Recombinant measles virus

Abstract

To provide a recombinant measles virus useful as a live vaccine against COVID-19, and to provide a vector used to produce the recombinant measles virus.SOLUTION: Provided are a recombinant measles virus in which the gene encoding the protein of coronavirus SARS-CoV-2 is inserted between the N and P gene regions in the measles virus genome; the recombinant measles virus in which the protein is the spike protein of SARS-CoV-2 or a partial protein thereof; and a DNA in which a gene encoding a SARS-CoV-2 protein is inserted into the region from the 1686th base to the 1694th base in the nucleotide sequence represented by SEQ ID NO:2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a recombinant measles virus useful as a vaccine against COVID-19. [Background technology]

[0002] COVID-19 is an infectious disease caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). Coronaviruses enter host cells when their spike glycoprotein interacts with angiotensin-converting enzyme, a receptor on the host cell surface.

[0003] COVID-19 has become a pandemic and a global threat. Since the first infection was reported in December 2019, the number of infections and deaths has been increasing and shows no signs of abating. The development of a treatment or vaccine is of paramount importance in bringing this pandemic under control, and there is a high global demand for the development of an effective vaccine. Furthermore, since vaccines developed in each country are supplied on a priority basis to their own countries, there is also a need for Japan to develop its own vaccine.

[0004] Vaccines are broadly classified into live vaccines and inactivated vaccines. Most of the COVID-19 vaccines currently under development in various countries are so-called inactivated vaccines, consisting of inactivated viruses, SARS-CoV-2 proteins, or nucleic acids (mRNA, DNA) to express those proteins in the body. These are primarily antibody-inducing vaccines that induce humoral immunity. These inactivated vaccines have a short duration of immunity, require frequent administration, and necessitate the preparation of large quantities of the vaccine. Furthermore, antibody-dependent enhancement (ADE) has been suggested to occur with COVID-19, and there are concerns about the side effect of severe illness due to the large amount of antibodies induced by frequent administration.

[0005] On the other hand, measles is caused by the measles virus, which belongs to the family Paramyxoviridae and the genus Morbillivirus. The outer envelope of the measles virus contains hemagglutinin (H) and fusion (F) proteins, while the inner capsid contains a helical negative RNA genome along with nucleocapsid (N) protein, phospho (P) protein, large (L) protein, and matrix (M) protein. The measles virus genome has a leader sequence and a trailer sequence at both ends, which are involved in viral replication, and between them, starting from the 3' end, are genes encoding the N, P, M, F, H, and L proteins. In addition to the P protein, the V and C proteins are also translated from the P gene region.

[0006] Measles is an acute systemic infection that can cause serious illnesses such as pneumonia and encephalitis. The measles virus is highly contagious and spreads through various routes, including airborne transmission, droplet transmission, and contact transmission. There is no cure for measles, and vaccination is the most effective preventive measure. The vaccine used for measles is a live vaccine in which the measles virus has been attenuated through cell culture.

[0007] Live measles vaccines possess excellent characteristics, such as inducing strong cellular immunity and providing extremely long-lasting immunity known as lifelong immunity. Furthermore, live measles vaccines have been used worldwide for over 50 years, and their safety in humans has been confirmed. Therefore, live vaccines for other diseases are being developed using genetically modified viral vectors in which genes from microorganisms that cause other diseases are inserted into the genomic RNA of attenuated measles virus strains useful as measles vaccines. For example, recombinant measles virus in which the F gene and G gene encoding the membrane protein of Nipah virus are inserted into the measles virus genome functions as a bivalent vaccine against both measles and Nipah virus infection (Patent Document 1). Also, recombinant measles virus in which the gene for the protein of the malaria parasite is inserted into the measles virus genome functions as a bivalent vaccine against both measles and malaria infection (Patent Document 2). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2010-115154 [Patent Document 2] Japanese Patent Publication No. 2010-099041 [Non-patent literature]

[0009] [Non-Patent Document 1] Rota et al, Virus Research, 1994, vol.31(3), p.317-330. [Overview of the project] [Problems that the invention aims to solve]

[0010] The primary objective of this invention is to provide a recombinant measles virus useful as a live vaccine against COVID-19, and a vector used for the production of said recombinant measles virus. [Means for solving the problem]

[0011] As a result of diligent research, the inventors of the present invention have discovered that recombinant measles virus, in which a gene encoding the SARS-CoV-2 spike protein or a partial protein thereof is inserted into the cDNA of the measles virus genome, is useful as a live vaccine against COVID-19, and have completed the present invention.

[0012] The recombinant measles virus, etc., according to the present invention are as follows: [1] Recombinant measles virus in which a gene encoding a protein or partial protein of the coronavirus SARS-CoV-2 is inserted between the N gene region and the P gene region of the measles virus genome. [2] The recombinant measles virus according to [1], wherein the protein is the spike protein or a partial protein thereof of SARS-CoV-2. [3] The recombinant measles virus according to [1] or [2], wherein the measles virus genome is an RNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by Sequence ID No. 1. [4] Genomic RNA of any recombinant measles virus described in [1] to [3] above. [5] RNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by Sequence ID No. 3. [6] DNA in which the gene encoding the SARS-CoV-2 protein is inserted in the region from base 1686 to base 1694 of the base sequence represented by Sequence ID No. 2. [7] DNA in which a gene encoding the SARS-CoV-2 protein or a partial protein thereof is inserted into the Fse I recognition sequence located between the 1686th and 1694th bases of the sequence represented by Sequence ID No. 1. [8] DNA consisting of the base sequence represented by Sequence ID No. 3. [9] A vector for the manufacture of a COVID-19 vaccine, comprising any of the DNAs described in [6] to [8] above.

[10] A plasmid vector containing DNA consisting of the nucleotide sequence represented by Sequence ID No. 1.

