-CoV-2 virus strain: hCoV-19 / Japan / AI / I-004 / 2020, which is registered on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) with GenBank accession number: LC521925.

[0022] Therefore, the genes contained in the recombinant vaccinia virus of the present invention, i.e., genes containing the non-structural protein region or structural protein region of SARS-CoV-2, can be obtained by conventional genetic engineering methods. For example, nucleic acid synthesis methods using DNA synthesis equipment, which are commonly used as genetic engineering methods, can be used. Alternatively, after isolating or synthesizing a template gene sequence, primers specific to each gene can be designed, and the gene sequence can be amplified using PCR or a cloning vector. The above methods can be carried out by those skilled in the art in accordance with "Molecular cloning 4th Edt. Cold Spring Harbor Laboratory Press (2012)," etc. Known methods can be used to purify the obtained PCR product.

[0023] In this invention, gene DNA encoding either a non-structural protein region or a structural protein region of the entire SARS-CoV-2 gene region is used to produce recombinant vaccinia virus. The non-structural protein region consists of the ORF1a, ORF1b, ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 regions, and in this invention, it is preferable to select and use, for example, the ORF1b region. The structural protein region consists of the spike (S), envelope (E), integral membrane (M), and nucleoprotein (N) regions, and in this invention, it is preferable to select and use, for example, the spike (S) protein region.

[0024] In this invention, the DNA sequence encoding the ORF1b region in the non-structural protein region derived from SARS-CoV-2 is shown in SEQ ID NO: 1, and the DNA sequence encoding the S protein region in the structural protein region derived from SARS-CoV-2 is shown in SEQ ID NO: 5. Furthermore, a variant DNA of the DNA sequence encoding the ORF1b region (in which six bases in the DNA sequence encoding the ORF1b region are replaced with other bases to improve gene expression efficiency within recombinant vaccinia virus) is shown in SEQ ID NO: 3, and a variant DNA of the DNA sequence encoding the S protein region (in which eight bases in the DNA sequence encoding the S protein region are replaced with other bases to improve gene expression efficiency within recombinant vaccinia virus) is shown in SEQ ID NO: 7. However, in addition to the DNA sequences shown in SEQ ID NOs: 1, 3, 5, and 7, the following DNAs can also be used in this invention.

[0025] DNA that has 80% or more identity (homology) to DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 1, and that encodes a non-structural protein derived from SARS-CoV-2 (a variant DNA of the ORF1b region). DNA having 80% or more identity (homology) with a base sequence complementary to the base sequence shown in Sequence ID No. 3, and encoding a non-structural protein derived from SARS-CoV-2 (mutant DNA of the region in which the aforementioned base substitution mutations are added to the ORF1b region).

[0026] DNA that has 80% or more identity (homology) with a base sequence complementary to the base sequence shown in Sequence ID No. 5, and that encodes a structural protein derived from SARS-CoV-2 (mutant DNA of the S protein region). DNA having 80% or more identity (homology) with a base sequence complementary to the base sequence shown in Sequence ID No. 7, and encoding a structural protein derived from SARS-CoV-2 (mutant DNA of the region in which the aforementioned base substitution mutations are added to the S protein region).

[0027] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 1, and that encodes a non-structural protein derived from SARS-CoV-2 (a variant DNA of the ORF1b region). DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 3, and that encodes a non-structural protein derived from SARS-CoV-2 (a mutant DNA of the region in which the aforementioned base substitution mutation is added to the ORF1b region).

[0028] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 5, and that encodes a structural protein derived from SARS-CoV-2 (mutant DNA of the S protein region). DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 7, and that encodes a structural protein derived from SARS-CoV-2 (a mutant DNA of the region in which the aforementioned base substitution mutation is added to the S protein region).

[0029] Here, "encoding a non-structural protein derived from SARS-CoV-2" means encoding a protein produced in cells when the virus replicates. Furthermore, the genes encoding these non-structural proteins include not only the full sequence but also partial sequences. Furthermore, "encoding a structural protein derived from SARS-CoV-2" means encoding a protein that makes up the outer shell of the virus. In addition, the gene encoding the above structural protein includes not only the full sequence but also a partial sequence.

[0030] The above-mentioned mutant DNA can be obtained by chemical synthesis, or by using DNA consisting of the base sequence represented by SEQ ID NOs: 1, 3, 5, or 7, or a fragment thereof, as a probe, and obtaining it from cDNA libraries and genomic libraries by known hybridization methods such as colony hybridization, plaque hybridization, and Southern blotting. Stringent conditions for the above hybridization include, for example, 0.1×SSC to 10×SSC, 0.1% to 1.0% SDS, and a temperature of 20°C to 80°C. More specifically, conditions include pre-hybridization at 37°C to 56°C for 30 minutes or more, followed by 1 to 3 washes in 0.1×SSC and 0.1% SDS at room temperature for 10 to 20 minutes each. For detailed procedures of the hybridization method, refer to "Molecular cloning 4th Edt. Cold Spring Harbor Laboratory Press (2012)," etc.

