Recombinant live attenuated herpes simplex virus type 1 vaccine

Abstract

The virus of the present disclosure is a multiple mutant virus in which two or more genes of herpes simplex virus type 1 (HSV-1) are modified, and the modification of the gene involves a loss or reduction in gene function.

Description

Live attenuated recombinant herpes simplex virus type 1 vaccine

[0001] The present invention relates to a recombinant herpes simplex virus type 1 and a recombinant live attenuated herpes simplex virus type 1 vaccine.

[0002] Human herpes simplex virus (HSV) is a pathogen that is widespread in humans. HSV, a dsDNA virus, belongs to the alphaherpesvirinae subfamily and has two serotypes, HSV-1 and HSV-2. HSV causes a variety of diseases in humans, including encephalitis, meningitis, herpes labialis, genital herpes, skin diseases, herpes cornea, and systemic neonatal herpes. As such, HSV is an extremely important virus in medical hygiene, and antiviral agents such as acyclovir and valacyclovir have actually been developed.

[0003] However, because anti-HSV agents developed to date inhibit viral DNA replication, their effectiveness against HSV, which is latent in the form of DNA and presents within the ganglia, is limited. Missing the dosing period due to drug suspension, dose reduction, or forgetting to take the drug can reduce the effectiveness of treatment, preventing fundamental disease control. To overcome this situation, it is necessary to develop a vaccine that is effective in preventing primary infection and recurrent disease, and is highly safe.

[0004] Pathogens that cause infectious diseases are broadly divided into Class I pathogens, for which conventional vaccines are sufficiently effective, and Class II pathogens, for which conventional vaccines or a history of pathogen infection do not provide sufficient protective immunity. The reason why Class II pathogens are difficult to protect against is thought to be their sophisticated immune evasion mechanisms. HSV is classified as a Class II pathogen, and this is thought to be because HSV has an immune evasion mechanism that allows it to cleverly evade the host's immune response. Previous attempts to develop HSV vaccines have focused on subunit vaccines, but the effectiveness of all of these has been insufficient (Non-Patent Documents 1 to 3).

[0005] It has been pointed out that the induction of CD4+ and CD8+ T cells is important for controlling the pathology, and the induction of tissue-resident memory T cells is also important (Non-Patent Documents 4 to 7). Also, it is known that live attenuated vaccines can potently induce humoral and cellular immunity (Non-Patent Documents 8 to 11).

[0006] The HSV-1 Us3 gene encodes the protein kinase Us3, which plays an important role in the expression of pathogenicity. Us3 mutants have been shown to have reduced pathogenicity (Non-Patent Documents 12-16 for deletions) and to be non-essential for viral growth in cultured cells (Non-Patent Documents 17-19 for deletions). Furthermore, HSV-1 Us3 has been shown to evade CTLs (cytotoxic T cells) by suppressing cell surface expression of MHC-I (major histocompatibility complex class I) (Non-Patent Document 20).

[0007] The HSV-1 gene UL13 encodes the protein kinase UL13 and plays an important role in the expression of pathogenicity. UL13 mutants have been shown to have reduced pathogenicity (Non-Patent Documents 21 and 22 for deletions), and it has been shown that UL13 is not essential for viral replication in cultured cells (Non-Patent Documents 17 and 22 for deletions). Furthermore, HSV-1 UL13 has been shown to suppress the expression of CXCL9 (chemokine ligand 9) and inhibit CTL infiltration into the infection site, thereby contributing to efficient viral replication (Non-Patent Document 22).

