Recombinant live attenuated herpes simplex virus type 2 vaccine

Abstract

A virus according to the present disclosure is a multiple mutant virus in which not fewer than 2 genes of herpes simplex virus type 2 (HSV-2) are modified, and the modification of said genes causes a loss or reduction of gene function.

Description

Live attenuated recombinant herpes simplex virus type 2 vaccine

[0001] The present invention relates to a recombinant live attenuated herpes simplex virus type 2 and a recombinant live attenuated herpes simplex virus type 2 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 12-15), and it has been shown that Us3 is not essential for viral growth in cultured cells (Non-Patent Documents 16 and 17). 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 18).

[0007] The HSV-1 gene UL41 encodes the virion host shut-off (VHS) RNase and plays an important role in pathogenicity. UL41 mutants have been shown to have reduced pathogenicity (Non-Patent Documents 19-23), and it has been shown to be dispensable for the growth of cultured cells (Non-Patent Documents 24-27). Furthermore, HSV-1 UL41 has been shown to evade host immunity by inhibiting protein kinase R activation, dendritic cell activation, cGAS / STING pathway activation, and suppressing inflammatory cytokine production (Non-Patent Documents 28-32).

[0008] The HSV-1 gene UL35 encodes the capsid protein VP26, which plays an important role in virulence expression. VP26 mutants have been shown to have reduced virulence (Non-Patent Document 33), and it has been shown that VP26 is not essential for growth in cultured cells (Non-Patent Document 33).

[0009] The HSV-1 gene UL50 encodes vdUTPase, a homolog of the host nucleic acid metabolic enzyme dUTPase, and plays an important role in the expression of pathogenicity. It has been shown that vdUTPase mutants have reduced pathogenicity (Non-Patent Document 34) and that vdUTPase is not essential for the growth of cultured cells (Non-Patent Document 15).

[0010] The HSV-1 gene UL27 encodes the envelope glycoprotein B (gB) and plays an important role in pathogenicity. gB mutants have been shown to have reduced pathogenicity (Non-Patent Document 35). gB has been shown to be essential for the growth of cultured cells, and gB-deficient cells lack the ability to proliferate, whereas gB point mutants can achieve a certain level of proliferation (Non-Patent Documents 36-39 for deletions, and Non-Patent Documents 13 and 35 for point mutants). Furthermore, HSV-1 gB has been shown to inhibit NKT cell function by suppressing the cell surface expression of CD1d (Non-Patent Document 40).

[0011] The functions of HSV-1 and HSV-2 are not necessarily conserved. For example, it has been shown that the function of Us3 differs between HSV-1 and HSV-2 (Non-Patent Document 12).

