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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
  • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
• • A—HUMAN NECESSITIES
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  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
  • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
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  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to herpes simplex virus vectors.
• the herpes simplex virus vector according to the present invention can be used as a vaccine.
• mRNA vaccines and viral vector vaccines are vaccines that administer part of the genetic information of a virus to the human body, and have a different mechanism from conventional vaccines that administer part of the protein of a virus. It is attracting attention as
• HSV-1 Herpes simplex virus type 1
• HSV-1 is one of the viruses used in viral therapy for cancer, and is a genetically modified virus that introduces artificial genetic modifications to the virus genome to improve safety and therapeutic efficacy.
• Oncolytic viruses have been developed (Patent Documents 1 to 4, Non-Patent Documents 1 to 5).
• Patent No. 4212897 Patent No. 4921669 WO2011/101912 WO2019/189643
• Viruses used for gene transfer as vaccines are: 1) able to insert large foreign genes into the viral genome; 2) the introduced foreign genes function as an epigenome without being integrated into the human genome; and 3) express one foreign gene.
• By mixing multiple types of viruses it is possible to achieve the same effect as expressing multiple types of foreign genes, and 4) the same expression effect can be achieved even after repeated administration without being inhibited by immunity against the virus itself. It is desirable to be able to demonstrate this every time.
• the present inventor has been proceeding with the clinical development of a third-generation herpesvirus G47 ⁇ for cancer treatment in which three viral genes have been modified in HSV-1.
• We have now completed the present invention by incorporating cDNA encoding the spike of the new coronavirus into this G47 ⁇ and discovering that it can be effectively used as a vaccine virus.
• the present invention provides the following.
• a pharmaceutical composition for inducing an immune response (a) ICP6 gene is deleted or inactivated (b) ⁇ 34.5 gene is deleted or inactivated (c) ⁇ 47 gene is deleted or inactivated
• Herpes simplex virus The pharmaceutical composition according to 1, wherein the vector has all the characteristics of (a) to (c).
• the pharmaceutical composition according to any one of 1 to 3 which is for producing a foreign polypeptide in non-cancerous cells.
• the pharmaceutical composition according to any one of 1 to 4 which is a subcutaneous or intramuscular injection.
• the pharmaceutical composition according to any one of 1 to 5, wherein the foreign polypeptide contains all or part of a protein derived from a pathogen.
• the pharmaceutical composition according to any one of 1 to 6, wherein the foreign polypeptide comprises all or part of the spike protein derived from SARS-CoV-2.
• the present invention provides the following. [8] Introducing a foreign polypeptide into non-cancerous cells containing a herpes simplex virus vector containing a region encoding a foreign polypeptide and having two or more characteristics selected from (a) to (c) below.
• a pharmaceutical composition for producing a peptide (a) ICP6 gene is deleted or inactivated (b) ⁇ 34.5 gene is deleted or inactivated (c) ⁇ 47 gene is deleted or inactivated (9)
• Herpes simplex virus 9 The pharmaceutical composition according to 8, wherein the vector has all the characteristics of (a) to (c).
• the pharmaceutical composition according to any one of 8 to 10 which is for producing a foreign polypeptide and inducing an immune response thereto.
• the pharmaceutical composition according to any one of 8 to 11 which is a subcutaneous or intramuscular injection.
• the pharmaceutical composition according to any one of 8 to 12 wherein the foreign polypeptide contains all or part of a protein derived from a pathogen.
• the pharmaceutical composition according to any one of 8 to 13, wherein the foreign polypeptide comprises all or part of the spike protein derived from SARS-CoV-2.
• a method for producing and inducing an immune response against a foreign polypeptide the method comprising administering to a subject.
• it For use in a method for producing a foreign polypeptide and inducing an immune response thereto, it contains a region encoding a foreign polypeptide and has two or more characteristics selected from the following (a) to (c).
• a herpes simplex virus vector containing a region encoding a foreign polypeptide and having two or more characteristics selected from the following (a) to (c) produces a foreign polypeptide and induces an immune response against it.
• Use in the manufacture of a composition for. (a) ICP6 gene is deleted or inactivated (b) ⁇ 34.5 gene is deleted or inactivated (c) ⁇ 47 gene is deleted or inactivated [16]
• Herpes simplex virus 16 The method, vector or composition comprising the same, or use according to 15, wherein the vector has all the characteristics of (a) to (c). [17] The method, vector, composition containing the same, or use according to 15 or 16, wherein the region encoding the foreign polypeptide is inserted into the IPC6 gene deletion site.
• a method of producing a foreign polypeptide in a non-cancerous cell comprising administering to a subject.
• a herpes simplex virus vector containing a region encoding a foreign polypeptide and having two or more characteristics selected from the following (a) to (c) produces the foreign polypeptide in non-cancerous cells.
• Use in the manufacture of a composition for. (a) ICP6 gene is deleted or inactivated (b) ⁇ 34.5 gene is deleted or inactivated (c) ⁇ 47 gene is deleted or inactivated
• Herpes simplex virus 23 The method, vector or composition comprising the same, or use according to 22, wherein the vector has all the characteristics of (a) to (c).
• a relatively large cDNA encoding a full-length spike protein or the like can be incorporated into the viral vector of the present invention.
• the immune response to eliminate the virus may promote vaccine efficacy.
• the virus-derived protein may act as an adjuvant.
• the viral vector vaccine provided by the present invention may not be attenuated in vaccine efficacy depending on the presence of anti-herpesvirus antibodies in the blood, and therefore repeated administration may be effective.
• the virus vector of the present invention which uses herpes simplex virus (HSV) as its backbone, has an established technology for inserting any cDNA easily and accurately, and therefore vaccines against new viruses can be produced relatively quickly.