[0031] In the preparation of the recombinant vaccinia virus of the present invention, there are no particular limitations, and any known method can be employed. For example, first, a nucleotide sequence DNA encoding a non-structural protein region (such as the ORF1b region) or structural protein region (such as the S protein region) of SARS-CoV-2 is inserted into a desired expression vector (plasmid) (e.g., a DIs strain homologous recombinant vector (plasmid) (Koji Ishii et al. Virology 2006)). Then, by transfecting cells infected with a desired vaccinia virus strain (e.g., attenuated vaccinia virus DIs strain) with this plasmid vector, homologous recombination is induced within the vaccinia virus genome, and a recombinant vaccinia virus expressing a desired region of the non-structural or structural protein of SARS-CoV-2 can be prepared. The expression vector is not particularly limited, but for example, a pSMART® vector having a DIs strain homologous gene sequence region can be used, and the expression promoter included in the recombinant vaccinia virus of the present invention can be any expression promoter that has been used for vaccinia virus gene expression in the past (e.g., the mH5 promoter).

[0032] The aforementioned attenuated vaccinia virus DIs strain is a highly attenuated host-range gene-deficient strain established from the Dalian strain (DIE), which was used as a smallpox vaccine, using the one-day egg passage method. It can only proliferate in Chick Embryo Fibroblast (CEF) cells and was developed by Dr. Isamu Tagaya of the National Institute of Preventive Health (now the National Institute of Infectious Diseases) (Tagaya et al. Nature, 192:381-382, 1961). Due to the large-scale gene deletion, it cannot proliferate in most mammalian cells, including those of mice, guinea pigs, rabbits, and humans. For this reason, its safety is guaranteed even if it is administered to immunocompromised or immunosuppressed patients.

[0033] The recombinant vaccinia virus produced can be used as a template for PCR with primers specific to the non-structural or structural protein region genes of SARS-CoV-2, allowing for confirmation of gene transfer to the desired non-structural or structural protein region. Furthermore, the expression of the desired non-structural or structural protein can be confirmed by Western blotting using animal cells infected with the recombinant vaccinia virus prepared as a sample. For the Western blotting method, for example, commercially available antibodies that specifically recognize the desired non-structural or structural protein region, or IgG purified with Protein G from antiserum prepared by immunizing with SARS-CoV-2 polypeptides, can be used. The recombinant vaccinia virus of the present invention exhibits high temperature stability and can maintain its stability as a SARS-CoV-2 vaccine even at room temperature (not limited to, but including a range of 10 to 40°C, with 10 to 20°C being preferred). Therefore, the vaccinia virus and the pharmaceutical compositions described later can be stored and transported at either refrigerated or room temperature, offering excellent practicality and convenience.

[0034] 3. Pharmaceutical compositions for the prevention or treatment of COVID-19 The present invention provides a pharmaceutical composition containing the recombinant vaccinia virus described above, more specifically, a pharmaceutical composition as a preventive and therapeutic agent for novel coronavirus (SARS-CoV-2) infection (COVID-19). The pharmaceutical composition of the present invention can be introduced into the body by any known method, such as injection into the muscle, intraperitoneal cavity, intradermal or subcutaneous tissue, or by inhalation through the nasal cavity, oral cavity or lungs, or by oral administration. Furthermore, the recombinant vaccinia virus contained in the pharmaceutical composition of the present invention can be used in combination with existing antiviral drugs (e.g., interferon). The manner of combination is not particularly limited, and the recombinant vaccinia virus of the present invention and the existing antiviral drug can be administered simultaneously, or they can be introduced into the body by administering one and then administering the other after a certain period of time has elapsed.

[0035] Furthermore, the pharmaceutical composition of the present invention can be mixed with known pharmaceutically acceptable carriers such as excipients, bulking agents, binders, lubricants, buffers, isotonic agents, chelating agents, colorants, preservatives, fragrances, flavoring agents, sweeteners, etc. The pharmaceutical composition of the present invention can be administered orally or parenterally, depending on its form, such as oral preparations like tablets, capsules, powders, granules, pills, liquids, and syrups, or parenteral preparations like injections, topical preparations, suppositories, eye drops, and nasal drops. Preferably, local injections into the skin, muscle, or abdominal cavity are examples of administration.