[11] A method for producing a recombinant measles virus, comprising transfecting a cell that expresses an RNA polymerase capable of transcribing RNA using the DNA as a template with a vector for producing a COVID-19 vaccine containing any one of the DNAs of [6] to [8] and an expression vector for the N protein, P protein, and L protein of the measles virus, and producing a recombinant measles virus in the cell.

[12] A pharmaceutical composition comprising any one of the recombinant measles viruses of [1] to [3] and a pharmaceutically acceptable carrier.

[13] The pharmaceutical composition of

[12] , which is used as a bivalent vaccine against measles and COVID-19.

Advantages of the Invention

[0013] The recombinant measles virus according to the present invention is useful as a vaccine against COVID-19. Further, the recombinant measles virus according to the present invention can be produced by the vector for producing a COVID-19 vaccine according to the present invention and the method for producing a recombinant measles virus using the same.

Brief Description of the Drawings

[0014] [Figure 1] FIG. schematically shows the genomic structure of a recombinant measles virus (MV-CoVS) having the spike protein (CoV-S) gene of SARS-CoV-2 in Example 1. [Figure 2] FIG. shows a transmission image of Vero cells in which CPE was observed (left figure) and an image of fluorescence immunostaining of the spike protein of SARS-CoV-2 (right figure) in Example 1. [Figure 3] FIG. shows the results of Western blotting analysis using an antibody (anti-MV-N antibody) that recognizes the N protein of the measles virus and an anti-CoV-S antibody against Vero cells not infected with the measles virus (non-infected cells), Vero cells infected with the measles virus MV-Ed (MV-Ed cells), and Vero cells infected with the measles virus MV-CoVS (MV-CoVS cells) in Example 1. [Figure 4] In Example 1, it is a figure showing the measurement results over time of the titer of anti-CoV-S antibody in serum after inoculating hamsters twice with measles virus MV-CoVS and measles virus MV-Ed, respectively. [Figure 5] In the pathogenicity comparison test among SARS-CoV-2 virus strains in Example 2, it is a figure showing the measurement results of the amount of virus grown in the lungs of hamsters inoculated with each virus strain. [Figure 6] In the efficacy test of the MV-SCoV2 vaccine in Example 2, it is a figure showing the measurement results over time of the body weights of hamsters inoculated with each virus strain. [Figure 7] In the efficacy test of the MV-SCoV2 vaccine in Example 2, it is a figure showing the results of measuring the amount of virus grown in the lungs collected during autopsy performed on the 3rd and 6th days after virus inoculation of hamsters inoculated with each virus strain. [Figure 8] In Example 3, it is a figure showing the measurement results of the neutral antibody titers against the Wuhan strain, α-1 strain, and δ strain of SARS-CoV-2 in the sera of hamsters after inoculation with the MV-SCoV2 vaccine (A) and on the 3rd and 6th days after inoculation with each virus strain (B). [Figure 9] In Example 4, it is a figure showing the results of measuring the amount of virus grown in the lungs collected during autopsy performed on the 3rd and 6th days after virus inoculation of hamsters inoculated with the δ strain. [Figure 10] In Example 5, it is a figure showing the measurement results of the amount of IFNγ in the sera of hamsters on the 3rd and 6th days after inoculation with each virus strain.

Mode for Carrying Out the Invention

[0015] In the present invention and this specification, "measles virus genome" means genomic RNA capable of producing infectious measles virus and that does not contain foreign genes encoding proteins. This may be genomic RNA of a field-epidemic measles virus, or genomic RNA of a weakened measles strain, or RNA that has been subjected to various genetic modification treatments other than the insertion of foreign genes encoding proteins.

[0016] In the present invention and this specification, "recombinant measles virus" means an infectious measles virus that expresses a foreign protein. The genome RNA of the recombinant measles virus has a foreign structural gene encoding a protein inserted into a region other than the gene region that encodes the measles virus protein.

[0017] The recombinant measles virus according to the present invention has a gene encoding the SARS-CoV-2 coronavirus protein or a partial protein thereof inserted between the N gene region and the P gene region of the measles virus genome. The recombinant measles virus, in which the gene encoding the SARS-CoV-2 protein or a partial protein thereof is inserted between the N gene region and the P gene region of the measles virus genome, expresses the SARS-CoV-2 protein or a partial protein thereof in infected cells and induces immunity against these proteins. Furthermore, since the recombinant measles virus maintains its function as a measles virus, it is also effective as a vaccine against measles.

[0018] The recombinant measles virus according to the present invention induces antibody production against SARS-CoV-2 proteins or partial proteins thereof expressed in infected cells. Therefore, this recombinant measles virus induces antibody production against SARS-CoV-2 and has a strong protective effect against COVID-19 and measles. Furthermore, similar to conventional live measles vaccines, it is expected to induce not only humoral immunity but also cellular immunity, and to provide long-lasting immunity, sometimes referred to as lifelong immunity. In addition, this recombinant measles virus is expected to have a low probability of causing adverse drug reactions (ADEs) upon administration. Similar to conventional live measles vaccines, it can be produced relatively inexpensively and can be manufactured as a lyophilized formulation, thus resolving cold chain issues.

[0019] The recombinant measles virus according to the present invention can be produced using, for example, the reverse genetics system used for the reconstruction of canine distemper virus as described in Japanese Patent Publication No. 2001-275684. Measles virus belongs to the (-)-strand RNA virus class, and modification of the viral genome and introduction of foreign genes can be performed using gene modification techniques commonly used for the cDNA of the measles virus genome. In the reverse genetics system, recombinant measles virus particles with infectious activity are produced from recombinant cDNA obtained by introducing a foreign gene into the cDNA of the measles virus genomic RNA. The genomic RNA of the produced recombinant measles virus is RNA consisting of a base sequence complementary to the recombinant cDNA used in production.