[0008] Kim,H.C.ら、Vaccines against Genital Herpes: Where Are We? Vaccines (Basel) 2020 8: 420Egan,K.ら、Vaccines to prevent genital herpes. Transl Res 2020 220: 138-152Truong,N.R.ら、Mechanisms of Immune Control of Mucosal HSV Infection: A Guide to Rational Vaccine Design. Front Immunol 2019 10: 373Chentoufi,A.A.ら、Towards a rational design of an asymptomatic clinical herpes vaccine: the old, the new, and the unknown. Clin Dev Immunol 2012 2012: 187585Zhu,J.ら、Persistence of HIV-1 receptor-positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition. Nat Med 2009 15: 886-92Zhu,J.ら、Virus-specific CD8+ T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation. J Exp Med 2007 204: 595-603Posavad,C.M.ら、Enrichment of herpes simplex virus type 2 (HSV-2) reactive mucosal T cells in the human female genital tract. Mucosal Immunol 2017 10: 1259-69Lauring,A.S.ら、Rationalizing the development of live attenuated virus vaccines.Nat Biotechnol 2010 28: 573-9Jang,Y.H.ら、Principles underlying rational design of live attenuated influenza vaccines. Clin Exp Vaccine Res 2012 1: 35-49Cote-Gravel,J.ら、Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphy-lococcus aureus. PLoS One 2019 14: e0227109Zhu,J.ら、Herpes Simplex Vaccines: Prospects of Live-attenuated HSV Vaccines to Combat Genital and Ocular infections. Curr Clin Microbiol Rep 2015 2: 125-36Koyanagi, N. ら、Role of herpes simplex virus 1 Us3 in viral neuroinvasiveness. Microbiol Immunol 2014 58: 31-7Morimoto, T. ら、Differences in the regulatory and functional effects of the Us3 protein kinase activities of herpes simplex virus 1 and 2. J Virol 2009 83: 11624-34Imai,T.ら、Effects of phosphorylation of herpes simplex virus 1 envelope glycoprotein B by Us3 kinase in vivo and in vitro.J Virol 2010 84: 153-62Sagou,K.ら、Regulation of the catalytic activity of herpes simplex virus 1 protein kinase Us3 by autophosphorylation and its role in pathogenesis. J Virol 2009 83: 5773-83Kato,A.ら、Phosphorylation of a herpes simplex virus 1 dUTPase by a viral protein kinase, Us3, dictates viral pathogenicity in the central nervous system but not at the periphery. J Virol 2014 88: 2775-85Gershburg, S. ら、The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature, Infectious Virions. PLos One 2015 10: e0131420Kato,A.ら、Identification of a physiological phosphorylation site of the herpes simplex virus 1-encoded protein kinase Us3 which regulates its optimal catalytic activity in vitro and influences its function in infected cells. J Virol 2008 82: 6172-89Shindo, K. ら、Characterization of a Herpes Simplex Virus 1 (HSV-1) Chimera in Which the Us3 Protein Kinase Gene Is Replaced with the HSV-2 Us3 Gene.J Virol 2015 90: 457-73Imai,T.ら、Us3 kinase encoded by herpes simplex virus 1 mediates downregulation of cell surface major histocompatibility complex class I and evasion of CD8+ T cells. PLoS One 2013 8: e72050Shibaki, T. ら、Participation of type I interferon in the decreased virulence of the UL13 gene-deleted mutant of herpes simplex virus type 1. J Interferon Cytokine Res 2001 21: 279-85Koyanagi, N. ら、Herpes simplex virus-1 evasion of CD8+ T cell accumulation contributes to viral encephalitis. J Clin Invest 2017 127: 3784-95Gordon, YJ. ら、HSV-1 thymidine kinase negative vaccine: pathogenicity, protection, and perils. Curr Eye Res 1987 6: 151-9Coen, DM. ら、Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate. Proc Natl Acad Sci U S A 1989 86: 4736-40Koshizuka, T. ら、Protection from lethal herpes simplex virus type 1 infection by vaccination with a UL41-deficient recombinant strain.Fukushima J Med Sci 2016 62: 36-42Shahnazaryan, D. ら、Herpes simplex virus 1 targets IRF7 via ICP0 to limit type I IFN induction. Sci Rep 2020 10: 22216Zhang, L. ら、HSV-1-encoded ICP0 degrades the host deubiquitinase BRCC36 to antagonize interferon antiviral response. Mol Immunol 2021 135: 28-35McMenamin, MM. ら、A gamma34.5 mutant of herpes simplex 1 causes severe inflammation in the brain. Neuroscience 1998 83: 1225-37Gobeil, PA. ら、Herpes simplex virus γ34.5 interferes with autophagosome maturation and antigen presentation in dendritic cells. mBio 2012 16: e00267-12Pyles, RB. ら、Evidence that the herpes simplex virus type 1 uracil DNA glycosylase is required for efficient viral replication and latency in the murine nervous system. J Virol 1994 68: 4963-72Jun, PY. ら、The UL4 gene of herpes simplex virus type 1 is dispensable for latency, reactivation and pathogenesis in mice. J Gen Virol 1998 79: 1603-11Fujii, H.