[0036] The dosage is appropriately selected depending on the type of active ingredient, route of administration, target population, patient's age, weight, sex, symptoms, and other conditions. However, the daily dose of recombinant vaccinia virus is approximately 1,000 to 1,000,000,000 PFU (plaque forming units) for oral administration, preferably approximately 1,000,000 to 1,000,000,000 PFU for parenteral administration, and approximately 100 to 1,000,000,000 PFU (plaque forming units) for parenteral administration, preferably approximately 1,000 to 1,000,000,000 PFU. The virus can be administered once a day or in several divided doses.

[0037] The recombinant vaccinia virus of the present invention can be used as a vaccine for the prevention or treatment of COVID-19, but it is preferable to measure the antibody titer or cellular immune activity as a vaccine in advance. For example, antibody titers against the recombinant vaccinia virus of the present invention, or the parental strain DIs, can be obtained by inoculating mice, rabbits, etc., with these virus strains, collecting serum over time, and measuring the ELISA titer or LIPS (luciferase immunoprecipitation system) titer for SARS-CoV-2 protein in the serum. This allows for confirmation of SARS-CoV-2 gene expression and the presence or absence of an immune response in the inoculated individuals. Mouse serum inoculated with the recombinant vaccinia virus of the present invention may show an increase in antibody titers against SARS-CoV-2 protein starting one week after inoculation.

[0038] Furthermore, cellular immune activity can be measured, for example, by inoculating mice with the recombinant vaccinia virus of the present invention or the parental strain DIs, then isolating spleen cells from the immunized mice and measuring whether CD4-positive and CD8-positive cells specific to SARS-CoV-2 non-structural and structural proteins have been induced and activated using FACS or ELISPOT assay. For example, by co-culturing spleen cells derived from BALB / c mice immunized with the recombinant vaccinia virus of the present invention with target cells expressing non-structural or structural proteins, antigen-specific activated CD4 and CD8-positive T cells can be detected.

[0039] The recombinant vaccinia virus according to the present invention can induce cellular immunity against SARS-CoV-2, and this effect is particularly pronounced in recombinant vaccinia viruses that can express SARS-CoV-2-derived non-structural proteins (preferably ORF1b). Furthermore, the recombinant vaccinia virus according to the present invention can induce neutralizing antibodies against SARS-CoV-2, and this effect is particularly pronounced in recombinant vaccinia viruses that can express SARS-CoV-2-derived structural proteins (preferably S protein). Furthermore, the recombinant vaccinia virus (SARS-CoV-2 vaccine) according to the present invention can be a vaccine with broad cross-reactivity, capable of responding not only to SARS-CoV-2, which is the vaccinia virus's direct target, but also to antigenic changes associated with mutations in SARS-CoV-2. In other words, in the pharmaceutical composition and SARS-CoV-2 vaccine for the treatment and prevention of SARS-CoV-2 infection according to the present invention, SARS-CoV-2 includes so-called mutant strains that have any mutations such as substitutions, deletions, or additions to its base sequence or amino acid sequence. Examples of SARS-CoV-2 mutant strains include SUMS2 (isolated from Shiga University of Medical Science), QHN001 (UK-type mutant), TY7-501 (Brazil-type mutant), and TY8-612 (South African-type mutant). Furthermore, the recombinant vaccinia virus according to the present invention can also be used as a pan-CoV infection preventive vaccine, exhibiting protective effects against unknown novel coronaviruses (CoV) that may emerge in the future.

[0040] The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0041] 1. Method (1) Production of a SARS-CoV-2 virus structural protein gene and non-structural protein gene recombinant vaccinia vaccine (i) Selection of SARS-CoV-2 virus strains to be used for genetic recombination Based on the gene sequence information (GenBank accession number: LC521925) (Sequence ID 9) of the SARS-CoV-2 virus strain: hCoV-19 / Japan / AI / I-004 / 2020, the following four genes were synthesized.

[0042] • Non-structural protein 1b (ORF1b) gene region nCoV-Japan-1b (8198 bp) (SEQ ID NO: 1) • Non-structural protein 1b (ORF1b) gene region mnCoV-Japan-1b GND (8198 bp) (SEQ ID NO: 3) • Spike protein gene region nCoV-Japan-S (3926 bp) (SEQ ID NO: 5) • Spike protein gene region mnCoV-Japan-S (3926 bp) (SEQ ID NO: 7)

[0043] Of the four genes mentioned above, the gene indicated by Sequence ID No. 1 is the wild-type ORF1b gene, and the gene indicated by Sequence ID No. 5 is the wild-type spike protein gene. On the other hand, the genes shown in Sequence IDs 3 and 7 are mutant genes optimized to improve gene expression efficiency in vaccinia virus, based on the genes shown in Sequence IDs 1 and 5, respectively. Specifically, the mutant gene shown in Sequence ID No. 3 has the thymine (T) at positions 2445, 2448, 4998, 5001, and 6000 of the base sequence shown in Sequence ID No. 1 replaced with cytosine (C), and the guanine (G) at position 2539 replaced with alanine (A) (a total of 6 base substitutions).