[0020] <recombinant cDNA> Specifically, first, a gene encoding the SARS-CoV-2 protein or a partial protein thereof is inserted between the N gene region and the P gene region of the measles virus genome cDNA to obtain recombinant cDNA.

[0021] The measles virus genome used in the production of recombinant measles virus according to the present invention is not particularly limited as long as it is a measles virus genomic RNA capable of inducing neutralizing antibodies against field-epidemic measles virus. The measles virus may be, for example, a natural virus strain such as the HL strain or the ICB strain, or an attenuated strain such as the Edmonston strain or the AIC strain, or it may be one of these that has been genetically modified to the extent that it does not inhibit the induction of neutralizing antibody production against measles virus.

[0022] As a recombinant measles virus according to the present invention, it is preferable that a gene encoding the SARS-CoV-2 protein or a partial protein thereof is incorporated into the genomic RNA of a weakened strain, or that a genetically modified strain thereof is used, because it is expected to have a higher protective effect against the onset of COVID-19.

[0023] Examples of attenuated measles virus strains include strains obtained by subculturing the Edmonston wild-type strain in cells derived from measles-insensitive animals. Table 1 shows the sequence identity of the full-length genomic RNA and the number of mismatched bases (Non-Patent Literature 1) of various known attenuated strains.

[0024] [Table 1]

[0025] The characteristics of the resulting recombinant measles virus are affected by the type of attenuated strain used as the host. The attenuated measles strain used as the host for producing the recombinant measles virus according to the present invention is preferably one that has a genomic RNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by Sequence ID No. 2. This genomic RNA differs from the full-length genomic RNA of Edmonston B strain (Rubeovax) (GenBank accession number: Z66517) by 30 nucleotides.

[0026] When producing recombinant measles virus according to the present invention, the cDNA of the measles virus genome used can be a DNA in which restriction enzyme recognition sequences for inserting foreign structural genes are appropriately placed between the N, P, M, F, H, and L genes that code for proteins constituting the virus. In particular, it is preferable to use a cDNA of the measles virus genome in which a restriction enzyme recognition sequence is placed between the N gene region and the P gene region (hereinafter sometimes referred to as "modified cDNA").

[0027] The restriction enzyme recognition sequence to be placed between the N gene region and the P gene region is not particularly limited, as long as it is a restriction enzyme recognition sequence that can be present only once between the N gene region and the P gene region throughout the entire length of the measles virus genome cDNA. Specifically, for example, a recognition sequence such as Fse I can be used. The insertion of the restriction enzyme recognition sequence between each gene encoding the proteins that make up the virus in the measles virus genome cDNA can be carried out, for example, by the methods described in Patent Documents 1 and 2.

[0028] The modified cDNA used in producing the recombinant measles virus according to the present invention is preferably a DNA consisting of a nucleotide sequence in which a restriction enzyme recognition sequence is placed between the N gene region and the P gene region in the nucleotide sequence represented by Sequence ID No. 2, more preferably a DNA consisting of a nucleotide sequence in which an Fse I recognition sequence is placed between the N gene region and the P gene region in the nucleotide sequence represented by Sequence ID No. 2, and even more preferably a DNA consisting of the nucleotide sequence represented by Sequence ID No. 1 (GenBank accession number: MT409882). The nucleotide sequence represented by Sequence ID No. 1 is a nucleotide sequence having an Fse I recognition sequence in the region from the 1686th base to the 1694th base between the N gene region and the P gene region in the nucleotide sequence represented by Sequence ID No. 2.

[0029] The SARS-CoV-2 protein or partial protein thereof can be any protein or partial protein present on the surface of SARS-CoV-2. The recombinant measles virus according to the present invention is preferably the SARS-CoV-2 spike protein or partial protein thereof, as it is expected to provide a higher protective effect against COVID-19. The cDNA of the gene encoding the SARS-CoV-2 spike protein or partial protein thereof can be obtained by a conventional method based on the base sequence of the SARS-CoV-2 genomic RNA (NCBI accession number: NC_045512.2). The gene region encoding the SARS-CoV-2 spike protein is the region from base 21563 to base 25384 of the genomic RNA.

[0030] For example, by performing RT-PCR using genomic RNA extracted from SARS-CoV-2 and primers designed based on the nucleotide sequence information of the genomic RNA, a cDNA fragment encoding the spike protein or a portion thereof can be synthesized. Primer design can generally be done using software used for designing PCR primers. When inserting a cDNA fragment encoding the spike protein or a portion thereof into a modified cDNA in which a restriction enzyme recognition sequence is placed between the N and P gene regions, RT-PCR is performed using a primer with the restriction enzyme recognition sequence placed at the 5' end. This allows obtaining a DNA fragment in which the restriction enzyme recognition sequence is placed at both ends of the cDNA encoding the spike protein or a portion thereof. In addition, full-length DNA fragments encoding the spike protein or a portion thereof, or DNA fragments with restriction enzyme recognition sequences placed at both ends of the cDNA, can also be produced by chemical synthesis.

[0031] A recombinant cDNA can be obtained by inserting a DNA fragment encoding a protein of SARS-CoV-2 or a partial protein thereof between the N gene region and the P gene region in the cDNA of the measles virus genome. When using a restriction enzyme recognition sequence, both a DNA fragment of a modified cDNA in which the restriction enzyme recognition sequence is arranged between the N gene region and the P gene region and a DNA fragment in which the restriction enzyme recognition sequence is arranged at both ends of a cDNA encoding a protein of SARS-CoV-2 or a partial protein thereof are treated with a restriction enzyme, and then these are ligated by a ligation reaction to obtain the desired recombinant cDNA.

[0032] The recombinant measles virus according to the present invention may be modified by insertion, deletion, substitution, or the like of a base at any site in the genomic RNA, as long as the ability to prevent the onset of COVID-19 and measles is maintained. For example, in order to inactivate a gene involved in immunogenicity or enhance the transcription efficiency or replication efficiency of RNA, a gene involved in the RNA replication of some measles viruses may be modified. Specifically, intervening sequences and modifications of the leader portion can be mentioned.