[0012] 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: 125-36Morimoto, 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. 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PLoS One 2013 8: e72050Leib, DA. ら、Interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo. J Exp Med 1999 189: 663-72Strelow, LI.ら、The virion host shutoff function of herpes simplex virus type 1 plays a role in corneal invasion and functions independently of the cell cycle. Virology 1997 231: 28-34Strelow, LI. ら、Analysis of conserved domains of UL41 of herpes simplex virus type 1 in virion host shutoff and pathogenesis. J Virol 1996 70: 5665-7Strelow, LI. ら、Role of the virion host shutoff (vhs) of herpes simplex virus type 1 in latency and pathogenesis. J Virol 1995 69: 6779-86Strand, SS. ら、Role of the VP16-binding domain of vhs in viral growth, host shutoff activity, and pathogenesis. J Virol 2004 78: 13562-72Sciortino, MT. ら、The virion host shutoff RNase plays a key role in blocking the activation of protein kinase R in cells infected with herpes simplex virus 1. J Virol 2013 87: 3271-6Cotter, CR. ら、The virion host shut-off (vhs) protein blocks a TLR-independent pathway of herpes simplex virus type 1 recognition in human and mouse dendritic cells. PLoS One 2010 5: e8684Cotter, CR.ら、The virion host shutoff protein of herpes simplex virus 1 blocks the replication-independent activation of NF-κB in dendritic cells in the absence of type I interferon signaling. J Virol 2011 85: 12662-72Su, C. ら、Herpes Simplex Virus 1 Abrogates the cGAS / STING-Mediated Cytosolic DNA-Sensing Pathway via Its Virion Host shutoff Protein, UL41. J Virol 2017 91: e02414-6Suzutani, T. ら、The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection. J Gen Virol 2000 81: 1763-71Sciotino, MT. ら、The virion host shutoff RNase plays a key role in blocking the activation of protein kinase R in cells infected with herpes simplex virus 1. J Virol 2013 87: 3271-6Cotter, CR. ら、The virion host shut-off (vhs) protein blocks a TLR-independent pathway of herpes simplex virus type 1 recognition in human and mouse dendritic cells. PLoS One 2010 5: e8684Cotter, CR.ら、The virion host shutoff protein of herpes simplex virus 1 blocks the replication-independent activation of NF-κB in dendritic cells in the absence of type I interferon signaling. J Virol 2011 85: 12662-72Su, C. ら、Herpes Simplex Virus 1 Abrogates the cGAS / STING-Mediated Cytosolic DNA-Sensing Pathway via Its Virion Host Shutoff Protein, UL41. J Virol 2017 91: e02414-6Kobayashi, 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: 6141-7Kato, A. ら、Herpes simplex virus 1 protein kinase Us3 phosphorylates viral dUTPase and regulates its catalytic activity in infected cells. J Virol 2014 88: 655-66Imai, 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: 5003-15Manservigi, R.ら、Cell fusion induced by herpes simplex virus is promoted and suppressed by different viral glycoproteins. Proc Natl Acad Sci U S A 1997 74: 3913-7Schaffer, PA. ら、Collaborative complementation study of temperature-sensitive mutants of herpes simplex virus types 1 and 2. J Virol 1978 27: 490-504Little, SP. ら、A virion-associated glycoprotein essential for infectivity of herpes simplex virus type 1. Virology 1981 115: 149-60Cai, WH. ら、Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion. J Virol 1988 62: 2596-604Rao, P. ら、Herpes simplex virus 1 glycoprotein B and US3 collaborate to inhibit CD1d antigen presentation and NKT cell function. J Virol 2011 85: 8093-104Da Costa, XJ. ら、Immunization against genital herpes with a vaccine virus that has defects in productive and latent infection. Proc Natl Acad Sci U S A 1999 96: 6994-8Shin, H. ら、A vaccine strategy that protects against genital herpes by establishing local memory T cells.Nature 2012 491: 463-7Sato, A. ら、Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease. J Virol 88: 13699-708Shahnazaryan, 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: 2359-64Koyanagi, N. ら、Herpes simplex virus-1 evasion of CD8+ T cell accumulation contributes to viral encephalitis. J Clin Invest 2017 127: 3784-95Jacobson, JG. ら、Importance of the herpes simplex virus UL24 gene for productive ganglionic infection in mice. Virology 1998 242: 161-9Samady, 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.Koyanagi, N. ら、Role of herpes simplex virus 1 Us3 in viral neuroinvasiveness. Microbiol Immunol 2014 58: 31-7Cabrera, 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.

[0013] As mentioned above, antiviral drugs such as acyclovir are used to treat HSV. However, these antiviral drugs 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 need for such a vaccine.

[0014] An object of the present invention is to provide a recombinant live attenuated vaccine that can be used for the prevention and treatment of HSV-2 infections, which has an increased immune-inducing ability of HSV-2 through the introduction of mutations and is highly safe.

[0015] Because the functions of HSV-1 and HSV-2 are not necessarily conserved, it is important to conduct a detailed evaluation of HSV-2 using the above information on HSV-1 as a reference. The present inventors comprehensively produced and evaluated multiple mutants of HSV-2, aiming to develop a live attenuated vaccine that is both highly effective and safe.