[0044] Furthermore, the mutant gene shown in Sequence ID No. 7 is one in which the thymine (T) at positions 99, 102, 2364, 2367, 3213, 3216, 3417, and 3420 of the base sequence shown in Sequence ID No. 5 is replaced with cytosine (C) (a total of 8 base substitutions). In this example, of the four genes mentioned above, the genes indicated by Sequence ID No. 3 and No. 7 were used to establish recombinant vaccinia virus.

[0045] (ii) Cloning two synthetic genes into the pSMART-DIs-L3 vector Two types of synthetic genes (genes shown in SEQ ID NOs: 3 and 7) were inserted into the recombinant vector for producing the DIs recombinant vaccine: pSMART-DIs-L3 (SEQ ID NO: 10) (Figures 6 and 7). Using these recombinant vectors, the establishment of recombinant vaccinia viruses (rDIs-1b-GND, rDIs-S) was carried out (Figure 4). "rDIs-1b-GND" is the recombinant vaccinia virus obtained using the vector with the gene shown in SEQ ID NO: 3 inserted (pSMART-DIs-L3-mnCoV-Japan-1b GND-GPTF), and is sometimes referred to as "rDIs-mnCoV-1b-GND". "rDIs-S" is the recombinant vaccinia virus obtained using the vector with the gene shown in SEQ ID NO: 7 inserted (pSMART-DIs-L3-mnCoV-Japan-S-GPTF), and is sometimes referred to as "rDIs-mnCoV-S".

[0046] (2) Evaluation of SARS-CoV-2 virus non-structural protein gene or structural protein recombinant vaccinia vaccine (i) After confirming the gene sequence inserted into the ORF1b non-structural protein recombinant DIs (rDIs-1b-GND), mRNA expression was confirmed by Real-Time Detection polymerase chain reaction (RTD-PCR) (Figure 8), and ORF1b constituent protein expression was confirmed by Western blotting (Figure 11B). The primers, probes, reaction mixture composition, and reaction conditions used in the above RTD-PCR are as follows.

[0047] <Primer / Probe> TIFF0007927590000001.tif55147

[0048] <Composition of reaction solution> Water (RNase-free) 8.5 μL 4x Reaction mix 5μL Forward Primer (10 μM) 1 μL Reverse Primer (10 μM) 1 μL Probe (10 μM) 0.5 μL RNA sample 4 μL Final reaction volume: 20 μL

[0049] <Reaction conditions> [Table 1]

[0050] (ii) After confirming the gene sequence inserted into the spike protein gene region recombinant DIs(rDIs-S), the expression of the spike protein was confirmed by Western blotting (Figure 9, Figure 11A).

[0051] 2.Results (1) After confirming the gene sequence inserted into the ORF1b non-structural protein recombinant DIs (rDIs-1b-GND), mRNA expression was confirmed by Real-Time Detection polymerase chain reaction (RTD-PCR) (Figure 8). Good mRNA expression was confirmed in 10 out of 12 clones. Furthermore, the expression of ORF1b constituent proteins in rDIs-1b-GND was confirmed by Western blotting (Figure 11B).

[0052] (2) After confirming the gene sequence inserted into the spike protein gene region recombinant DIs(rDIs-S), the expression of the spike protein was confirmed by Western blotting (Figure 9, Figure 11A). Good spike protein expression was confirmed in all 9 clones.

[0053] 3. Discussion A vaccine against the SARS-CoV-2-derived ORF1b non-structural protein (rDIs-1b-GND) may be an effective prophylactic vaccine that induces cellular immunity. Furthermore, a vaccine against the SARS-CoV-2-derived spike protein (rDIs-S) may be an effective prophylactic vaccine that induces neutralizing antibodies (Figure 10). [Examples]

[0054] rDIs-1b-GND was inoculated into human ACE2-expressing transgenic mice (hACE2-expressing Tg mice), and the SARS-CoV-2-specific T cell response in these mice was analyzed. Specifically, as shown in Figure 12, rDIs-1b-GND or DIs (control) was inoculated twice at 3-week intervals, and one week later, SARS-CoV-2 attack infection was induced. The activation of antigen-specific T cells against SARS-CoV-2 6 days after infection was analyzed by interferon-γ ELISpot assay. As a result, activation of SARS-CoV-2-specific T cells (ORF1b antigen (RdRp)-specific T cells) (production of interferon-γ) was observed in the rDIs-1b-GND inoculation group (Figure 12). [Examples]

[0055] The efficacy and safety of rDIs-S were evaluated using cynomolgus monkeys. Specifically, as shown in Figure 13, rDIs-S or DIs (control) was intradermally inoculated into cynomolgus monkeys twice at 3-week intervals. One week later, attack infection with SARS-CoV-2 was induced, and autopsy was performed 7 days after infection. The evaluation criteria and results for each evaluation are described below in (1) to (5).