[0033] The recombinant measles virus according to the present invention may have any other foreign gene inserted at any site in the genomic RNA, as long as the ability to prevent the onset of COVID-19 and measles is maintained. Examples of other foreign genes to be inserted include genes encoding various proteins that cause pathogenicity in viruses, bacteria, and parasites that can be expressed in a host, genes encoding various cytokines, genes encoding various peptide hormones, genes encoding antigen recognition sites within antibody molecules, and the like. For example, genes encoding various proteins such as influenza virus, mumps virus, HIV, dengue virus, diphtheria, Leishmania, and the antigen recognition site gene of a monoclonal antibody against a cancer-specific marker molecule can be mentioned.

[0034] <Vector for manufacturing COVID-19 vaccine> A vector containing DNA in which the recombinant cDNA is linked downstream of a specific promoter can be used in the production of recombinant measles virus according to the present invention. The recombinant measles virus according to the present invention is effective as a vaccine against COVID-19, and the vector can be used as a vector for the production of a COVID-19 vaccine. The vector for the production of a COVID-19 vaccine is preferably a plasmid vector, but it may also be a linear DNA vector.

[0035] For example, a vector for producing a COVID-19 vaccine can be manufactured by ligating a DNA fragment containing a promoter sequence with a DNA fragment containing recombinant cDNA such that the recombinant cDNA is connected downstream of the promoter sequence, and then incorporating this ligature into a plasmid. The ligation of DNA fragments and insertion into the plasmid can be performed using genetic recombination technology.

[0036] In a vector for the production of a COVID-19 vaccine, the promoter upstream of the recombinant cDNA can be appropriately selected from among promoters recognized by known DNA-dependent RNA polymerases. Examples of such promoters include the T7 promoter, T3 promoter, and SP6 promoter.

[0037] Specifically, a plasmid vector is preferred in which a DNA fragment with a promoter ligated upstream of any of the following DNA sequences is incorporated: DNA in which a gene encoding the SARS-CoV-2 protein is inserted in the region from the 1686th to the 1694th base of the base sequence represented by Sequence ID No. 2; DNA in which a gene encoding the SARS-CoV-2 protein is inserted in the Fse I recognition site from the 1686th to the 1694th base of the base sequence represented by Sequence ID No. 1; or DNA consisting of the base sequence represented by Sequence ID No. 3. A plasmid vector is more preferred in which a DNA fragment with a T7 promoter, T3 promoter, or SP6 promoter ligated upstream of DNA consisting of the base sequence represented by Sequence ID No. 3 is incorporated.

[0038] <Genetically modified vectors> The COVID-19 vaccine vector can be manufactured more easily by using a plasmid vector containing a modified cDNA in which a restriction enzyme recognition sequence is positioned between the N gene region and the P gene region. In particular, a plasmid vector in which a modified cDNA with a restriction enzyme recognition sequence positioned between the N gene region and the P gene region is attached downstream of the promoter is useful as a recombinant vector for manufacturing bivalent vaccines against measles and other diseases.

[0039] The insertion of modified cDNA or promoter fragments into plasmid vectors can be carried out using gene modification techniques such as ligation reactions. The promoter connected upstream of the modified cDNA can be appropriately selected from known promoters recognized by DNA-dependent RNA polymerases, such as the T7 promoter, T3 promoter, and SP6 promoter.

[0040] The modified cDNA to be inserted into the recombinant vector is preferably a DNA sequence in which a restriction enzyme recognition sequence is placed in the region from base 1686 to base 1694 of the sequence represented by Sequence ID No. 2, and more preferably a DNA sequence represented by Sequence ID No. 1. A plasmid vector containing a sequence in which the sequence represented by Sequence ID No. 1 is linked downstream of the promoter sequence is very useful as a recombinant vector.

[0041] For example, a COVID-19 vaccine vector can be produced by genetically engineering a plasmid vector containing a sequence of bases where the base sequence represented by Sequence ID No. 1 is linked downstream of a promoter sequence. This involves inserting a DNA fragment consisting of a sequence in which Fse I recognition sequences are positioned at both ends of a gene encoding a spike protein or a partial protein into the Fse I recognition sequence in the sequence represented by Sequence ID No. 1. Alternatively, by replacing the DNA fragment containing the gene encoding the spike protein or a partial protein with a DNA fragment containing the gene encoding a protein that induces an immune response effective in preventing COVID-19 and other diseases besides measles, a vector capable of producing recombinant measles virus effective as a vaccine against both measles and the other disease can be produced.

[0042] <Method for producing recombinant measles virus> The aforementioned COVID-19 vaccine production vector is transfected into cells that possess an RNA polymerase capable of transcribing RNA using the measles virus genome cDNA incorporated into the vector as a template, and all the enzymes necessary for the transcription and replication of the measles virus. Within these cells, the polymerase synthesizes recombinant measles virus genomic RNA using the measles virus genome cDNA incorporated into the vector as a template. Within these cells, recombinant measles virus is produced by the genomic RNA and the enzymes necessary for the transcription and replication of the measles virus. In other words, the genomic RNA of the produced recombinant measles virus is RNA with a base sequence complementary to the recombinant cDNA used in production.

[0043] An example of an RNA polymerase used to transcribe RNA using the cDNA of the measles virus genome as a template is a DNA-dependent RNA polymerase that acts on a promoter connected upstream of the cDNA of the measles virus genome incorporated into a COVID-19 vaccine vector. If the promoter connected upstream of the cDNA of the measles virus genome is the T7 promoter, then, for example, T7 RNA polymerase can be used as the DNA-dependent RNA polymerase. Examples of cells that express this RNA polymerase include cells that have been previously infected with recombinant vaccinia virus that expresses T7 RNA polymerase, or T7 RNA polymerase constitutive expression strains in which the T7 RNA polymerase gene has been incorporated into the chromosome of cultured cells.