• the viral vector vaccine provided by the present invention has established methods for mass production and purification at high titers, and is suitable for clinical application.
• A T-Cov2SSP
• B T-Cov2SSPF
• C T-Cov2STH
• D T-Cov2S1Fc
• mock T-01: 1/100 dilution.
• Residual amount of virus in muscle after 8 weeks Pathological image after 8 weeks.
• HE staining and immunostaining were performed using anti-HSV-1 antibody (green), anti-CD8 antibody (brown), and anti-CD4 antibody (red). The results of a representative 1x10 7 pfu administration are shown. Change in body weight of vaccinated mice Changes in the expression level of anti-Cov2spike protein antibodies Changes in the expression level of Cov2-spike protein.
• IHC was performed using HE staining and anti-HSV-1 antibody (green), anti-CD8 antibody (brown), and anti-CD4 antibody (red). Many CD8 and CD4 positive cells were observed, probably due to inflammation at the injection site. Almost no HSV-1 positivity was observed. Images of muscles administered with each virus (day 5, 1x10 6 pfu administered). IHC was performed using HE staining and anti-HSV-1 antibody (green), anti-CD8 antibody (brown), and anti-CD4 antibody (red). Many CD8 and CD4 positive cells were observed. Almost no HSV-1 positivity was observed. Time course of viral DNA content in muscle. The amount of virus in the muscle at the injection site remained almost the same from week 1 to week 4, with a trace number of copies.
• herpes simplex virus In this embodiment, a herpes simplex virus (HSV) containing a region encoding a foreign polypeptide is used.
• HSV herpes simplex virus
• viruses When viruses are used as tools to transfer genetic material into nucleic acids, they are sometimes referred to as viral vectors. Viruses that have been genetically manipulated are sometimes called mutant or genetically modified viruses.
• Herpes simplex virus is classified into the genus Simplevirus, family Herpesviridae, subfamily Alphaherpesvirinae. Two viruses have been isolated so far: type 1 (HSV-1) and type 2 (HSV-2). In this embodiment, HSV-1 is preferably used.
• HSV-1 is enveloped and mature particles are 100-150 nm in size. There is a tegument inside the envelope, and a capsid inside the tegument, and the viral DNA exists inside the capsid.
• the genome is double-stranded DNA.
• HSV-1 is known to have the following characteristics. Regarding this, please refer to Attachment 2. (Reference 1). 1) It is capable of infecting all types of human cells. 2) The life cycle and genome sequence of the virus have been elucidated. 3) The functions of most of the viral genes are known, and they can be genetically manipulated. 4) Since the virus genome size is large (approximately 152 kb), large size genes and multiple genes can be integrated.
• HSV-1 has the following advantages that make it suitable for clinical applications. 5) Antiviral drugs exist that suppress proliferation 6) Vaccine efficacy may not be attenuated depending on the presence of anti-HSV-1 antibodies in the blood, and repeated administration is possible 7) Susceptibility to HSV-1 Because mice and monkeys exist, preclinical evaluation of safety and efficacy can be done in animals. 8) Viral DNA is not incorporated into the genome of host cells and exists outside the chromosome. 9) Large-scale production and purification methods are not available. established
• HSV-1 genome consists of two unique sequence regions: 82% long unique region ( UL ) and 12% short unique region (Us), and terminal repeat (TR) and inverted repeat (TR) located at both ends of each region.
• IR Table 1, Todo, T. (2008). a journal and virtual library 13, 2060-2064. Since the two regions L and S can each independently take two orientations, HSV-1 genomic DNA consists of four isomers. This genome has a total of 84 genes in RL1 and RL2 on TR L , U L 1 to U L 56 on U L , R S 1 on TR S , and U S 1 to U S 12 on US. The genes are encoded in a unidirectional manner, and about half of these genes are unnecessary for virus replication. By deleting these non-essential gene parts, pathogenicity can be attenuated or genes can be introduced (Carson, J. et al. (2010). Drugs of the future 35, 183-195).
• the virus used in this embodiment is an HSV-1 mutant, it may have any one or more of the following characteristics.
• ⁇ By inactivating enzymes involved in viral DNA synthesis such as thymidine kinase (TK), ribonucleotide reductase (RR), uracil-N-glycosylase (UNG or UDG), etc.
• loss of viral replication capacity in non-cancerous cells - Deletion of virus replication ability in non-cancerous cells by deleting gene ⁇ 34.5, which encodes ICP34.5, a protein involved in the pathogenesis of HSV-1.
• genes to prevent reversion to wild type and increase safety e.g., endogenous ⁇ 34.5 gene, ⁇ 47 gene, ⁇ 0 gene (ICP0 gene), U L 41 gene (vhs gene) ), deletion or inactivation of the U L 56 gene).
• - Enhancement of immunity and prolongation of survival by expressing immune stimulating genes IL-4, IL-10, GM-CSF, IL-12, soluble B7.1, etc.
• the virus used is an HSV-1 mutant that preferably has at least one, more preferably two or more, and even more preferably all of the following characteristics selected from (a) to (c).
• the ICP6 gene is deleted or inactivated.
• the ⁇ 34.5 gene is deleted or inactivated.
• ⁇ 47 gene is deleted or inactivated.
• ICP6 a large subunit of RR, which is an important enzyme for nucleotide metabolism in non-dividing cells and viral DNA synthesis, and/or phosphorus produced during viral infection.
• ⁇ 34.5 which antagonizes the function of oxidized PKR
• the virus becomes unable to proliferate in normal cells.