[0056] (1) Changes in body temperature after SARS-CoV-2 infection were measured. In the control (DIs) group (dashed line), a rise in body temperature of 1 degree or more was observed 2 days after SARS-CoV-2 infection, but no rise in body temperature was observed in the rDIs-S vaccination group (solid line) (Figure 14).

[0057] (2) The time course of infectious virus load in nasal cavity and airway swabs was measured. Viral load in nasal swabs: In the DIs-vaccinated group, infectious virus was detected up to 7 days after infection. In the rDIs-S-vaccinated group, it was detected only 1 day after infection and was rapidly eliminated thereafter. In one of the four rDIs-S-vaccinated animals, the virus was below the detection limit even 1 day after infection (Figure 15A). Viral load in respiratory swabs: In the DIs-vaccinated group, infectious virus was detected in 1 out of 4 dogs up to 7 days after infection. In the rDIs-S-vaccinated group, in 2 out of 4 dogs, the levels were below the detection limit even 1 day after infection, and in the 2 dogs where viral load was detected, the levels were remarkably low (Figure 15B).

[0058] (3) The amount of viral RNA in the lungs was measured 7 days after SARS-CoV-2 infection. In the DIs-vaccinated group, high levels of viral RNA were detected in most lung lobes of all four animals, while in the rDIs-S-vaccinated group, viral RNA levels in the lung lobes were almost below the detection limit, demonstrating a good elimination effect (Figure 16).

[0059] (4) Pathological findings and pathological scores of the lungs 7 days after SARS-CoV-2 infection were examined. In the DIs vaccination group, pneumonia with interstitial thickening was observed. On the other hand, in the rDIs-S vaccination group, almost no lesions were observed, indicating that pneumonia could be reduced (Figure 17).

[0060] (5) Neutralizing antibody induction effect in the rDIs-S vaccinated group (NT 50 The titer was confirmed. In the rDIs-S vaccinated group, neutralizing antibodies were detected in all four animals (0 dpi), and a rapid increase in antibody titers due to SARS-CoV-2 attack infection (7 dpi) was observed (Figure 18). [Examples]

[0061] The efficacy of rDIs-S was evaluated using human ACE2-expressing transgenic mice (hACE2-expressing Tg mice) and C57BL / 6. The evaluation criteria and results are described below in (1) and (2).

[0062] (1) SARS-CoV-2 infection protection test in rDIs-S inoculated hACE2-expressing transgenic mice As shown in Figure 19, rDIs-S or DIs (control) were administered twice to hACE2-expressing transgenic mice at 3-week intervals, and an attack infection experiment with SARS-CoV-2 was conducted one week later. In mice inoculated with DIs, rapid weight loss occurred and death occurred, but in mice inoculated with rDIs-S, almost no weight loss was observed and a 100% survival rate was observed (Figure 19).

[0063] (2) Evaluation of in vivo activity of antigen-specific cytotoxic T cells in rDIs-S-inoculated C57BL / 6 mice As shown in Figure 20, C57BL / 6 mice were inoculated twice with rDIs-S or DIs (control) at 3-week intervals. One week later, a 1:1 mixture of SARS-CoV-2 S protein-stimulated and unstimulated splenocytes was intravenously transferred into the mice. The following day, the spleens were collected, and the effect of eliminating antigen-stimulated cells in the transplanted mice was analyzed using flow cytometry. In mice inoculated with DIs, S protein peptide-stimulated cells could not be eliminated, but in mice inoculated with rDIs-S, S protein-derived peptide-stimulated cells were rapidly eliminated (Figure 20). This confirmed that rDIs-S induces SARS-CoV-2 S protein-specific cytotoxic T cells. [Examples]

[0064] The antibody-inducing ability of rDIs-S was evaluated using C57BL / 6J mice. The evaluation criteria and results are described below in (1) and (2).

[0065] (1) As shown in Figure 21, C57BL / 6J mice were inoculated with rDIs-S or DIs (control) twice at 3-week intervals, and blood samples were collected weekly from before inoculation until 4 weeks after. Serum samples were diluted 100-fold with a diluent (1% BSA, 0.5% Tween 20, 2.5 mM EDTA-containing PBS(-)), and the production of SARS-CoV-2 S protein-specific binding antibodies (immunoglobulin G; IgG) was measured by ELISA. In rDIs-S inoculated individuals (n=3), S protein-specific antibodies were detected from 1 week after rDIs-S inoculation. Furthermore, additional rDIs-S inoculation 3 weeks after the initial inoculation increased the amount of S protein-specific IgG in the serum 1 week later.