[0044] The enzymes necessary for the transcription and replication of the measles virus include the N protein, P protein, and L protein. Cells that possess all the enzymes necessary for the transcription and replication of the measles virus include, for example, cells that have been transfected with an expression vector containing the gene encoding the N protein (N protein expression vector), an expression vector containing the gene encoding the P protein (P protein expression vector), and an expression vector containing the gene encoding the L protein (L protein expression vector).

[0045] For example, cultured cells that have been previously infected with recombinant vaccinia virus expressing T7 RNA polymerase are transfected together with a COVID-19 vaccine production vector, an N protein expression vector, a P protein expression vector, and an L protein expression vector. Within these transfected cells, recombinant measles virus is constructed, with a genome consisting of RNA with a base sequence complementary to the recombinant cDNA used for production.

[0046] The recombinant measles virus produced can be used as a vaccine against COVID-19. Specifically, the recombinant measles virus replicates within infected cells, and subsequently expresses the SARS-CoV-2 spike protein or a partial protein thereof, inducing the production of antibodies against that protein. Therefore, this recombinant measles virus functions as both a vaccine against measles and a vaccine against COVID-19.

[0047] <Pharmaceutical Compositions> By appropriately mixing the recombinant measles virus according to the present invention with a pharmaceutically acceptable carrier, a pharmaceutical composition useful as a bivalent vaccine against measles and COVID-19 can be produced. The pharmaceutical composition containing the recombinant measles virus and the pharmaceutically acceptable carrier can be produced by methods commonly used in the field of pharmaceutical manufacturing, with the use of additives as appropriate.

[0048] A pharmaceutically acceptable carrier is a diluent, excipient, binder, solvent, etc., that does not cause harmful physiological reactions in the target patient and does not cause harmful interactions with other components such as the active ingredient. Specifically, such carriers include, for example, water, physiological saline, and various buffer solutions. Other additives that can be used include adjuvants, diluents, excipients, binders, stabilizers, isotonic agents, buffers, solubilizers, suspending agents, preservatives, freeze-drying protectants, freeze-protection agents, and bacteriostatic agents.

[0049] For adjuvants used to enhance immunogenicity, those commonly used in the pharmaceutical field can be used. Specifically, examples include bacterial cells and cell walls such as BCG and Propionibacterium acnes, bacterial components such as trehalose dimycolate (TDM), endotoxins of Gram-negative bacteria such as lipopolysaccharides (LPS) and lipid A fraction, synthetic compounds such as β-glucan, N-acetylmuramyl dipeptide (MDP), pestatin, and levamisole, proteins and peptide substances derived from biological components such as thymic hormones, thymic factors, and tuftosin, as well as Freund's incomplete adjuvants and Freund's complete adjuvants.

[0050] Vaccine dosage forms may be liquid, frozen, or lyophilized. A liquid vaccine can be manufactured by collecting a culture medium obtained by culturing cells in a suitable culture medium or cultured cell culture medium, adding additives such as stabilizers, dispensing it into small bottles or ampoules, and then sealing them tightly. A frozen vaccine is obtained by gradually lowering the temperature after dispensing and freezing it, and adding freeze-damage prevention agents or freeze-protection agents. A lyophilized vaccine can be obtained by freezing the dispensed containers in a freeze-dryer, then vacuum-drying them, and then sealing them tightly either as is or after filling them with nitrogen gas. Liquid vaccines can be used as is or diluted with physiological saline. Frozen vaccines and dried vaccines require a dissolving solution to dissolve the vaccine at the time of use. Dissolving solutions include various buffers and physiological saline.

[0051] The vaccine can be administered subcutaneously, intramuscularly, intravenously, etc. The dosage varies depending on the subject's age, weight, sex, and method of administration, and is not particularly limited, but is usually 10mg per dose. 4 ~10 7 TCID 50 A range of at least 10 is preferred. 5 TCID 50 This is particularly preferable. Administration is preferably carried out in the same manner as the measles vaccine.

[0052] Furthermore, the recombinant measles virus according to the present invention can be used to infect various animals, including humans, and then antiserum and other substances can be obtained from bodily fluids collected from those animals for treatment and diagnosis. [Examples]

[0053] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0054] [Example 1] We created a recombinant measles virus (MV-CoVS) that possesses the SARS-CoV-2 spike protein (CoV-S) gene.

[0055] <Preparation of plasmid pMV-CoVS containing cDNA of genomic RNA of recombinant measles virus carrying CoV-S gene> First, genomic RNA was extracted from a measles virus, which is an attenuated measles strain with genomic RNA being RNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 2. Using the obtained RNA as a template, RT-PCR was performed, and the full-length genomic RNA was divided into several partial regions for RT-PCR. The obtained amplified fragments were ligated, and a plasmid pMV-Ed was obtained by incorporating a DNA fragment consisting of the nucleotide sequence represented by SEQ ID NO: 1 into plasmid pMDB1 so that it was ligated downstream of the T7 promoter.

[0056] Using the total RNA extracted from SARS-CoV-2-infected Vero cells, cDNA of the CoV-S gene was obtained by RT-PCR. Using the obtained cDNA as a template, PCR was performed using a primer set having an Fse I recognition sequence at the 5' end, and a DNA fragment encoding the spike protein (CoV-S fragment: SEQ ID NO: 4) having Fse I recognition sequences at both ends was obtained. Plasmid pMV-Ed and the CoV-S fragment were digested with Fse I, ligated together by ligation, and inserted into the Fse I recognition site of pMV-Ed to obtain plasmid pMV-CoVS having cDNA with the genomic structure shown in FIG. 1.