ら、Role of the nuclease activities encoded by herpes simplex virus 1 UL12 in viral replication and neurovirulence. J Virol 2014 88: 2359-64Jacobson, JG. ら、Importance of the herpes simplex virus UL24 gene for productive ganglionic infection in mice. Virology 1998 242: 161-9Imai, T. ら、Role of the herpes simplex virus 1 Us3 kinase phosphorylation site and endocytosis motifs in the intracellular transport and neurovirulence of envelope glycoprotein B. J Virol 2011 85: 5003-15Kobayashi, R. ら、Function of the Herpes Simplex Virus 1 Small Capsid Protein VP26 Is Regulated by Phosphorylation at a Specific Site. J Virol 2015 89: 6141-7Samady, L. ら、Deletion of the virion host shutoff protein (vhs) from herpes simplex virus (HSV) relieves the viral block to dendritic cell activation: potential of vhs- HSV vectors for dendritic cell-mediated immunotherapy. J Virol 2003 77: 3768-76Paludan, SR. ら、Recognition of herpesviruses by the innate immune system. Nat Rev Immunol 2011 11: 143-54Everly, DN Jr.ら、mRNA degradation by the virion host shutoff (Vhs) protein of herpes simplex virus: genetic and biochemical evidence that Vhs is a nuclease. J Virol 2002 76: 8560-71Friedman, HM. ら、Immune evasion properties of herpes simplex virus type 1 glycoprotein gC. J Virol 1996 70:4253-60Lubinski, JM. ら、Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo. J Virol 1998 72: 8257-63Visalli, RJ. ら、Mutation of the herpes simplex virus 1 KOS UL45 gene reveals dose dependent effects on central nervous system growth. Arch Virol 2002 147: 519-32Deschamps, T. ら、Evasion of the STING DNA-Sensing Pathway by VP11 / 12 of Herpes Simplex Virus 1. J Virol 2017 91: e00535-17You, H. ら、Herpes Simplex Virus 1 Tegument Protein UL46 Inhibits TANK-Binding Kinase 1-Mediated Signaling. mBio 2019 10: e00919-19Ariza, ME. ら、Human herpesviruses-encoded dUTPases: a family of proteins that modulate dendritic cell function and innate immunity. Front Microbiol 2014 5: 504Kato, A.ら、Identification of a herpes simplex virus 1 gene encoding neurovirulence factor by chemical proteomics. Nat Commun 2020 11: 4894Berkowitz, C. ら、Herpes simplex virus type 1 (HSV-1) UL56 gene is involved in viral intraperitoneal pathogenicity to immunocompetent mice. Arch Virol 1994 134: 73-83Zheng, ZQ. ら、Herpes simplex virus protein UL56 inhibits cGAS-Mediated DNA sensing to evade antiviral immunity. Cell Insight 2022 1: 100014Lu, X. ら、The Us2 Gene Product of Herpes Simplex Virus 2 modulates NF-κB activation by targeting TAK1. Sci Rep 2017 7: 8396.Cabrera, JR. ら、Secreted herpes simplex virus-2 glycoprotein G modifies NGF-TrkA signaling to attract free nerve endings to the site of infection. PLoS Pathog 2015 11: e1004571.Martinez-Martin, N. ら、Herpes simplex virus particles interact with chemokines and enhance cell migration. J Gen Virol 2016 97: 3007-16Zhou, G.ら、Glycoprotein D or J delivered in trans blocks apoptosis in SK-N-SH cells induced by a herpes simplex virus 1 mutant lacking intact genes expressing both glycoproteins. J Virol 2000 74: 11782-91Jerome, KR. ら、HSV and glycoprotein J inhibit caspase activation and apoptosis induced by granzyme B or Fas. J Immunol 2001 167: 3928-35Kato, A. ら、Roles of Us8A and Its Phosphorylation Mediated by Us3 in Herpes Simplex Virus 1 Pathogenesis. J Virol 2016 90: 5622-35Polcicova, K. ら、Herpes keratitis in the absence of anterograde transport of virus from sensory ganglia to the cornea. Proc Natl Acad Sci U S A 2005 102: 11462-7Jerome, KR. ら、Herpes simplex virus inhibits apoptosis through the action of two genes, Us5 and Us3. J Virol 1999 73: 8950-7Peters, GA. ら、Inhibition of PACT-mediated activation of PKR by the herpes simplex virus type 1 Us11 protein. J Virol 2002 76: 11054-64Schust, DJ. ら、Herpes simplex virus blocks intracellular transport of HLA-G in placentally derived human cells.J Immunol 1996 157: 3375-80Galocha, B. et al., The active site of ICP47, a herpes simplex virus-encoded inhibitor of the major histocompatibility complex (MHC)-encoded peptide transporter associated with antigen processing (TAP), maps to the NH2-terminal 35 residues. J Exp Med 1997 185: 1565-72Jugovic, P. et al., Inhibition of major histocompatibility complex class I antigen presentation in pig and primate cells by herpes simplex virus type 1 and 2 ICP47. J Virol 1998 72: 5076-84.