[0066] (2) Using the same serum sample as in (1) above, the neutralizing antibody titer against SARS-CoV-2 (TY / WK-521: early epidemic strain) was evaluated by the plaque reduction neutralization test. Specifically, the serum was diluted 10-fold with culture medium, and then further serially diluted 4-fold. 50 μL of serum diluted 10-fold, 40-fold, 160-fold, 640-fold, 2560-fold, and 10240-fold were mixed with 50 PFU / 50 μL of virus solution, and the mixtures were incubated at 37°C for 1 hour. Subsequently, 100 μL of the serially diluted serum / virus mixture was inoculated into VeroE6 / TMPRSS2 cells susceptible to SARS-CoV-2 infection and adsorbed for 1 hour. After removing the solution and washing the cells with culture medium, 0.6% agarose-containing medium was added and the cells were cultured for 48 hours. The "50% neutralization titer" was calculated as the reciprocal of the serum dilution ratio at which the number of plaques was halved after serum addition, with the number of plaques formed in serum-free samples after 48 hours being set to 100%. As shown in Figure 22, it was revealed that neutralizing antibodies were induced from one week after rDIs-S vaccination in rDIs-S-vaccinated individuals (n=3). Furthermore, the neutralizing antibody titer was enhanced by a booster vaccination three weeks after the initial vaccination. [Examples]

[0067] In Example 3, plasma samples from cynomolgus monkeys that had been inoculated with rDIs-S (intradermal inoculation twice at 3-week intervals) were used to measure neutralizing antibody titers against the early SARS-CoV-2 strain (TY / WK-521) and variant strains (SUMS2: Shiga University of Medical Science isolate, QHN001: UK variant, TY7-501: Brazilian variant) using the TCID50 method. In this example, plasma was collected at the time of booster immunization 3 weeks after the initial rDIs-S inoculation (day-7 in the graph in Figure 23), at the time of SARS-CoV-2 attack infection (day 0 in the graph in Figure 23), and at autopsy (day 7 in the graph in Figure 23). As shown in Figure 23, it was found that neutralizing antibodies against both the early SARS-CoV-2 strain and all variant strains were induced by two doses of the vaccine. [Examples]

[0068] hACE2-expressing transgymatured mice were inoculated twice with either rDIs-S or DIs (control) at 3-week intervals. One week after booster immunization, they were infected via the respiratory tract with 100 PFU of SARS-CoV-2 South African variant (TY8-612). Survival rates were monitored daily, and necropsies were performed 7 days after infection. As shown in Figure 24, 2 out of 4 mice in the DIs-vaccinated (control) group died, while all 5 mice in the rDIs-S-vaccinated group survived. The protective effect of rDIs-S vaccination against this variant (TY8-612) was also confirmed.

[0069] Related information / papers (1) Masahiro Kitabatake, Shingo Inoue, Fumihiko Yasui, Shoji Yokochi, Masaaki Arai, Kouichi Morita, Hisatoshi Shida, Minoru Kidokoro, Fukashi Murai, Mai Quynh Le, Kouji Matsushima and Michinori Kohara. SARS-CoV spike protein recombinant vaccinia virus efficiently induces neutralizing antibodies in spite of pre-immunization with vaccinia virus. Vaccine 25:630-637 (2007). (2) Fumihiko Yasui, Chieko Kai, Masahiro Kitabatake, Shingo Inoue, Misako Yoneda, Shoji Yokochi, Ryoichi Kase, Satoshi Sekiguchi, Kouichi Morita, Tsunekazu Hishima, Hidenori Suzuki, Katsuo Karamatsu, Yasuhiro Yasutomi, Hisatoshi Shida, Minoru Kidokoro, Kyosuke Mizuno, Kouji Matsushima, Michinori Kohara. Prior immunization with SARS-CoV nucleocapsid protein causes severe pneumonia in mice infected with SARS-CoV. J. Immunology 181(9):6337-48(2008).