[0057] <Reconstitution of MV-CoVS> For measles virus replication, we used the following vectors: pGEM-NP, an N protein expression vector in which the measles virus N protein gene was inserted into a plasmid pGEM; pGEM-P, a P protein expression vector in which the measles virus P protein gene was inserted into a pGEM; pGEM-L, an L protein expression vector in which the measles virus L protein gene was inserted into a pGEM; and pMV-CoVS. A nucleic acid solution was prepared by adding 2 μg of pGEM-NP, 2 μg of pGEM-P, 0.2 μg of pGEM-L, and 2 μg of pMV-CoVS to a 1.5 mL sampling tube containing 1 mL of Opti-MEM (Thermo Fisher). 1 mL of Opti-MEM was transferred to another sampling tube, and the transfection reagent "Lipofectamin LTX" (Thermo Fisher) was added and mixed. The mixture was then allowed to stand at room temperature for 5 minutes. Subsequently, the transfection reagent solution was mixed with the nucleic acid solution and allowed to stand at room temperature for at least 15 minutes before being used as the plasmid solution.

[0058] In a 6-well plate, 5 × 10⁶ cells of the BHK-T7 cell line, which has been treated with standard trypsin, were placed in each well. 5 Each well was added to 2 mL of culture medium (DMEM medium containing 2% fetal bovine serum), and incubated at 5% CO2 and 37°C for 6 hours. Subsequently, 200 μL of plasmid solution was added dropwise to each well.

[0059] The cells in the aforementioned 6-well plate were cultured for 2 days under conditions of 5% CO2 and 37°C. After removing the culture medium on the second day, Vero cells suspended in VP-SFM (Gibco) were added to 5 × 10⁶ cells per well. 5 The cells were seeded individually and layered on top of BHK-T7 cells. When the plate was further cultured under conditions of 5% CO2 and 37°C, cytotoxic effects (CPE) were observed in Vero cells. This CPE indicates that recombinant measles virus MV-CoVS, reconstituted within BHK-T7 cells, proliferated in Vero cells infected with MV-CoVS.

[0060] RT-PCR was performed using RNA extracted from Vero cells in which CPE was observed as a template, and sequencing analysis of the resulting PCR fragments confirmed that recombinant measles virus MV-CoVS was reconstituted within the Vero cells.

[0061] Furthermore, immunofluorescence staining was performed on Vero cells in which CPE was observed, using an antibody against CoV-S (anti-CoV-S antibody) as the primary antibody. First, recombinant measles virus MV-CoVS was infected into Vero cells, and after 48 hours, the cells were fixed with 4% paraformaldehyde and permeostomized with 0.2% TritonX-100. Next, anti-CoV-S antibody (mouse serum) diluted 1000-fold was added and incubated at room temperature for 1 hour. After removing the anti-CoV-S antibody and washing three times with PBS, Alexa488-labeled anti-mouse IgG antibody diluted 500-fold was added and incubated at room temperature for 30 minutes. After removing the Alexa488-labeled anti-mouse IgG antibody and washing five times with PBS, MV-CoVS-infected Vero cells were observed using a confocal laser microscope.

[0062] Figure 2 shows transmission images of MV-CoVS-infected Vero cells (left) and images of SARS-CoV-2 spike protein immunofluorescence staining (right). As shown in Figure 2, Alexa488 fluorescence was not observed in Vero cells not infected with MV-CoVS, but it was observed in MV-CoVS-infected Vero cells. From these results, it was confirmed that the CoV-S protein is expressed in Vero cells infected with recombinant measles virus MV-CoVS, that is, recombinant measles virus expressing the CoV-S protein could be created.

[0063] Western blotting analysis was performed on Vero cells that were not infected with measles virus (uninfected cells), Vero cells infected with measles virus MV-Ed (MV-Ed cells), and Vero cells infected with measles virus MV-CoVS (MV-CoVS cells) using antibodies that recognize the N protein of measles virus (anti-MV-N antibody) and anti-CoV-S antibody. The results of Western blotting using anti-MV-N antibody and anti-rabbit IgG antibody are shown in Figure 3 (left), and the results of Western blotting using anti-CoV-S antibody and anti-mouse IgG antibody are shown in Figure 3 (right). Bands for the MV-N protein were confirmed in both MV-Ed cells and MV-CoVS cells (Figure 3 (left), asterisk), but bands for the CoV-S protein were confirmed only in MV-CoVS cells (Figure 3 (right), asterisk).

[0064] <Induction of antibody production by measles virus MV-CoVS> Furthermore, we investigated whether antibody production was induced by inoculating hamsters with measles virus MV-CoVS. Specifically, hamsters were inoculated twice intraperitoneally (IP) with measles virus MV-CoVS, and blood samples were collected over time to measure the titer of anti-CoV-S antibodies in the serum. The second viral inoculation was performed 5 weeks (35 days) after the first inoculation. As a control, hamsters were inoculated with measles virus MV-Ed in the same manner. The titer of anti-CoV-S antibodies in the serum was measured over time. The titer of anti-CoV-S antibodies in serum was measured by ELISA using serum.

[0065] Figure 4 shows the time-course measurement of serum anti-CoV-S antibody titer from the first vaccination (day 0). In hamsters inoculated with measles virus MV-Ed, no production of serum anti-CoV-S antibodies was observed. In contrast, in hamsters inoculated twice with measles virus MV-CoVS, serum anti-CoV-S antibody titer increased, indicating that the production of anti-CoV-S antibodies was induced by the two vaccinations. As these results show, measles virus MV-CoVS has immune-inducing potential and is a useful candidate for a COVID-19 vaccine.

[0066] [Example 2] The effectiveness of the measles virus vaccine MV-CoVS produced in Example 1 against each virus strain of SARS-CoV-2 was examined.