[0016] Based on the hypothesis that introducing similar mutations into HSV-2 for virulence factors and immune evasion factors identified in HSV-1 would enable the construction of a highly safe, recombinant live attenuated vaccine with enhanced immune induction potential, the inventors created various mutant viruses and evaluated them in animal experiments. As a result, they found that the following double mutant virus, in which a mutation (inactivation) was introduced into a second gene of herpes simplex virus type 2 (HSV) containing a mutation (inactivation) in either the Us3 or UL41 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 live attenuated virus ΔTk (thymidine kinase deficient) of the same origin. ΔTk was chosen as a comparative target because its safety and efficacy have been suggested in non-patent literature (Non-patent literature 41-43). Here, for example, the designation "Us3 / UL2" indicates a double mutant virus of Us3 and UL2. *Double mutant viruses: Us3 / UL2, Us3 / UL3, Us3 / UL4, Us3 / UL13, Us3 / UL27, Us3 / UL35, Us3 / UL41, Us3 / UL44, Us3 / UL45, Us3 / UL46, Us3 / UL50, Us3 / UL56, Us3 / Us4, Us3 / Us5, Us3 / Us8A, Us3 / Us9, Us3 / Us11 UL41 / UL4, UL41 / UL12, UL41 / UL12.5, UL41 / UL24, UL41 / UL27, UL41 / ICP34.5, UL41 / UL35, UL41 / UL43, UL41 / UL56, UL41 / Us2, UL41 / Us4, UL41 / Us5, UL41 / Us8A, UL41 / Us9

[0017] Furthermore, among the above, Us3 / UL3, Us3 / UL4, Us3 / UL27, Us3 / UL35, Us3 / UL44, Us3 / UL46, Us3 / UL56, Us3 / Us4, Us3 / Us5, Us3 / Us8A, UL41 / UL4, and UL41 / Us5 had survival rates higher than those of the Us3 single mutant attenuated live virus inoculation group or the UL41 single mutant attenuated live virus inoculation group, indicating attenuated pathogenicity.

[0018] Furthermore, among the above, Us3 / UL27, Us3 / UL35, Us3 / Us4, and UL41 / Us5 showed a relapse prevention effect of 40% or more in a guinea pig relapse model, and of these, Us3 / UL35 showed a relapse prevention effect of 80% or more.

[0019] 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 2, 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.

[0020] 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 2 (HSV-2) have been modified, and the modifications of the genes result in a loss or reduction of gene function.

[0021] According to one aspect of the present invention, when immunity is induced by a recombinant live attenuated herpes simplex virus type 2 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.

[0022] FIG. 1 is a schematic diagram of the mouse primary infection protection test in Example 4. FIG. 2 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 3 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 4 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 5 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 6 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 7 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 8 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 9 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 10 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 11 is a diagram showing the virus shedding inhibitory effect in the mouse primary infection protection test in Example 4. FIG. 1 is a graph showing the effect of suppressing virus excretion in the primary mouse protection test of Example 4. FIG. 2 is a graph showing the effect of suppressing virus excretion in the primary mouse protection test of Example 4. FIG. 3 is a graph showing the effect of suppressing virus excretion in the primary mouse protection test of Example 4. FIG. 4 is a graph showing the effect of suppressing virus excretion in the primary mouse protection test of Example 4. FIG. 5 is a graph showing the effect of suppressing virus excretion in the primary mouse protection test of Example 4. FIG. 6 is a graph showing the effect of suppressing virus excretion in the primary mouse protection test of Example 4. FIG. 7 is a graph showing the effect of suppressing virus excretion in the primary mouse protection test of Example 4. FIG. 8 is a graph showing the effect of suppressing virus excretion in the primary mouse protection test of Example 4. FIG. 9 is a graph showing the effect of suppressing virus excretion in the primary mouse protection test of Example 4.FIG. 1 is a graph showing the effect of suppressing viral excretion in the primary mouse protection test of Example 4. FIG. 2 is a graph showing the effect of suppressing viral excretion in the primary mouse protection test of Example 4. FIG. 3 is a graph showing the effect of suppressing viral excretion in the primary mouse protection test of Example 4. FIG. 4 is a graph showing the pathology score in the primary mouse protection test of Example 4. FIG. 5 is a graph showing the pathology score in the primary mouse protection test of Example 4. FIG. 6 is a graph showing the pathology score in the primary mouse protection test of Example 4. FIG. 7 is a graph showing the pathology score in the primary mouse protection test of Example 4. FIG. 8 is a graph showing the pathology score in the primary mouse protection test of Example 4. FIG. 9 is a graph showing the pathology score in the primary mouse protection test of Example 4. FIG. 10 is a graph showing the pathology score in the primary mouse protection test of Example 4. FIG. 1 shows the pathology scores in the primary mouse protection test for Example 4. FIG. 2 shows the pathology scores in the primary mouse protection test for Example 4. FIG. 3 shows the pathology scores in the primary mouse protection test for Example 4. FIG. 4 shows the pathology scores in the primary mouse protection test for Example 4. FIG. 5 shows the pathology scores in the primary mouse protection test for Example 4. FIG. 6 shows the pathology scores in the primary mouse protection test for Example 4. FIG. 7 shows the pathology scores in the primary mouse protection test for Example 4. FIG. 8 shows the pathology scores in the primary mouse protection test for Example 4. FIG. 9 shows the pathology scores in the primary mouse protection test for Example 4. FIG. 10 shows the pathology scores in the primary mouse protection test for Example 4. 1 is a schematic diagram of the mouse intracerebral infection test in Example 5. FIG. 2 is a diagram showing the survival rate in the mouse intracerebral infection test in Example 5.FIG. 1 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 2 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 3 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 4 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 5 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 6 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 7 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 8 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 9 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 10 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 5. FIG. 11 is a diagram showing the survival rate in the mouse intracerebral infection test of Example 6. FIG. 12 is a diagram showing the survival rate in the mouse intranasal infection test of Example 6. FIG. 13 is a diagram showing the survival rate in the mouse intranasal infection test of Example 6. FIG. 14 is a diagram showing the survival rate in the mouse intranasal infection test of Example 6. FIG. 15 is a diagram showing the survival rate in the mouse intranasal infection test of Example 6. 1 is a diagram showing the recurrence-inhibiting effect in the guinea pig recurrence-inhibiting test of Example 7. FIG. 2 is a diagram showing the recurrence-inhibiting effect in the guinea pig recurrence-inhibiting test of Example 7. FIG. 3 is a diagram showing the recurrence-inhibiting effect in the guinea pig recurrence-inhibiting test of Example 7.