[0070] (3)Jin-Won Youn,Yu-Wen Hu,Nancy Tricoche,Wolfram Pfahler,Mohammed Tarek Shata,Marlene Dreux,Francois-Loic Cosset,Antonella Folgori,Dong-Hun Lee,Betsy Brotman,and Alfred M.Prince Immunization with Replicating Recombinant Vaccinia Virus.JOURNAL OF VIROLOGY,Nov.2008,82(21):10896-10905.doi:10.1128 / JVI.01179-08 (4)FRANCOIS HABERSETZER,GERALDINE HONNET,CHRISTINE BAIN,MARIANNE MAYNARD-MUET,VINCENT LEROY,JEAN-PIERCE ZARSKI,CYRILLE FERAY,THOMAS F.BAUMERT,JEAN-PIERRE BRONOWICKI,MICHEL DOFFOEL,CHRISTIAN TREPO,DELPHINE A Poxvirus Vaccine Is Safe, Induces T-Cell Responses, and Decreases Viral Load in Patients With Chronic Hepatitis C.GASTROENTEROLOGY 2011;141:890–899.doi:10.1053 / j.gastro.2011.06.009

[0071] (5) Satoshi Sekiguchi, Kiminori Kimura, Tomoko Chiyo, Takahiro Ohtsuki, Yoshimi Tobita, Yuko Tokunaga, Fumihiko Yasui, Kyoko Tsukiyama-Kohara, Takaji Wakita, Toshiyuki Tanaka, Masayuki Miyasaka, Kyosuke Mizuno, Yukiko Hayashi, Tsunekazu Hishima, Kouji Matsushima, and Michinori Kohara. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver. PLoS ONE 7(12):e51656(2012). (6)Takeshi Wada, Michinori Kohara and Yasuhiro Yasutomi.DNA vaccine expressing the non-structural proteins of hepatitis C virus diminishes the expression of HCV proteins in a mouse model.Vaccine 31(50):5968-74,doi:10.1016 / j.vaccine.2013.10.037(2013). (7) Takahiro Ohtsuki, Kiminori Kimura, Yuko Tokunaga, Kyoko Tsukiyama-Kohara, Chise Tateno, Yukiko Hayashi, Tsunekazu Hishima, and Michinori Kohara. M2 macrophages play critical roles in progression of inflammatory liver disease in hepatitis C virus transgenic mice. J. Virology 2015 Oct 14;90(1):300-7. doi:10.1128 / JVI.02293-15. [Industrial applicability]

[0072] The recombinant vaccinia virus used as a COVID-19 preventive vaccine (SARS-CoV-2 vaccine) according to the present invention, and the pharmaceutical compositions using the same, are extremely useful as a SARS-CoV-2 vaccine that strongly induces immunity and can maintain immunity for a long period of time, making them useful for the prevention or treatment of COVID-19 infection. Furthermore, the recombinant vaccinia virus of the present invention can maintain its stability as a SARS-CoV-2 vaccine even at room temperature, and its storage and transportation temperatures can be refrigerated or at room temperature, making it highly practical. [Sequence Listing Free Text]