[0067] <Virus strains of SARS-CoV-2> As SARS-CoV-2, one Wuhan strain (JPN / Ty / WK-521 strain), two alpha strains (QHN001 strain (alpha-1), QK002 strain (alpha-2)), one beta strain (TY8-612 strain), and two gamma strains (TY7-501 strain (gamma-1), TY7-503 strain (gamma-2)) were used. All of these virus strains were provided by the National Institute of Infectious Diseases.

[0068] <Pathogenicity comparison test among virus strains of SARS-CoV-2> First, each virus strain was infected into hamsters to compare their pathogenicity. Specifically, hamsters implanted with a body temperature measurement chip were inoculated nasally with 1×10 5 TCID 50 / animal (50 μL) of the virus. After inoculation, body weight measurement, throat swab collection, and nasal wash collection were performed over time. In addition, necropsy was performed on the 3rd or 6th day after inoculation, and the virus amount in the lungs (right upper lobe, right middle lobe, right lower lobe, left lobe) and trachea was analyzed by RT-qPCR using the RNA extracted from each tissue as a template.

[0069] The results of measuring the virus amount grown in the lungs of hamsters inoculated with each virus strain are shown in Fig. 5. It was confirmed that all strains grew in the lungs of hamsters. Among them, in the beta strain, the virus growth was suppressed earlier than in other virus strains.

[0070] <Effectiveness test of MV-SCoV2 vaccine> As SARS-CoV-2, the Wuhan strain, alpha-1 strain, beta strain, and gamma-1 strain were used. First, hamsters were given two intraperitoneal (IP) inoculations of the MV-SCoV-2 vaccine. The second viral inoculation was performed 4 weeks after the first inoculation (day 0) (28 days after inoculation). Subsequently, 30 days after the first vaccination, a thermometer chip was implanted in each hamster. Then, 35 days after the first vaccination, the hamsters were inoculated with a virus strain in the same manner as in the "comparative study of pathogenicity between SARS-CoV-2 virus strains." Weight measurements, pharyngeal swabs, and nasal lavage fluids were collected over time. Autopsies were performed 3 or 6 days after viral inoculation (38 or 41 days after inoculation) to analyze the viral load in the lungs and trachea. In addition, histopathological analysis of each tissue was performed. As a control, hamsters that had not been vaccinated with the MV-SCoV-2 vaccine were also inoculated with the virus strain in the same manner and analyzed.

[0071] Figure 6 shows the weight changes of each hamster. Figure 6(A) shows the results for the unvaccinated group, and Figure 6(B) shows the results for the unvaccinated group. In the unvaccinated group, weight decreased by about 10% over 6 days (Figure 6(A)). In contrast, no significant weight loss was observed in the vaccinated group (Figure 6(B)).

[0072] Macroscopic examination of lungs collected during autopsies performed six days after viral inoculation revealed that the lungs of the unvaccinated group showed significant signs of pneumonia. In contrast, the lungs of the vaccinated group were almost normal or showed only a few hemorrhagic spots. In other words, the MV-SCoV2 vaccine demonstrated excellent protective efficacy in strongly suppressing pneumonia caused by SARS-CoV2. Furthermore, the protective effect of the vaccine against the virus was similarly obtained against all variants of SARS-CoV2.

[0073] Figure 7 shows the results of measuring the amount of replicated virus in the lungs collected during autopsies performed on days 3 and 6 after virus inoculation. Figure 7(A) shows the results for the unvaccinated group on day 3 after virus inoculation, and Figure 7(B) shows the results for the vaccinated group on day 3 after virus inoculation. Figure 7(C) shows the results for the unvaccinated group on day 6 after virus inoculation, and Figure 7(D) shows the results for the vaccinated group on day 6 after virus inoculation. High viral replication was observed in the lungs of the unvaccinated group (Figures 7(A) and (C)). In contrast, viral replication was strongly suppressed in the vaccinated individuals (Figures 7(B) and (D)). In other words, the MV-SCoV2 vaccine strongly suppressed SARS-CoV2 replication in the lungs, and this viral replication suppression effect was obtained similarly for all mutant strains.

[0074] [Example 3] The ability of the measles virus vaccine MV-CoVS, manufactured in Example 1, to induce neutralizing antibody production after SARS-CoV-2 infection was investigated.

[0075] For SARS-CoV-2, the Wuhan strain, α-1 strain, and δ strain (TY11-927 strain) were used. The δ strain was provided by the National Institute of Infectious Diseases.

[0076] First, hamsters were given two intraperitoneal (IP) inoculations of the MV-SCoV-2 vaccine. The second viral inoculation was performed three weeks after the first inoculation (day 0 after vaccination) (day 21 after vaccination). Serum was collected from each hamster 28 days after the first vaccination, and neutralizing antibody titers against the Wuhan strain, α-1 strain, and δ strain of SARS-CoV-2 were measured. The measurement results are shown in Figure 8(A).

[0077] Subsequently, 30 days after the initial vaccination, a thermometer chip was implanted in each hamster, and then 35 days after the initial vaccination, the virus strain was inoculated in the same manner as in Example 2. Serum was then collected from the hamsters 3 or 6 days after virus inoculation (38 or 41 days after vaccination), and the neutralizing antibody titers against the Wuhan, α-1, and δ strains of SARS-CoV-2 were measured. The measurement results are shown in Figure 8(B). In Figure 8(B), "Day 3" and "Day 6" represent the serum results from hamsters 3 and 6 days after virus inoculation, respectively.

[0078] As shown in Figure 8(A), it was revealed that hamster serum after two doses of the MV-SCoV-2 vaccine showed neutralizing antibody titers ranging from 10 to 160 times against all three strains: Wuhan, α-1, and δ. Furthermore, as shown in Figure 8(B), hamsters that had been further infected with SARS-CoV-2 after two doses of the MV-SCoV-2 vaccine showed even higher neutralizing antibody titers against all three strains compared to before virus inoculation.