• the protein encoded by the ⁇ 47 gene has the effect of escaping from host immune surveillance by inhibiting TAP and suppressing the expression of MHC class I on the host cell surface. Deleted HSV-1 is expected to stimulate immune cells more strongly by maintaining MHC class I expression in host cells.
• the deletion of ⁇ 47 simultaneously deletes the US11 late promoter whose genome overlaps with ⁇ 47, so the expression of the US11 gene is ⁇ 47 immediate-
• the expression timing is accelerated, and it has the effect of restoring the virus replication ability, which has been attenuated by ⁇ 34.5 deletion, only in tumor cells.
• deletion or inactivation of a gene refers to deletion or inactivation of the gene through deletion of all or part of the gene, substitution of some bases, modification, insertion of unnecessary sequences, etc. It means to suppress the expression.
• Gene deletion or inactivation can be performed by known methods.
• the HSV containing deletion or inactivation of the ⁇ 34.5 gene, ICP6 gene and/or ⁇ 47 gene described in (a) to (c) above includes deletion of both copies of the ⁇ 34.5 gene, and the ICP6 gene.
• Examples include G207, which has an inactivated gene, G47 ⁇ , which has two copies of the ⁇ 34.5 gene deleted, the ICP6 gene inactivated, and the ⁇ 47 gene deleted. Therefore, the virus used in this embodiment can also be produced by further modifying G207 or G47 ⁇ .
• the virus used has G47 ⁇ as its basic skeleton.
• G47 ⁇ is known as a restricted propagation type recombinant HSV-1 that expresses the E. coli lacZ gene and has the ⁇ 34.5 gene, ICP6 gene ( UL39 gene), and ⁇ 47 gene (ICP47 gene) deleted or inactivated. It is being Due to its wide therapeutic range, it is possible to safely administer high doses into the human brain. , product name: Approved as Deritact Note).
• G47 ⁇ was produced from G207 derived from a wild-type virus (HSV-1 F strain) (Todo, T. et al. (2001). Proc Natl Acad Sci USA 98: 6396-6401).
• a region encoding a foreign polypeptide described below be inserted into the site where the IPC6 gene is deleted or inactivated.
• a certain region encodes a polypeptide it refers to the case where the base sequence of that region encodes the amino acid sequence of the polypeptide, and the case where the complementary sequence of the base sequence of that region encodes the polypeptide. This includes cases where the amino acid sequence is coded for.
• a virus having the above feature (a) and having a feature in which a region encoding a foreign polypeptide is inserted into the site where the IPC6 gene is deleted or inactivated means that the virus has, for example, A sequence that has high identity with the base sequence of SEQ ID NO: 5 in the sequence listing except for Location 5349..9170, and a sequence encoding an immunogenic polypeptide or its complementary sequence in place of Location 5349..9170. It means to have an inserted sequence.
• SEQ ID NO: 5 and FIG. 14 show the LacZ-fused T-Cov2 spike (A) gene insertion site and the nucleotide sequences before and after it of the virus used in the experiment described in the Examples section.
• the region encoding the foreign polypeptide, that is, the insertion site is Location 5349..9170 in SEQ ID NO: 5, and in Figure 14, from the doublet TCA to the doublet CAT (the doublet TCA is from beginning to end It is a complementary sequence of the codon, and the double line CAT is a complementary sequence of the start codon.).
• a virus having the characteristic (b) above means, for example, that all or part of Location 88..834 is deleted, or some bases are substituted or modified in the base sequence of SEQ ID NO: 6 in the sequence listing. , which has a sequence in which an unnecessary sequence has been inserted, thereby suppressing the expression of the ⁇ 34.5 gene.
• all or part of the base sequence in IRL corresponding to Location 88..834 of SEQ ID NO: 6 is deleted, or a sequence in which some bases are replaced, modified, or an unnecessary sequence is inserted. This means that the expression of the ⁇ 34.5 gene is suppressed.
• a virus having the characteristic (b) above means, for example, that it has a sequence with high identity to the sequence obtained by removing Location 177..1128 from the base sequence of SEQ ID NO: 6 in the sequence listing.
• SEQ ID NO: 6 and FIG. 15 show the ⁇ 34.5 gene of the strain from which G47 ⁇ is derived and the base sequences before and after it (inside the TR L region).
• Location 88..834 is the coding region of the ⁇ 34.5 gene from the start codon to the stop codon, which is shown as shaded in FIG.
• the deleted site in the basic skeleton of G47 ⁇ (nucleotide numbers 576 to 1527 of GU734771.1) is Location 177..1128 in SEQ ID NO: 6, and is the underlined portion in FIG. 15.
• the above sequence of TR L and the complementary sequence around the coding region of the ⁇ 34.5 gene of IRL base numbers 124349 to 125798 of GU734771.1) are completely identical.
• a virus having the characteristic (c) above means, for example, that in the base sequence of SEQ ID NO: 7 in the sequence listing, all or part of Location 218..484 is deleted, some bases are substituted or modified, It has a sequence in which an unnecessary sequence has been inserted, thereby suppressing the expression of the ⁇ 47 gene.
• a virus having the characteristic (c) above means, for example, that it has a sequence that is highly identical to the base sequence of SEQ ID NO: 7 in the sequence listing, excluding Locations 230..540.
• SEQ ID NO: 7 and FIG. 16 show the ⁇ 47 gene of the strain from which G47 ⁇ is derived and the nucleotide sequences before and after it.
• the ⁇ 47 gene is encoded on the complementary strand side.
• Location 218..484 from the start codon to the stop codon shown as shaded in FIG. 16, is the coding region of the ⁇ 34.5 gene.
• the site sequence (nucleotide numbers 576 to 1527 of GU734771.1) that is deleted in the basic skeleton of G47 ⁇ is Location 177..1128 in the sequence of SEQ ID NO: 6, and is the underlined portion in FIG.