[0009] As mentioned above, antiviral agents such as acyclovir are used to treat HSV. However, these antiviral agents cannot completely eliminate the virus, and there is a risk of the virus reactivating due to factors such as decreased adherence. Therefore, there is a need for the development of a preventive vaccine that protects against HSV infection itself or a therapeutic vaccine that alleviates recurrent symptoms. However, there is currently no effective vaccine, and there is a high unmet medical need.

[0010] The present invention aims to provide a recombinant live attenuated vaccine that can be used to prevent and treat HSV-1 infections, which has a high level of safety by introducing mutations into the viral gene responsible for pathogenicity while enhancing the immune-inducing ability of HSV-1 through mutation introduction.

[0011] Based on the hypothesis that introducing mutations into HSV-1 virulence factors and immune evasion factors would enable the construction of a recombinant attenuated live vaccine with high safety and enhanced immune induction potential, the inventors created various mutant viruses and evaluated them in animal experiments. As a result, they found that a Us3 / UL13 double mutant virus, in which a mutation (inactivation) was introduced into the Us3 gene of herpes simplex virus type 1 and an additional mutation (inactivation) was introduced into the UL13 gene, exhibited superior primary infection prophylactic efficacy (viral shedding suppression or onset prevention) in a mouse primary genital herpes infection model compared to a group vaccinated with the conventional vaccine prototype attenuated live virus ΔTk (thymidine kinase deficient) and a group vaccinated with a Us3 single mutant attenuated live virus, both of which share the same origin. ΔTk was chosen as a comparison subject because its safety and efficacy have been suggested in Non-Patent Documents 23-25.

[0012] Based on the above findings, the present inventors have discovered that by introducing mutations (inactivation) into two or more genes of herpes simplex virus type 1, it is possible to obtain a recombinant attenuated live vaccine with improved safety by increasing immune induction ability and reducing pathogenicity, thereby completing the present invention.

[0013] That is, a virus according to one embodiment of the present invention is a multiple mutant virus in which two or more genes of herpes simplex virus type 1 (HSV-1) have been modified, and the modifications of the genes result in a loss or reduction of gene function.

[0014] According to one aspect of the present invention, when immunity is induced by a recombinant live attenuated herpes simplex virus type 1 and a vaccine containing the same, a superior preventive effect against primary infection can be exhibited compared to when immunity is induced by a conventional live attenuated virus ΔTk of the same origin, and therefore a high preventive effect against HSV infection can be expected.

[0015] FIG. 1 is a schematic diagram of a mouse primary infection protection test in Example 4. FIG. 2 is a diagram showing the virus excretion inhibitory effect in a mouse primary infection protection test in Example 4. FIG. 3 is a diagram showing the pathology score in a mouse primary infection protection test in Example 4. FIG. 4 is a schematic diagram of a mouse intracerebral infection test in Example 5. FIG. 5 is a diagram showing the survival rate in a mouse intracerebral infection test in Example 5. FIG. 6 is a schematic diagram of a mouse nasal infection test in Example 6. FIG. 7 is a diagram showing the survival rate in a mouse nasal infection test in Example 6.

[0016] In this specification, "A to B" means A or more and B or less unless otherwise specified.

[0017] [Multiple Mutant Virus] A multiple mutant virus according to one embodiment of the present invention (hereinafter, sometimes referred to as a "genetically recombinant live attenuated herpes simplex virus type 1") is a virus in which two or more genes of herpes simplex virus type 1 (HSV-1) have been modified.

[0018] As used herein, gene modification refers to the loss or reduction of gene function. Examples of loss or reduction of gene function include loss or reduction of gene function due to the lack of expression of a protein encoded by a target gene caused by gene deletion, start codon substitution, or introduction of a stop codon immediately downstream of the start codon, and loss or reduction of gene function due to amino acid substitution caused by a change in the base sequence of the gene (e.g., deletion, substitution, or insertion of one to several tens of bases in the coding region).

[0019] The HSV-1 may be a laboratory strain or a clinical isolate.

[0020] In terms of high immune induction ability and high safety, it is preferable that the genetically modified live attenuated herpes simplex virus type 1 has a modified gene associated with immune escape or pathogenicity expression. Examples of genes associated with immune escape or pathogenicity expression include RL1 (Non-Patent Documents 26 and 27), RL2 (Non-Patent Documents 28 and 29), UL2 (Non-Patent Document 30), UL3, UL4 (Non-Patent Document 31), UL12 (Non-Patent Document 32), UL12.5, UL13 (Non-Patent Document 21), UL24 (Non-Patent Document 33), UL27 (point mutants: Non-Patent Documents 17 and 34), UL35 (deleted forms and point mutants: Non-Patent Document 35), UL41 (deleted forms: Non-Patent Document 36, function review: Non-Patent Document 37, point mutants: Non-Patent Document 38), UL43, UL44 ( Non-Patent Documents 39, 40), UL45 (Non-Patent Document 41), UL46 (Non-Patent Documents 42, 43), UL50 (non-patent document 44 reporting the function, non-patent document 45 of point mutants), UL56 (non-patent documents 46, 47), Us2 (non-patent document 48), Us3 (non-patent document 15 of deletion bodies, non-patent documents 16-20 of point mutants), Us4 (non-patent documents 49, 50), Us5 (non-patent documents 51, 52), Us8A (non-patent document 53), Us9 (non-patent document 54), Us11 (non-patent documents 55, 56), Us12 (non-patent documents 57-59), etc. It is more preferable that at least one gene of Us3 and UL13 is modified, and it is even more preferable that at least Us3 and UL13 are modified.