[0073] Sequence ID 3: Synthetic DNA Sequence ID 4: Synthetic construct Sequence ID 7: Synthetic DNA Sequence ID 8: Synthetic construct Sequence ID 10: Synthetic DNA Sequence ID 11: Synthetic DNA Sequence ID 12: Synthetic DNA Sequence ID 13: Synthetic DNA [Sequence List] TIFF0007927590000003.tif255149TIFF0007927590000004.tif255149TIFF0007927590000005.tif255149TIFF0007927590000006.tif255149TIFF0007927590000007.tif255149TIFF0007927590000008.tif255149TIFF0007927590000009.tif255149TIFF0007927590000010.tif255149TIFF0007927590000011.tif255149TIFF0007927590000012.tif255149TIFF0007927590000013.tif255149TIFF0007927590000014.tif255149TIFF0007927590000015.tif255149TIFF0007927590000016.tif255150TIFF0007927590000017.tif255149TIFF0007927590000018.tif255149TIFF0007927590000019.tif255149TIFF0007927590000020.tif255148TIFF0007927590000021.tif255149TIFF0007927590000022.tif255149TIFF0007927590000023.tif255149TIFF0007927590000024.tif255150TIFF0007927590000025.tif255149TIFF0007927590000026.tif255149TIFF0007927590000027.tif255149TIFF0007927590000028.tif255149TIFF0007927590000029.tif255150TIFF0007927590000030.tif255150TIFF0007927590000031.tif255149TIFF0007927590000032.tif255149TIFF0007927590000033.tif255149TIFF0007927590000034.tif255150TIFF0007927590000035.tif255149TIFF0007927590000036.tif255149TIFF0007927590000037.tif255149TIFF0007927590000038.tif255149TIFF0007927590000039.tif255149TIFF0007927590000040.tif255149TIFF0007927590000041.tif255149TIFF0007927590000042.tif255149TIFF0007927590000043.tif255149TIFF0007927590000044.tif255149TIFF0007927590000045.tif255149TIFF0007927590000046.tif255149TIFF0007927590000047.tif255149TIFF0007927590000048.tif255149TIFF0007927590000049.tif255149TIFF0007927590000050.tif255148TIFF0007927590000051.tif255148TIFF0007927590000052.tif255149TIFF0007927590000053.tif255149TIFF0007927590000054.tif255149TIFF0007927590000055.tif255149TIFF0007927590000056.tif255149TIFF0007927590000057.tif255149TIFF0007927590000058.tif255148TIFF0007927590000059.tif255149TIFF0007927590000060.tif255148TIFF0007927590000061.tif255149TIFF0007927590000062.tif255149TIFF0007927590000063.tif255149TIFF0007927590000064.tif255149TIFF0007927590000065.tif255149TIFF0007927590000066.tif255149TIFF0007927590000067.tif255149TIFF0007927590000068.tif255148TIFF0007927590000069.tif255149TIFF0007927590000070.tif255149TIFF0007927590000071.tif255149TIFF0007927590000072.tif255148TIFF0007927590000073.tif255148TIFF0007927590000074.tif255149TIFF0007927590000075.tif255149TIFF0007927590000076.tif255149TIFF0007927590000077.tif255149TIFF0007927590000078.tif255149TIFF0007927590000079.tif255149TIFF0007927590000080.tif255148TIFF0007927590000081.tif255149TIFF0007927590000082.tif255149TIFF0007927590000083.tif255149TIFF0007927590000084.tif255148TIFF0007927590000085.tif255148TIFF0007927590000086.tif255149TIFF0007927590000087.tif255149TIFF0007927590000088.tif255149TIFF0007927590000089.tif255150TIFF0007927590000090.tif255148TIFF0007927590000091.tif255148TIFF0007927590000092.tif255149TIFF0007927590000093.tif255149TIFF0007927590000094.tif255148TIFF0007927590000095.tif255148TIFF0007927590000096.tif255149TIFF0007927590000097.tif255149TIFF0007927590000098.tif255149TIFF0007927590000099.tif255149TIFF0007927590000100.tif255149TIFF0007927590000101.tif255149TIFF0007927590000102.tif255149TIFF0007927590000103.tif255149TIFF0007927590000104.tif255148TIFF0007927590000105.tif255149TIFF0007927590000106.tif255149TIFF0007927590000107.tif255149TIFF0007927590000108.tif255149TIFF0007927590000109.tif255149.

Claims

1. A recombinant vaccinia virus comprising cDNA encoding a non-structural or structural protein derived from SARS-CoV-2 and an expression promoter, The cDNA encoding the aforementioned non-structural protein is the DNA of either (a) or (b) below: (a) DNA consisting of the base sequence shown in Sequence ID No. 3, (b) DNA having more than 90% identity with the DNA consisting of the base sequence shown in Sequence ID No. 3, encoding a non-structural protein derived from SARS-CoV-2, and exhibiting improved gene expression in vaccinia virus compared to DNA consisting of the base sequence shown in Sequence ID No. 1 (wherein the DNA of (b) is such that the bases corresponding to the 2445th, 2448th, 4998th, 5001st, and 6000th bases of the base sequence shown in Sequence ID No. 3 are cytosine, and the base corresponding to the 2539th base of the base sequence shown in Sequence ID No. 3 is adenine). And, The cDNA encoding the aforementioned structural protein is the DNA of (c) or (d) below: (c) DNA consisting of the base sequence shown in Sequence ID No. 7, (d) DNA that has 90% or more identity with the DNA consisting of the base sequence shown in Sequence ID No. 7, encodes a structural protein derived from SARS-CoV-2, and exhibits improved gene expression in vaccinia virus compared to DNA consisting of the base sequence shown in Sequence ID No. 5 (wherein the DNA of (d) is DNA in which the bases corresponding to the 99th, 102nd, 2364th, 2367th, 3213th, 3216th, 3417th, and 3420th bases of the base sequence shown in Sequence ID No. 7 are cytosine). That is, The aforementioned recombinant vaccinia virus.

2. The recombinant vaccinia virus according to claim 1, wherein the vaccinia virus is the DIs strain.

3. The recombinant vaccinia virus according to claim 1 or 2, which can maintain stability even at room temperature.

4. A pharmaceutical composition comprising the recombinant vaccinia virus described in any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, which is a preventive agent for SARS-CoV-2 infection.

6. The pharmaceutical composition according to claim 4, which is a therapeutic agent for SARS-CoV-2 infection.

7. A vaccine for SARS-CoV-2 comprising the recombinant vaccinia virus described in any one of claims 1 to 3.

8. The vaccine according to claim 7, which is used for the prevention or treatment of SARS-CoV-2 infection.

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