• PCR gene amplification method
• FA highly sensitive fluorescent antibody assay
• the herpes simplex virus vector contains a region encoding a foreign polypeptide.
• Foreign means originating from something other than herpes simplex virus.
• the polypeptide encoded by the region contained in this embodiment can be produced by expression of the region in a subject to which the virus has been administered, and the size and type of the polypeptide can be particularly determined as long as it induces an immune response thereto. Not limited. Inducing an immune response means, unless otherwise specified, that at least one of antibody production and cellular immunity is induced in the administered subject.
• the property of a polypeptide to induce antibody production or cell-mediated immunity is sometimes referred to as immunogenicity.
• the polypeptide applied to this embodiment is all or part of a protein derived from a pathogen.
• Pathogens can be viruses, bacteria, fungi, rickettsiae, parasites, prions.
• Pathogen-derived includes not only polypeptides constituting pathogens themselves such as viruses and bacteria, but also toxin proteins produced by pathogens.
• pathogens are: New coronavirus (SARS-CoV-2), influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza virus, Streptococcus pneumoniae, Clostridium diphtheria, Clostridium tetani, measles, mumps, rubella, rabies virus, yellow Staphylococcus, Clostridium difficile, Mycobacterium tuberculosis, Candida albicans, Haemophilus influenzae B (HiB), poliovirus, hepatitis B virus, human papillomavirus (L1, L2, E6, E7), human immunodeficiency virus, Helicobacter pylori, yellow Staphylococcus, pertussis toxin, poliovirus, Staphylococcus aureus, Bordetella pertussis (toxin), poliovirus VP1-4, malaria parasite.
• SARS-CoV-2 New coronavirus
• influenza A virus influenza B virus
• the polypeptide is derived from a cancer-causing pathogen.
• Infection with certain pathogens is known to cause cancer.
• liver cancer caused by hepatitis B and C viruses
• cervical cancer penile cancer
• vulvar cancer vaginal cancer
• vaginal cancer anal cancer
• oral cancer and pharynx cancer caused by human papillomavirus (HPV).
• Cancer gastric cancer caused by Helicobacter pylori; Burkitt's lymphoma, Hodgkin's lymphoma, and pharyngeal cancer caused by Epstein-Barr virus (EBV); adult T-cell leukemia-lymphoma caused by human T-cell leukemia virus type I (HTLV-1), etc.
• EBV Epstein-Barr virus
• HTLV-1 human T-cell leukemia virus type I
• the polypeptide can be a cancer antigen.
• a cancer antigen refers to a protein, etc. that is expressed more highly in cancer cells than in normal cells, or that is specifically expressed in cancer cells.
• the use of cancer-derived peptide epitopes, personalized cancer antigen parts, or cancer hotspot antigen parts to treat cancer and suppress cancer recurrence is being considered (e.g., WO2020/ 097291).
• the foreign polypeptide of this embodiment may be relatively large in size. For example, it may be 500 amino acid residues long, 1000 amino acid residues long, or 1500 amino acid residues long. Because herpes simplex virus has a large genome size, it can incorporate regions encoding large polypeptides or regions encoding multiple polypeptides.
• the site where the region encoding the foreign polypeptide is introduced is not particularly limited, and for example, the region encoding the foreign polypeptide can be introduced into a site where a non-essential gene of the virus has been deleted or inactivated.
• the virus is HSV-1
• the non-essential genes shown in Table 1 above can be deleted and a region encoding a foreign polypeptide can be introduced.
• a region encoding a foreign polypeptide is inserted into the site where the IPC6 gene has been deleted or inactivated. Expression of the introduced region can be confirmed using known techniques, as shown in Examples below.
• the G47 ⁇ -BAC system For insertion of a region encoding a foreign polypeptide into herpes simplex virus, for example, the G47 ⁇ -BAC system, T-BAC system, described in Non-Patent Documents 1 to 5, Patent Documents 1 to 4, and WO2005/103237, Or something similar to these can be used.
• the T-BAC system is a modification of the G47 ⁇ -BAC system (non-patent document 4 cited above), and it produces HSV-1 that has a higher replication ability than the G47 ⁇ -BAC product and has the same replication ability as G47 ⁇ . can.
• G47 ⁇ This is a technology for producing recombinant HSV-1 that facilitates the maintenance and amplification of the G47 ⁇ genome by inserting it into the ICP6 deletion site of the G47 ⁇ genome.
• This T-BAC contains loxP and FRT sequences, and can be mixed with a shuttle vector plasmid that also has loxP and FRT sequences to utilize the two recombinase systems of Cre/loxP and FLP/FRT. Insertion of the foreign gene carried in the shuttle vector plasmid and excision of the BAC occur, making it possible to create recombinant HSV-1 in which the foreign gene is inserted into the basic skeleton of G47 ⁇ .
• a region encoding a foreign polypeptide is introduced into a herpes simplex virus for production in non-cancerous cells.
• Non-cancerous cells refer to cells that are not cancer cells. Those skilled in the art can appropriately determine whether or not the cells are non-cancerous. Production of the target polypeptide in non-cancerous cells and whether an immune response to it is induced is determined by HSV-1-sensitive mice such as A/J mice, DBA/2 mice, and BALB/c mice. It can be evaluated using experimental animals.
• the foreign polypeptide is derived from the novel coronavirus (SARS-CoV-2). More specifically, the foreign polypeptide comprises the wild-type leader sequence from SARS-CoV-2, the full length spike, and a double proline (PP) mutation for conformational stabilization of the spike.