[0021] Us3 is a gene encoding a protein kinase. An example of the amino acid sequence of the protein encoded by Us3 is the amino acid sequence shown in SEQ ID NO: 1 (NCBI Reference Sequence: GU734771.1).

[0022] UL13 is a gene encoding a protein kinase. An example of the amino acid sequence of the protein encoded by UL13 is the amino acid sequence shown in SEQ ID NO: 2 (NCBI Reference Sequence: GU734771.1).

[0023] In terms of high immune induction ability and high safety, the modification of Us3 is preferably a modification of the lysine residue at position 220 (K220) in the amino acid sequence of SEQ ID NO: 1. Furthermore, the modification of UL13 is preferably a modification of the lysine residue at position 176 (K176) in the amino acid sequence of SEQ ID NO: 2. It is more preferable that the recombinant live attenuated herpes simplex virus type 1 has modifications at K220 in SEQ ID NO: 1 and K176 in SEQ ID NO: 2.

[0024] As used herein, "modification of an amino acid residue" refers to deletion or substitution of an amino acid residue. An example of a modification of K220 in SEQ ID NO: 1 is substitution of a lysine residue with a methionine residue. An example of a modification of K176 in SEQ ID NO: 2 is substitution of a lysine residue with a methionine residue.

[0025] Genetically modified live attenuated herpes simplex virus type 1 can be obtained by known gene modification methods, such as a gene modification method utilizing homologous recombination in cultured cells, a gene modification method using cosmid, or a gene modification method using a BAC (bacterial artificial chromosome) system.

[0026] [HSV-1 Vaccine] An HSV-1 vaccine according to one embodiment of the present invention is a vaccine comprising the recombinant live attenuated herpes simplex virus type 1 as an antigen. This HSV-1 vaccine can be used for treating or preventing HSV-1 infection. As used herein, "treatment or prevention of HSV-1 infection" includes alleviating or preventing the worsening of one or more symptoms associated with HSV-1 infection, reducing the occurrence of symptoms after HSV-1 infection, preventing, delaying, or stopping HSV-1 infection of cells in vivo, reducing the number of HSV-1 in vivo, etc.

[0027] Examples of conditions associated with HSV-1 infection include herpes labialis, herpes cornea, herpes genitalis, and systemic neonatal herpes, as well as stomatitis, skin diseases, encephalitis, meningitis, and myelitis caused by HSV-1, and neurodegenerative diseases that may be caused by HSV-1.

[0028] The HSV-1 vaccine according to one embodiment of the present invention is superior in safety or efficacy to a vaccine containing, as an antigen, HSV-1 in which only Us3 has been modified.

[0029] The recombinant live attenuated herpes simplex virus type 1 contained in the HSV-1 vaccine according to one embodiment of the present invention may be one type or two or more types.

[0030] The HSV-1 vaccine according to one embodiment of the present invention may contain a pharmaceutically acceptable carrier depending on the purpose, use, route of administration, etc. The carrier may be a carrier that is commonly used in the production of vaccines. Examples of such carriers include adjuvants, solvents, thickeners, binders, colorants, stabilizers, pH adjusters, diluents, excipients, buffers, isotonicity agents, soothing agents, preservatives, antioxidants, etc.

[0031] Examples of dosage forms of the HSV-1 vaccine according to one embodiment of the present invention include powder formulations such as freeze-dried and vacuum-dried formulations, liquid formulations, capsules, injections, sprays, aerosols, suppositories, nasal drops, etc. In terms of track record as a vaccine dosage form and the mechanism by which it exerts its effect, preferred dosage forms of the HSV-1 vaccine according to one embodiment of the present invention are injections, sprays, and nasal drops.

[0032] The amount of the recombinant live attenuated herpes simplex virus type 1 contained in the HSV-1 vaccine according to one embodiment of the present invention can be determined appropriately depending on the type, age, weight, condition, treatment period, etc. of the subject to be administered.

[0033] The dose of the recombinant live attenuated herpes simplex virus type 1 contained in the HSV-1 vaccine according to one embodiment of the present invention is 10 ３ pfu ~ 10 ８ pfu, preferably 10 ４ pfu ~ 10 ７ More preferably, 10 pfu ５ pfu ~ 10 ６ More preferably, it is pfu.