[0023] In this specification, "A to B" means A or more and B or less unless otherwise specified.

[0024] [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 2") is a virus in which two or more genes of herpes simplex virus type 2 (HSV-2) have been modified.

[0025] 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).

[0026] The HSV-2 may be a laboratory strain or a clinical isolate.

[0027] From the viewpoint of high immune induction ability and high safety, it is preferable that the genetically modified live attenuated herpes simplex virus type 2 has a gene associated with immunogenicity or immune escape modified. Examples of genes associated with immunogenicity or immune escape include RL1 (Non-Patent Documents 44, 45), RL2 (Non-Patent Documents 46, 47), UL2 (Non-Patent Document 48), UL3, UL4 (Non-Patent Document 49), UL12 (Non-Patent Document 50), UL12.5, UL13 (Non-Patent Document 51), UL24 (Non-Patent Document 52), UL27 (point mutants: Non-Patent Documents 13, 35), UL35 (deletion forms and point mutants: Non-Patent Document 33), UL41 (deletion forms: Non-Patent Document 53, function review: Non-Patent Document 54, point mutants: Non-Patent Document 55), UL43, UL44 (Non-Patent Document 56, 57), UL45 (Non-Patent Document 58), UL46 (Non-Patent Documents 59, 60), UL50 (Non-Patent Document 61 reporting the function, Non-Patent Document 62 reporting a point mutant), UL56 (Non-Patent Documents 63, 64), Us2 (Non-Patent Document 65), Us3 (Non-Patent Document 66 reporting a deletion, Non-Patent Documents 12, 13, 14, 15, 18 reporting point mutants), Us4 (Non-Patent Documents 67, 68), Us5 (Non-Patent Documents 69, 70), Us8A (Non-Patent Document 71), Us9 (Non-Patent Document 72), Us11 (Non-Patent Documents 73, 74), Us12 (Non-Patent Documents 75-77), and the like.

[0028] From the viewpoint of high immune-inducing ability and high safety, it is more preferable that the recombinant live attenuated herpes simplex virus type 2 has two or more genes, including Us3, modified. Furthermore, it is even more preferable that at least Us3 and UL2, Us3 and UL3, Us3 and UL4, Us3 and UL13, Us3 and UL27, Us3 and UL35, Us3 and UL41, Us3 and UL44, Us3 and UL45, Us3 and UL46, Us3 and UL50, Us3 and UL56, Us3 and Us4, Us3 and Us5, Us3 and Us8A, Us3 and Us9, or Us3 and Us11 have been modified.