• SARS-CoV-2 novel coronavirus
• PP double proline
• polypeptide is, for example, any one of the following polypeptides (i) to (iii): (i) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; (ii) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added in the amino acid sequence set forth in SEQ ID NO: 1, which corresponds to positions 986 and 987 of SEQ ID NO: 1.
• the foreign polypeptide comprises the wild-type leader sequence from SARS-CoV-2, the full-length spike, a double proline (PP) mutation for conformational stabilization of the spike, and the host cell.
• PP double proline
• Such a polypeptide is, for example, any one of the following polypeptides (iv) to (vi): (iv) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2; (v) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added to the amino acid sequence set forth in SEQ ID NO: 2, and positions 683, 685, 986, and 987 of SEQ ID NO: 2 A polypeptide consisting of an amino acid sequence in which the amino acids corresponding to are A, A, P, and P in order, and is capable of inducing an immune response; (vi) an amino acid sequence having at least 80% identity with the amino acid sequence set forth in SEQ ID NO: 2, in which the amino acids corresponding to positions 683, 685, 986, and 987 of SEQ ID NO: 2 are sequentially A, A, A polypeptide consisting of the amino acid sequence P and P, and capable of inducing an immune response.
• deletion of an amino acid sequence recognized and cleaved by a certain enzyme means deletion of all or part of the amino acid sequence, substitution, modification, or unnecessary deletion of some amino acids. This refers to the insertion of a specific sequence into the enzyme that causes it to no longer be recognized or cleaved by the enzyme.
• the foreign polypeptide is derived from SARS-CoV-2 and contains a mutation-containing leader sequence, only the spike extracellular domain (ECD), a PP mutation, a furin cleavage site deletion, a C Contains a trimerization domain at the end.
• Such a polypeptide is, for example, any one of the following polypeptides (vii) to (ix): (vii) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3; (viii) In the amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 3, the region excluding the region consisting of amino acids 1 to 20 and the region consisting of amino acids 1219 to 1256 of SEQ ID NO: 1 (i.e., the region consisting of amino acids 21 to 1218) A polypeptide consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added (a portion consisting of), and is capable of inducing an immune response; (ix) Consists of an amino acid sequence having at least 80% identity with the amino acid sequence set forth in SEQ ID NO: 3, excluding the region consisting of amino acids 1 to 20 and the region consisting of amino acids 1219 to 1256 (i.e., A polypeptide that can induce an immune response in the region consist
• the foreign polypeptide comprises the wild-type leader sequence, spike subunit 1 (S1), from SARS-CoV-2, and comprises the Fc region of human IgG at the C-terminus.
• a polypeptide is, for example, any one of the following polypeptides (x) to (xii): (x) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4; (xi) a polypeptide consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added to the amino acid sequence set forth in SEQ ID NO: 4, and is capable of inducing an immune response; (xii) A polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence set forth in SEQ ID NO: 4 and capable of inducing an immune response.
• the amino acid may be a natural amino acid, or a derivative thereof, an artificial amino acid, or a non-natural amino acid.
• the amino acids of this embodiment are preferably natural amino acids.
• A is alanine
• C is cysteine
• D is aspartic acid
• E is glutamic acid
• F is phenylalanine
• G is Glycine
• I is isoleucine
• K is lysine
• L leucine
• M methionine
• N is asparagine
• P proline
• Q is glutamine
• R arginine
• S is serine
• T threonine
• U is selenocysteine.
• V represents valine
• W represents tryptophan
• Y represents tyrosine.
• any protein is not particularly limited as long as the protein consisting of the amino acid sequence has the desired function, but may be 1 to 250, 1 to 200, 1 to 150, or 1 to 100. 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 9, or 1 to 4, or by substitution with amino acids with similar properties. If so, there may be many more replacements.
• Means for preparing polynucleotides or proteins according to such amino acid sequences are well known to those skilled in the art.
• identity with respect to base sequences (sometimes referred to as nucleotide sequences) or amino acid sequences refers to “identity” in any base sequence or amino acid sequence, unless otherwise specified. It refers to the percentage of identical nucleotides or amino acids shared between two sequences when the sequences are optimally aligned. That is, identity can be calculated as (number of matched positions/total number of positions) ⁇ 100, and can be calculated using a commercially available algorithm. Such algorithms have also been incorporated into the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol. 215 (1990) 403-410.
• searches and analyzes regarding the identity of base sequences or amino acid sequences can be performed using algorithms or programs (eg, BLASTN, BLASTP, BLASTX, ClustalW) well known to those skilled in the art. Parameters when using a program can be appropriately set by those skilled in the art, and default parameters for each program may be used. Specific techniques for these analysis methods are also well known to those skilled in the art. Gene information processing software GENETIX (registered trademark) (Genetics Co., Ltd.) may be used to calculate identity. Note that if the target sequence for which % identity is to be determined has an additional sequence such as a tag sequence at the end that does not exist in the compared sequence, the additional sequence portion is not included in the calculation of % identity.
• GENETIX registered trademark
• nucleotide sequence or an amino acid sequence when referring to a nucleotide sequence or an amino acid sequence as having a high degree of identity, unless otherwise specified, in each case, at least 50%, for example 60% or more, 70% or more, preferably 70% or more. Sequence identity of 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97.5% or more, and still more preferably 99% or more.
• Polypeptides, proteins, polynucleotides, DNA, and genes used in the present invention and this embodiment can be produced by methods well known to those skilled in the art.
• composition relates to a pharmaceutical composition containing a virus used as the vector of this embodiment described above.
• the pharmaceutical composition may be used to produce a foreign polypeptide in non-cancerous cells, more preferably to induce an immune response against the foreign polypeptide in the administered subject. It can be used to induce cell-mediated immunity in a subject to produce antibodies against a foreign polypeptide.