[0034] (Administration Method / Administration Route of HSV-1 Vaccine) The administration method (administration route) of the HSV-1 vaccine according to one embodiment of the present invention can be appropriately determined depending on the age, condition, treatment period, etc. of the recipient. Specifically, either oral or parenteral administration may be used, with parenteral administration being preferred. Examples of administration routes for parenteral administration include intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, nasal administration, transdermal administration, rectal administration, respiratory administration, vaginal administration, and ophthalmic administration. In terms of the track record as a vaccine dosage form and the mechanism by which it exerts its effect, the preferred administration route for the HSV-1 vaccine according to one embodiment of the present invention is nasal administration. A vaccine for nasal administration is administered via the nasal mucosa, for example, by spraying, applying, or dripping the vaccine onto the nasal mucosa.

[0035] The subjects to which the HSV-1 vaccine according to one embodiment of the present invention is administered are those infected with the HSV-1 virus or at risk of infection with the HSV-1 virus. Examples of subjects to which the vaccine is administered include mammals, birds, reptiles, amphibians, etc., with mammals being preferred. Mammals include humans and non-human animals. Non-human animals include livestock such as cows, horses, pigs, and sheep, as well as pets or laboratory animals such as dogs, cats, rats, mice, hamsters, monkeys, and rabbits. Humans are preferred. Birds include poultry such as chickens, ducks, and geese.

[0036] The number of doses and timing of administration of the HSV-1 vaccine according to one embodiment of the present invention can be appropriately determined depending on the type, age, weight, condition, etc. of the subject to be administered.

[0037] Another aspect of the present invention is a method for treating or preventing HSV-1 infection, which comprises the step of administering an HSV-1 vaccine according to one aspect of the present invention to a subject.

[0038] To date, no vaccine has been developed that is both safe and effective, and past findings suggest the importance of live attenuated vaccines that can potently induce humoral and cellular immunity. This invention is innovative in that it has made it possible to develop a live attenuated vaccine that is highly safe and effective by using genetic recombination technology.

[0039] [Use as a Vaccine Vector] A recombinant live attenuated herpes simplex virus type 1 according to one embodiment of the present invention can also be used as a vaccine vector against pathogens other than HSV-1, such as influenza virus and SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2).

[0040] [Summary] The virus according to aspect 1 of the present invention is a multiple mutant virus in which two or more genes of herpes simplex virus type 1 (HSV-1) have been modified, and the modifications of the genes result in a deletion or reduction of gene function.

[0041] The virus according to aspect 2 of the present invention may be the virus according to aspect 1 of the present invention, wherein the modifications of the two or more genes are modifications of two or more genes selected from genes associated with immunogenicity or immune escape.

[0042] A virus according to Aspect 3 of the present invention may be the virus according to Aspect 2 of the present invention, wherein the genes associated with immunogenicity or immune evasion include Us3 and UL13.

[0043] The virus according to Aspect 4 of the present invention may be any of Aspects 1 to 3 of the present invention, in which the lysine residue at position 220 (K220) in the amino acid sequence of SEQ ID NO: 1 is modified, and the lysine residue at position 176 (K176) in the amino acid sequence of SEQ ID NO: 2 is modified.

[0044] A vaccine according to Aspect 5 of the present invention is a herpes simplex virus type 1 (HSV-1) vaccine comprising the virus according to any one of Aspects 1 to 4 of the present invention as an antigen.

[0045] The vaccine according to Aspect 6 of the present invention is superior in safety or efficacy to the vaccine according to Aspect 5 of the present invention, which contains, as an antigen, HSV-1 in which only Us3 has been modified.

[0046] The vaccine according to Aspect 7 of the present invention may be for nasal administration in Aspect 5 or 6 of the present invention.

[0047] A vaccine according to aspect 8 of the present invention is a vaccine according to any one of aspects 5 to 7 of the present invention, wherein the dose of the virus is 10 ５ pfu ~ 10 ６ It may also be pfu.

[0048] A use according to Aspect 9 of the present invention is the use of the virus according to any one of Aspects 1 to 4 of the present invention as a vaccine vector against a pathogen other than herpes simplex virus type 1 (HSV-1).

[0049] The use according to aspect 10 of the present invention may be the use according to aspect 9 of the present invention, wherein the pathogen is influenza virus or SARS-CoV-2.

[0050] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents described in this specification are incorporated by reference.

[0051] Example 1 Genetic Recombination in Escherichia coli A recombinant cassette carrying a kanamycin resistance gene was introduced by electroporation into Escherichia coli (carrying a chloramphenicol resistance gene) carrying an HSV-BACmid containing the genetic sequence of the HSV-1 wild-type virus F strain. The cells were cultured in 1 mL of LB medium at 32°C for 30 minutes with shaking at 200 rpm, and the cells were plated on LB agar medium containing chloramphenicol and kanamycin and cultured overnight at 32°C.