[0079] [Example 4] The efficacy of the measles virus vaccine MV-CoVS manufactured in Example 1 against the δ strain of SARS-CoV-2 was investigated. First, hamsters were given two intraperitoneal (IP) inoculations of the MV-SCoV2 vaccine. The second viral inoculation was performed 4 weeks after the first inoculation (day 0 after inoculation) (day 28 after inoculation). Subsequently, 30 days after the first vaccination, a thermometer chip was implanted in each hamster, and then 35 days after the first vaccination, the hamsters were inoculated with the virus strain in the same manner as in Example 2. Autopsies were then performed 3 or 6 days after viral inoculation (day 38 or 41 after inoculation) to analyze the viral load in the lungs and trachea. In addition, histopathological analysis of each tissue was also performed. As a control, hamsters that had not been vaccinated with the MV-SCoV2 vaccine were also inoculated with the virus strain in the same manner and analyzed.

[0080] Macroscopic examination of lungs collected during autopsies performed six days after viral vaccination revealed that the lungs of the unvaccinated group showed significant signs of pneumonia. In contrast, the vaccinated group showed almost no bleeding, edema, or other abnormalities observed in the unvaccinated group. In other words, the MV-SCoV2 vaccine demonstrated excellent protective efficacy in strongly suppressing pneumonia caused by the SARS-CoV2δ strain.

[0081] Figure 9 shows the results of measuring the amount of virus that proliferated in the lungs, collected during autopsies performed on the 3rd and 6th days after virus inoculation. Figure 9(A) shows the results for hamsters on the 3rd day after virus inoculation, and Figure 9(B) shows the results for hamsters on the 6th day after virus inoculation. High viral proliferation was observed in all lung lobes in the unvaccinated group, but viral proliferation was significantly suppressed in the vaccinated group compared to the unvaccinated group. In other words, the MV-SCoV2 vaccine strongly suppressed the proliferation of the SARS-CoV2δ strain in the lungs.

[0082] [Example 5] The ability of the measles virus vaccine MV-CoVS, manufactured in Example 1, to induce cellular immunity after SARS-CoV-2 infection was investigated.

[0083] The SARS-CoV-2 strains used were the Wuhan strain, α-1 strain, β strain, and γ-1 strain. First, hamsters were given two intraperitoneal (IP) inoculations of the MV-SCoV2 vaccine. The second viral inoculation was performed 3 weeks after the first inoculation (day 0 after inoculation) (day 21 after inoculation). On day 24 after the first vaccination, a thermometer chip was implanted in each hamster, and then on day 28 after the first vaccination, the virus strain was inoculated in the same manner as in Example 2. Subsequently, serum was collected from the hamsters on day 3 or 6 after viral inoculation (day 31 or 34 after inoculation), and the amount of IFNγ in the serum, one of the indicators of cellular immunity, was measured by ELISA. As a control, thermometer chips were implanted in hamsters that had not been vaccinated with the MV-SCoV2 vaccine in the same manner, and the amount of IFNγ in their serum was measured on day 3 or 6 after viral inoculation.

[0084] Figure 10 shows the results of measuring serum IFNγ levels in each hamster. In hamsters not immunized with the MV-SCoV2 vaccine, IFNγ was not detected in the serum after the attack test (vaccination), and no induction of IFNγ production was observed. In contrast, in hamsters immunized with the MV-SCoV2 vaccine, 10-30 pg / mL of IFNγ was detected in the serum regardless of which virus strain was inoculated. These results indicate that the MV-SCoV2 vaccine induces cellular immunity after SARS-CoV-2 infection, and that this ability to induce cellular immunity is exerted against all virus strains.

[0085] The measles virus induces both humoral and cellular immunity in infected individuals. Measles virus infection is thought to particularly strongly induce cellular immunity. Critical T cells of cellular immunity recognize the spike protein even in SARS-CoV-2 strains that have mutations in the amino acids of the antibody-binding site of the spike protein that evade the binding of neutralizing antibodies. Therefore, the MV-SCoV2 vaccine is expected to induce cellular immunity and exert an antiviral effect against any variant of SARS-CoV-2. In fact, in this experiment, the MV-SCoV2 vaccine showed similarly high protective efficacy against attacks by all variants. Therefore, even if various antibody-evading variants emerge in the future, the MV-SCoV2 vaccine can be expected to maintain high efficacy against SARS-CoV-2.

Claims

1. Between the N gene region and the P gene region of the measles virus genome, the gene encoding the SARS-CoV-2 coronavirus protein is inserted. The measles virus genome is an RNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by Sequence ID No.

1. Recombinant measles virus, wherein the protein is the spike protein or a partial protein thereof of SARS-CoV-2.

2. The recombinant measles virus genomic RNA according to claim 1.

3. RNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by Sequence ID No.

3.

4. DNA in which the gene encoding the SARS-CoV-2 protein is inserted into the region from base 1686 to base 1694 of the base sequence represented by Sequence ID No.

2.

5. This DNA has a gene encoding the SARS-CoV-2 protein inserted into the Fse I recognition sequence located between bases 1686 and 1694 of the base sequence represented by Sequence ID No.

1.

6. DNA consisting of the base sequence represented by Sequence ID No.

3.

7. A vector for producing a COVID-19 vaccine, comprising the DNA described in any one of claims 4 to 6.

8. A plasmid vector containing DNA consisting of the base sequence represented by Sequence ID No.

1.

9. A method for producing recombinant measles virus, comprising transfecting cells expressing RNA polymerase capable of transcribing RNA using the DNA as a template with a COVID-19 vaccine production vector containing DNA according to any one of claims 4 to 6 and expression vectors for the N protein, P protein, and L protein of measles virus, and producing recombinant measles virus within the cells.

10. A pharmaceutical composition comprising the recombinant measles virus described in claim 1 and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, to be used as a bivalent vaccine against measles and COVID-19.

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