• the pharmaceutical composition of this embodiment is suitable for the treatment of the infectious disease caused by the pathogen, preferably for the prevention of the infectious disease. It can be used as a vaccine for
• Infectious diseases include viral infections (e.g., novel coronavirus infection, influenza, acquired immunodeficiency syndrome (AIDS), adult T-cell leukemia, Ebola hemorrhagic fever, viral hepatitis, viral meningitis, yellow fever, and common cold).
• viral infections e.g., novel coronavirus infection, influenza, acquired immunodeficiency syndrome (AIDS), adult T-cell leukemia, Ebola hemorrhagic fever, viral hepatitis, viral meningitis, yellow fever, and common cold).
• the pharmaceutical composition of this embodiment can be used as a vaccine for the prevention of specific cancers.
• Applicable cancers include liver cancer, cervical cancer, penile cancer, vulvar cancer, vaginal cancer, anal cancer, oral cancer, pharyngeal cancer, stomach cancer, Burkitt lymphoma, Hodgkin lymphoma, Includes adult T-cell leukemia-lymphoma.
• the pharmaceutical composition of this embodiment can be used therapeutically, preferably as a prophylactic vaccine, for the treatment of a specific cancer. It can be used as a vaccine for the prevention of recurrence, as well as for prevention of recurrence.
• Cancers that can be expected to be applied include, but are not limited to, the following.
• treatment in relation to a disease or condition includes prevention, reduction of the risk of onset, treatment, suppression of progression, and prevention of recurrence, unless otherwise specified.
• the pharmaceutical composition of this embodiment contains an effective amount of a virus as an active ingredient.
• an effective amount is an amount that, when administered to a subject, produces the polypeptide of interest in non-cancerous cells in the subject, and an amount that induces an immune response in the subject against the foreign polypeptide. and the amount by which antibodies against the foreign polypeptide are produced in a subject.
• the specific dosage can be appropriately determined by those skilled in the art, depending on the severity of symptoms, the age, sex, body weight, sensitivity difference of the subject, administration method, administration timing, administration interval, nature of the preparation, strength of the promoter, etc. can.
• the pharmaceutical composition of this embodiment may contain, for example, about 10 1 to about 10 12 plaque forming units (pfu), preferably about 10 7 to about 10 10 pfu, more preferably about 10 8 to about 5 ⁇ 10 9 pfu. , each can be administered once or in several divided doses by injection.
• the number of administrations of the pharmaceutical composition of this embodiment can be appropriately set depending on the target patient and target disease. For example, an initial dose can be administered followed by a second dose 2 to 5 weeks later. Further, if necessary, the administration can be repeated every 1 to 8 months, from the third time onwards, as many times as necessary.
• the method of administering the pharmaceutical composition of this embodiment is not particularly limited, and examples include intravenous, intraarterial, intraventricular, intraperitoneal, intrathoracic, intraspinal, subcutaneous, intradermal, intraepidermal, intramuscular, and on mucosal surfaces. (eg, ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, urinary tract surfaces).
• the pharmaceutical composition is a vaccine for the prevention of infectious diseases, it is preferably administered by subcutaneous or intramuscular injection. If the pharmaceutical composition is a vaccine for the prevention of cancer, it may be administered by subcutaneous or intramuscular injection.
• the pharmaceutical composition when the pharmaceutical composition is for the treatment of cancer, it can be administered directly to the cancer (intratumoral administration). By intratumoral administration, high efficacy can be expected with a smaller dose.
• the pharmaceutical composition of this embodiment can be formulated by a known formulation method.
• it may contain an adjuvant or any carrier, or it may simply be diluted with a physiologically acceptable solution such as sterile saline or sterile buffered saline without the addition of an adjuvant or carrier.
• a physiologically acceptable solution such as sterile saline or sterile buffered saline without the addition of an adjuvant or carrier.
• It may also be a frozen preparation, a dried preparation, a lyophilized preparation, etc. suitable for long-term storage.
• the pharmaceutical composition of this embodiment may contain pharmaceutically acceptable additives.
• additives include buffering agents, tonicity agents, preservatives, antioxidants, stabilizers, absorption enhancers, excipients, binders, lubricants, disintegrants, colorants, flavorings, Examples include emulsifiers, surfactants, solubilizing agents, suspending agents, and the like.
• the pharmaceutical composition When in the form of a subcutaneous or intramuscular injection, the pharmaceutical composition contains, for example, in one vial (5 mL), in addition to the active ingredient, 6 mg of L-histidine, 2 mg of L-histidine hydrochloride hydrate, 10 mg of sodium chloride, It can be a formulation containing 1 mg of magnesium, 0.2 mg of sodium edetate hydrate, 375 mg of refined white sugar, 20 mg of absolute ethanol, and 805 mg of polysorbate.
• the pharmaceutical composition of this embodiment can further contain other active ingredients, or can be used in combination with a pharmaceutical composition containing other active ingredients. Furthermore, the pharmaceutical composition of this embodiment can be used in combination with other therapies.
• A is for T-Cov2SPP virus production and includes the wild-type leader sequence derived from SARS-CoV-2, the full length spike, and a double proline (PP) mutation to stabilize the spike conformation.
• B is for T-Cov2SPP virus production, and in A, a deletion site (furin cleavage site) consisting of the amino acid sequence RRAR (SEQ ID NO: 8) that is recognized and cleaved by furin, a host cell protease, is added. Including losses.
• C is for T-Cov2STH virus production, with mu-phosphatase leader sequence, spike extracellular domain (ECD) only, PP mutation, deletion of furin cleavage site, and trimerization domain at the C-terminus.