[0029] From the viewpoint of high immune-inducing ability and high safety, it is more preferable that the recombinant live attenuated herpes simplex virus type 2 has two or more genes, including UL41, modified.Moreover, it is even more preferable that at least UL41 and UL2, UL41 and UL3, UL41 and UL4, UL41 and UL12, UL41 and UL12.5, UL41 and UL24, UL41 and UL27, UL41 and ICP34.5, UL41 and UL35, UL41 and UL43, UL41 and UL56, UL41 and Us2, UL41 and Us4, UL41 and Us5, UL41 and Us8A, or UL41 and Us9 have been modified.

[0030] Us3 is a gene encoding the protein kinase Us3. 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: NC_001798.2).

[0031] 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.

[0032] 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.

[0033] RL1 is a gene encoding ICP34.5 (also known as γ34.5). An example of the amino acid sequence of the protein encoded by RL1 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the ICP34.5 variant is preferably an expression-deficient form of ICP34.5.

[0034] UL2 is a gene encoding uracil DNA glycosidase UL2. An example of the amino acid sequence of the protein encoded by UL2 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. In terms of high immune induction ability and high safety, the UL2 variant is preferably an expression deletion variant of UL2. As used herein, "expression deletion variant" refers to a mutant in which the protein encoded by each gene is not expressed, due to gene deletion, start codon substitution, introduction of a stop codon immediately downstream of the start codon, or the like.

[0035] UL3 is a gene that encodes the phosphorylated protein UL3, which is primarily present in the nucleus. An example of the amino acid sequence of the protein encoded by UL3 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the UL3 variant is preferably an expression-deleted UL3 variant.

[0036] UL4 is a gene encoding UL4, a viral particle-forming protein distributed in the nucleus. An example of the amino acid sequence of the protein encoded by UL4 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the UL4 variant is preferably an expression-deleted UL4 variant.

[0037] UL12 is a gene encoding alkaline nuclease UL12. An example of the amino acid sequence of the protein encoded by UL12 is the amino acid sequence shown in SEQ ID NO: 2 (NCBI Reference Sequence: NC_001798.2). From the viewpoints of high immune induction ability and high safety, the modification of UL12 is preferably a modification of the tyrosine residue at position 361 (Y361) in the amino acid sequence of SEQ ID NO: 2. An example of a modification of Y361 in SEQ ID NO: 2 is a substitution of the tyrosine residue with a phenylalanine residue.

[0038] UL12.5 is a gene encoding alkaline nuclease UL12.5. An example of the amino acid sequence of the protein encoded by UL12.5 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. In terms of high immune induction ability and high safety, the UL12.5 variant is preferably an expression deletion variant of UL12.5.

[0039] UL13 is a gene encoding the protein kinase UL13. An example of the amino acid sequence of the protein encoded by UL13 is the amino acid sequence shown in SEQ ID NO: 3 (NCBI Reference Sequence: NC_001798.2). From the viewpoints of high immune induction ability and high safety, the modification of UL13 is preferably a modification of the 176th lysine residue (K176) in the amino acid sequence of SEQ ID NO: 3. An example of a modification of K176 in SEQ ID NO: 3 is a substitution of the lysine residue with a methionine residue.

[0040] UL24 is a gene encoding UL24, a viral particle-forming protein present in the membrane. An example of the amino acid sequence of the protein encoded by UL24 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the UL24 variant is preferably an expression-deleted UL24 variant.

[0041] UL27 is a gene encoding the glycoprotein gB. An example of the amino acid sequence of the protein encoded by UL27 is the amino acid sequence shown in SEQ ID NO: 4 (NCBI Reference Sequence: NC_001798.2). From the viewpoints of high immune induction ability and high safety, the modification of UL27 is preferably a modification of the tyrosine residue at position 889 (Y889) in the amino acid sequence of SEQ ID NO: 4. Examples of modifications of Y889 in SEQ ID NO: 4 include substituting the tyrosine residue with an alanine residue.

[0042] UL35 is a gene encoding the capsid protein VP26. An example of the amino acid sequence of the protein encoded by UL35 is the amino acid sequence shown in SEQ ID NO: 5 (NCBI Reference Sequence: NC_001798.2). From the viewpoints of high immune induction ability and high safety, the modification of UL35 is preferably a modification of the threonine residue at position 111 (T111) in the amino acid sequence of SEQ ID NO: 5. Examples of modifications of T111 in SEQ ID NO: 5 include substituting the threonine residue with an alanine residue.