[0052] PCR was performed on the grown clones to select clones into which the target sequence had been introduced. The selected clones were cultured overnight in 1 mL of LB medium at 32°C and 200 rpm with shaking. 100 μL of the culture solution was added to 2 mL of chloramphenicol-containing LB medium and cultured with shaking at 32°C and 200 rpm for 2.5 hours. 400 μL of 10% arabinose was added and cultured with shaking at 32°C and 200 rpm for 1 hour. The culture was cultured in a hot bath at 42°C and 50 rpm for 30 minutes, and then cultured with shaking at 32°C and 200 rpm for 1.5 hours. 10 μL of the culture solution was diluted with 1 mL of ultrapure water, and 10 μL of the diluted solution was diluted with 1 mL of ultrapure water. 150 μL was then spread on chloramphenicol-containing LB agar medium and cultured overnight at 32°C. The grown clones were streaked onto chloramphenicol-containing LB agar medium and chloramphenicol and kanamycin-containing LB agar medium, and cultured overnight at 32° C. Clones that grew only on the chloramphenicol-containing LB medium were selected by PCR.

[0053] [Example 2] BACmid Purification The selected clone was inoculated into 200 mL of chloramphenicol-containing LB medium and cultured overnight at 32°C and 200 rpm. The culture was centrifuged at 7,000 rpm at 4°C for 5 minutes, and the supernatant was removed. The pellet was suspended in 5 mL of Sol I (0.9% D-glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA), and 10 mL of Sol II (0.1% SDS, 0.2 N NaOH) was added. After mixing by inversion, the mixture was incubated at room temperature for 10 minutes. 7.5 mL of Sol III (29.43% potassium acetate, 11.5% glacial acetic acid) was added and mixed by inversion. The mixture was centrifuged at 15,000 rpm at 4°C for 20 minutes, and the supernatant was filtered. 0.6 volumes of 2-propanol was added, and the mixture was mixed by inversion. The mixture was centrifuged at 5,000 rpm at 4°C for 5 minutes, and the supernatant was removed. The pellet was suspended in 4 mL of TE (Tris-EDTA), and 5.4 g of cesium chloride was added. 180 μL of a 10 mg / mL ethidium bromide solution was added, and the mixture was ultracentrifuged overnight at 70,000 rpm at 20°C.

[0054] The bands formed by ultracentrifugation were collected using a 1 mL syringe with an 18G needle, and an equal volume of 99.5% ethanol was added. After mixing by inversion, the mixture was centrifuged at 15,000 rpm at 4°C for 2 minutes, and the supernatant was removed. The pellet was suspended in 400 μL of TE, and 500 μL of an equal volume phenol:chloroform mixture was added. After mixing by inversion, the mixture was centrifuged at 15,000 rpm at 4°C for 3 minutes. The aqueous layer was transferred to a new tube and the same process was repeated. An equal volume of diethyl ether was added, mixed by inversion, and centrifuged at 15,000 rpm at 4°C for 3 minutes. The ether layer was removed, and the same process was repeated three times. The ether layer was removed, and 40 μL of 3 M sodium acetate (pH 5.2) and 1 mL of 99.5% ethanol were added and mixed by inversion. The mixture was centrifuged at 15,000 rpm at 4°C for 10 minutes, and the supernatant was removed. 1 mL of 70% ethanol was added, mixed by inversion, and centrifuged at 15,000 rpm at 4°C for 2 minutes, and the supernatant was removed. The mixture was centrifuged again under the same conditions, and the supernatant was removed.

[0055] [Example 3] Virus reconstitution in rabbit skin cells. The DNA solution was suspended in 250 μL of sterile ultrapure water, and 35 μL of 2.2 M calcium chloride solution was added and mixed by tapping. 250 μL of 2x HBS (1.6% sodium chloride, 0.074% potassium chloride, 0.027% sodium hydrogen phosphate dihydrate, 0.2% dextrose, 1% HEPES (pH 7.05)) was added and mixed by tapping (DNA solution). After 10 minutes, the medium was removed from the rabbit skin cells cultured in a T-25 flask and washed with 5 mL of PBS. 5 mL of 0.04% DEAE-dextran was added, and the cells were incubated at 37°C and 5% CO ２ The cells were incubated for 10 minutes under reduced pressure. DEAE-dextran was removed, and 5 mL of TEN (0.2 M sodium chloride, 20 mM Tris-HCl (pH 8.0), 2 mM EDTA) was added and removed. 5 mL of 5% FCS-containing DMEM medium was added, and the DNA solution was added. After 4 hours, the medium was removed, and 5 mL of 5% FCS-containing DMEM medium was added and removed. 5 mL of 5% FCS-containing DMEM medium was added, and the cells were incubated at 37°C, 5% CO ２ The medium was removed, 5 mL of 199V medium was added, and the mixture was incubated at 37°C, 5% CO ２ Incubated under

[0056] The presence or absence of plaques was observed as an indicator of virus production. Once virus production was confirmed, the virus was infected into Vero cells at the appropriate time and expanded. The virus titer was measured by plaque assay. A "genetically modified live attenuated HSV-1 vaccine" (hereinafter referred to as "HSV-1 vaccine") was produced using the obtained virus as an antigen and used in experiments.