• ECD spike extracellular domain
• D is for T-Cov2S1Fc virus production and contains a wild-type leader sequence, spike subunit 1 (S1), and a human IgG Fc region at the C-terminus.
• the structure of the virus used in the Examples is shown in Figure 1-2.
• the virus has two deletions of ⁇ 34.5 present in TR L and IRL , inactivation by inserting the LacZ gene into ICP6 present in UL , and ⁇ 47 present in US and the US11 promoter region overlapping with it.
• G47 ⁇ which has a deletion in , was used as the basic skeleton.
• the T-BAC system described above was used for virus production.
• the virus contains G47 ⁇ at the deletion site of the ICP6 gene in the basic skeleton, LacZ, polyA of SV40 (SV40 pA), the reverse cytomegalovirus promoter (PCMV), and four types A to D downstream of it. It was created by inserting one selected from the following (indicated as SARS-CoV-2 Spike in Figure 1-2) and BGH polyA (BGH pA).
• virus T-01 in which only LacZ and PCMV were inserted without inserting a foreign gene into the deletion site of the IPC6 gene was used as a control.
• the LacZ-fused T-Cov2 spike (A) gene insertion site used in the experiment and the nucleotide sequences before and after it are shown in SEQ ID NO: 5 in the sequence listing and in FIG. 14. The characteristics of the array are shown below.
• the dotted line (Location 1..226 of SEQ ID NO: 5) is the upstream sequence of the UL 39 gene coding region, which is reported to include the UL 39 gene promoter, and the underline (Location 227.. 1544) is the amino acid of the UL 39 gene.
• the coding region (base number ⁇ 87656 of GU734771.1), after the wavy line (from Location 1545) is the inserted sequence, the wavy line is the sequence generated due to genetic recombination (Note 1: including the loxP sequence), and the broken line is the lacZ gene
• the underlined line is the plasmid vector-derived sequence (Note 2: 3' sequence of lacZ derived from pcDNA6-E/Uni-lacZ), and the dotted line is the plasmid vector-derived sequence (Note 2: pVP22/Uni-lacZ-derived sequence).
• TGA is the stop codon of the ICP6-lacZ fusion protein
• the double wavy line is the SV40 polyA sequence derived from pVP22/myc-His2
• the dashed-double line is pVP22/myc-His2
• the dotted line is the sequence derived from the plasmid vector (SV40 polyA derived from pVP22/myc-His2 and the surrounding sequence of BGH polyA)
• the underlined line is the amino acid coding region of the T-Cov2 spike protein (A).
• the wavy line is the complementary sequence of the adapter sequence and FRT sequence used for genetic recombination, and the part after the dotted line (from base number 88551 of GU734771.1) is the 894bp deletion part (base number of GU734771.1).
• E. coli ⁇ -galactosidase is expressed as a fusion protein with the N-terminus of ICP6.
• the parts (9 places) where this sequence differs from GU734771.1 are indicated by ⁇ .
• the ⁇ 34.5 gene of HSV-1 from which G47 ⁇ is derived and the base sequences before and after it (within the TR L region) are shown in SEQ ID NO: 6 of the sequence listing and FIG. 15.
• the region from the start codon to the stop codon shown by the shaded area is the coding region of the ⁇ 34.5 gene (Location 88..834 of SEQ ID NO: 6).
• the deleted site base numbers 576 to 1527 of GU734771.1 in the produced virus is underlined (Location 177..1128 of SEQ ID NO: 6).
• the non-coding region on the 3' side of the ⁇ 34.5 gene is translated as a meaningless amino acid sequence up to the stop codon (double underlined) that occurs 130 bp downstream.
• the above sequence of TR L (around the coding region of the ⁇ 34.5 gene; base numbers 400 to 1849 of GU734771.1) and the complementary sequence around the coding region of the ⁇ 34.5 gene of IRL (base numbers 124349 to 125798 of GU734771.1) ) is an exact match.
• the deletion site of the ⁇ 34.5 gene in IRL is base number 124671 to 125622 (complementary sequence) of GU734771.1. There is no difference between this sequence and GU734771.1.
• the ⁇ 47 gene of HSV-1 from which G47 ⁇ is derived and the nucleotide sequence before and after it are shown in SEQ ID NO: 7 of the sequence listing and FIG. 16.
• the characteristics of the array are shown below.
• the ⁇ 47 gene is encoded on the complementary strand.
• the area between the stop codon and the start codon shown in shaded areas is the coding region of the ⁇ 47 gene (Location 218..484 of SEQ ID NO: 7).
• the deleted site in the produced virus is underlined (Location 230..540 of SEQ ID NO: 7).
• the ⁇ 47 gene is deleted from the translation initiation site, and no new ORF is generated.
• the sequence of this part is a gene that expresses the fused human IL-12 gene and E. coli lacZ gene and inactivates the ⁇ 34.5 gene, U L 39 gene, and ⁇ 47 gene approved on May 31, 2019.
• Biodiversity impact assessment report for “Recombinant human herpes simplex virus type 1 (derived from strain F) (T-hIL12)” https://www.biodic.go.jp/bch/download/lmo/R1.5.31_iyaku_ap1. pdf), 3. Methods for preparing genetically modified organisms, etc., (2) Methods for transferring nucleic acids into hosts, this sequence is derived from HSV-1 strain 17, and this sequence has the base number JN555585.1.
• Example 3 In vivo experiment, Prepare 1x10 7 pfu or 1x10 6 pfu * /100 ⁇ l of each virus and administer intramuscularly to the hind foreleg of 6-week-old A/J mice (female) using a 27G needle (100 ⁇ l per mouse to both legs). It was administered in divided doses (50 ⁇ l per site). Another dose was given 4 weeks (4w) later. Serum was collected over time after 2, 4, 6, and 8 weeks (see the table below), and the expression level of anti-Cov2-spike protein antibody was measured by ELISA (Cov2-spike protein immobilized, HRP-labeled anti-mouse IgG antibody). Detection). (*Benzonaze-treated virus. During virus production, the recovered virus is enzyme-treated with Benzonase to remove DNA and RNA derived from Vero cells used as host cells.)
• Figure 4-2 shows changes over time in the amount of antibody produced by individual mice. Here, it is indicated by the OD value in ELISA. OD3 or higher is beyond the measurement limit. A7-2 died after 4w administration. Viruses A, C, and D showed a lot of variation among individuals, while viruses B were relatively stable. In C, C7-1, 5 were positive, and in D, D7-1, 4 were positive. A false positive was observed at T-01 (T7-3).
• FIG. 7 shows the typical results of 1x10 7 pfu administration, and almost the same results were obtained with 1x10 6 pfu. There was no staining with anti-HSV-1 antibody, anti-CD4 antibody, and anti-CD8 antibody.
• Body weight change Figure 8 shows the change in body weight of individual mice over time.
• A One T-Cov2SPP died due to the effects of anesthesia in both cases of administration of 1x10 6 pfu and administration of 1x10 7 pfu.
• weights were measured under anesthesia for virus administration or blood sampling. The body weight appeared to be lower when no anesthesia was given. During the observation period, there were no abnormalities in behavior or coat appearance. There were no mice that walked strangely.
• T-Cov2SPPF virus was prepared at 1x10 6 pfu or 1x10 7 pfu/100 ⁇ l and administered intramuscularly to the front thigh of the hind legs of 6-week-old A/J mice (female) (100 ⁇ l per mouse was administered to both legs). (administered in separate doses). Serum was collected over time after 6, 12, and 18 days, and the expression level of anti-Cov2-spike protein antibody was quantified by ELISA (Cov2-spike protein immobilized and detected using HRP-labeled anti-mouse IgG antibody).
• Example 4 In vivo experiment, Prepare each virus at 1x10 7 pfu/100 ⁇ l and administer intramuscularly to the front thigh of the hind limb of 6-week-old or 19-week-old A/J mice (female) (administer 100 ⁇ l per mouse divided into both legs). did. Muscle and serum were collected after 0,1,2,3 days, and the expression level of Cov2-spike protein (in blood and muscle) was quantified by ELISA (immobilized anti-spikeRBD antibody, HRP-labeled anti-spikeRBD antibody) Detection) and further quantified the amount of virus in the muscle using real-time PCR. As viruses, A:T-Cov2SPP, T-01 and mock were used. A was used because it had the highest expression level in AD.
• Cov2-spike protein expression level The results are shown in Figure 10-1. Expression is observed within muscle. It was found that the expression level was higher in young mice (6 weeks old) than in old mice (19 weeks old). The expression peaks on the first day of administration, and the expression level decreases over time.
• T-Cov2SPPF virus was prepared at 1x10 6 pfu or 1x10 7 pfu/100 ⁇ l and administered intramuscularly to the front thigh of the hind limb of 6-week-old or 17-week-old A/J mice (female) (per mouse). (100 ⁇ l was administered in divided doses to both legs). After 1 and 4 days, muscle and whole blood were collected, and the amount of virus in the blood and muscle was quantified by real-time PCR. (B was used because it produced the highest amount of antibodies)
• Example 5 In vivo experiment, Prepare each virus at 1x10 7 pfu or 1x10 6 pfu/100 ⁇ l and administer intramuscularly to the front thigh of the hind limb of 7-week-old A/J mice (female) (administer 50 ⁇ l per mouse divided between both legs). did. Muscles were collected 0, 1, and 5 days later, and HE staining and IHC were performed using anti-HSV-1 antibody (green), anti-CD8 antibody (brown), and anti-CD4 antibody (red). As viruses, A:T-Cov2SPP, T-01 and mock were used.
• T-Cov2SPPF virus was prepared at 1x10 6 pfu/100 ⁇ l and administered intramuscularly to the front thigh of the hind leg of a 7- to 8-week-old DBA/2 mouse (female) (100 ⁇ l per mouse was divided between both legs). administration), and the following measurements were performed.
• Muscles were collected after 1, 2, 3, and 4 weeks, and the amount of virus in the muscles was quantified using real-time PCR.
• Serum was collected after 1, 2, 3, and 4 weeks, and the expression level of anti-Cov2-spike protein antibody was quantified by ELISA (Cov2-spike protein immobilized and detected using HRP-labeled anti-mouse IgG antibody). .
• B:T-Cov2SPPF virus was administered intratumorally at 2x10 5 pfu/20 ⁇ l to tumor-bearing mice in which subcutaneous tumors were formed by subcutaneously transplanting the clone M3 cell line (melanoma) into DBA/2 mice. After 3 weeks, serum was collected, and the expression level of anti-Cov2-spike protein antibody was quantified using the above-mentioned ELISA.
• Both A/J mice and DBA/2 mice are mouse strains that are relatively susceptible to HSV-1, but the vaccine efficacy was similarly obtained even if the mouse strains were different ( Figure 13-1 left) .
• Intratumoral administration (IT) resulted in a blood antibody concentration that was approximately 35 times higher than that of intramuscular administration (IM) when compared over the same 3 weeks ( Figure 13). -2 right, table below).
• the present invention provides a new viral vector.
• This viral vector can be used as a vaccine, and has potential applications in important industrial fields such as pharmaceutical research and development (including regenerative medicine products), pharmaceutical manufacturing, and medical care.

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