[0057] [Example 4] Primary infection protection test in mice. 3 pfu of HSV-1 vaccine was administered intranasally. Three weeks later, 8.35 mg / mL of Depo-Gestin was administered subcutaneously. One week later, 1 × 10 wild-type HSV-1 F strain was administered subcutaneously. ７ The mice were challenged with pfu intravaginally. The vaginal lesion score was assessed for two weeks, and vaginal washes were collected and the virus titer was measured by plaque assay. The vaginal lesion score was scored as follows: 0 (no symptoms), 1 (slight erythema and edema of the genitals), 2 (hair loss, erythema, and edema of the genitals), 3 (severe hair loss, erythema, and edema of the genitals), 4 (hind leg paralysis), and 5 (death).

[0058] A schematic diagram of the primary mouse infection protection test is shown in Figure 1. The HSV-1 vaccine containing Us3KM / UL13KM as an antigen suppressed virus shedding more than the group vaccinated with ΔTk, a conventional vaccine prototype live-attenuated virus (Figure 2). Here, "Us3KM" refers to a virus in which the 220th lysine of Us3 was converted to methionine, "UL13KM" refers to a virus in which the 176th lysine of UL13 was converted to methionine, and "Us3KM / UL13KM" refers to a double mutant virus of Us3KM and UL13KM.

[0059] Vaginal lesion symptoms were also significantly reduced in the Us3KM / UL13KM-vaccinated group compared with the ΔTk-vaccinated group, demonstrating a favorable protective effect against the onset of the disease (FIG. 3).

[0060] [Example 5] Mouse intracerebral infection test 1 × 10 ５ The mice were intracerebrally inoculated with pfu of HSV-1 vaccine, and the survival rate was monitored for two weeks.

[0061] A schematic diagram of the mouse intracerebral infection test is shown in Figure 4. The survival rate after administration of the HSV-1 vaccine containing Us3KM / UL13KM as the antigen was higher than that of the wild-type virus F strain and the Us3KM single mutant virus inoculation groups, confirming that the pathogenicity was attenuated (Figure 5).

[0062] [Example 6] Mouse nasal infection test 1 × 10 ６ The mice were intranasally inoculated with pfu of HSV-1 vaccine, and the survival rate was observed for two weeks.

[0063] A schematic diagram of the mouse nasal infection test is shown in Figure 6. The survival rate after administration of the HSV-1 vaccine using Us3KM / UL13KM as the antigen was higher than that of the group inoculated with the wild-type virus F strain, confirming that the pathogenicity was attenuated (Figure 7).

[0064] The HSV-1 vaccine of the present invention, in which mutations have been introduced into two genes of herpes simplex virus type 1, Us3 and UL13, is expected to be industrially useful as a vaccine that is both highly safe and effective.

Claims

1. A vaccine for herpes simplex virus type 1 (HSV-1) comprising a multiple mutant virus as an antigen, wherein the multiple mutant virus has two or more genes of HSV-1 modified, The aforementioned gene modification results in a deficiency or reduction of gene function. A vaccine comprising the two or more genes mentioned above, including Us3 and UL13.

2. The aforementioned multimutant virus is The lysine residue at position 220 (K220) in the amino acid sequence of Sequence ID No. 1 has been modified. The vaccine according to claim 1, wherein the 176th lysine residue (K176) in the amino acid sequence of SEQ ID NO: 2 is modified.

3. The vaccine according to claim 1 or 2, which has superior safety or efficacy compared to a vaccine containing only US3 modified HSV-1 as an antigen.

4. The vaccine according to claim 1 or 2, for intranasal administration.

5. The dose of the aforementioned virus is 10 ５ pfu~10 ６ The vaccine according to claim 1 or 2, wherein it is a pneumococcal fungicide (PFU).

6. Two or more genes of herpes simplex virus type 1 (HSV-1) are modified, The aforementioned gene modification results in a deficiency or reduction of gene function. The use of a multimutant virus containing the two or more genes Us3 and UL13 as a vaccine vector against pathogens other than herpes simplex virus type 1 (HSV-1).

7. The use as a vaccine vector according to claim 6, wherein the pathogen is influenza virus or SARS-CoV-2.