[0043] UL41 is a gene encoding an RNase (VHS; virion host shut-off). An example of the amino acid sequence of the protein encoded by UL41 is the amino acid sequence shown in SEQ ID NO: 6 (NCBI Reference Sequence: NC_001798.2). From the viewpoints of high immune induction ability and high safety, the modification of UL41 is preferably a modification of the aspartic acid residue at position 215 (D215) in the amino acid sequence of SEQ ID NO: 6. Examples of modifications of D215 in SEQ ID NO: 6 include substituting an aspartic acid residue with an asparagine residue.

[0044] UL43 is a gene encoding the membrane protein UL43. An example of the amino acid sequence of the protein encoded by UL43 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the UL43 variant is preferably an expression-deficient UL43 variant.

[0045] UL44 is a gene encoding glycoprotein gC. An example of the amino acid sequence of the protein encoded by UL44 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the UL44 variant is preferably an expression-deficient UL44 variant.

[0046] UL45 is a gene encoding the membrane protein UL45. An example of the amino acid sequence of the protein encoded by UL45 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the UL45 variant is preferably an expression deletion variant of UL45.

[0047] UL46 is a gene encoding VP11 / 12. An example of the amino acid sequence of the protein encoded by UL46 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the UL46 variant is preferably an expression-deleted UL46 variant.

[0048] UL50 is a gene encoding vdUTPase, a homolog of the host's nucleic acid metabolic enzyme dUTPase. An example of the amino acid sequence of the protein encoded by UL50 is the amino acid sequence shown in SEQ ID NO: 7 (NCBI Reference Sequence: NC_001798.2). From the viewpoints of high immune induction ability and high safety, the modification of UL50 is preferably a modification of the 97th aspartic acid residue (D97) in the amino acid sequence of SEQ ID NO: 7. An example of a modification of D97 in SEQ ID NO: 7 is substitution of the aspartic acid residue with an alanine residue.

[0049] UL56 is a gene encoding the membrane protein UL56. An example of the amino acid sequence of the protein encoded by UL56 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the UL56 variant is preferably an expression-deficient UL56 variant.

[0050] Us2 is a gene encoding the tegument protein Us2. An example of the amino acid sequence of the protein encoded by Us2 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the Us2 variant is preferably an expression-deficient Us2 variant.

[0051] Us4 is a gene encoding the glycoprotein gG. An example of the amino acid sequence of the protein encoded by Us4 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the modified Us4 is preferably an expression-deleted Us4 mutant.

[0052] Us5 is a gene encoding the glycoprotein gJ. An example of the amino acid sequence of the protein encoded by Us5 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the modified Us5 is preferably an expression-deleted Us5 mutant.

[0053] Us8A is a gene predicted to encode the membrane protein Us8A. An example of the amino acid sequence of the protein encoded by Us8A is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the modified Us8A is preferably an expression-deleted Us8A mutant.

[0054] Us9 is a gene encoding the tegument protein Us9. An example of the amino acid sequence of the protein encoded by Us9 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. From the viewpoints of high immune induction ability and high safety, the Us9 variant is preferably an expression-deficient Us9 variant.

[0055] Us11 is a gene encoding the tegument protein Us11. An example of the amino acid sequence of the protein encoded by Us11 is the amino acid sequence registered as NCBI Reference Sequence: NC_001798.2. In terms of high immune induction ability and high safety, the Us11 variant is preferably an expression-deleted Us11 variant.

[0056] Genetically modified live attenuated herpes simplex virus type 2 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.

[0057] [HSV-2 Vaccine] An HSV-2 vaccine according to one embodiment of the present invention is a vaccine comprising the recombinant live attenuated herpes simplex virus type 2 as an antigen. This HSV-2 vaccine can be used for treating or preventing HSV-2 infection. As used herein, "treatment or prevention of HSV-2 infection" includes alleviating or preventing the worsening of one or more symptoms associated with HSV-2 infection, suppressing the onset of symptoms after HSV-2 infection, preventing, delaying, or stopping HSV-2 infection of cells in the body, reducing the number of HSV-2 in the body, etc.

[0058] Examples of conditions associated with HSV-2 infection include herpes labialis, herpes cornea, herpes genitalis, and systemic neonatal herpes, as well as stomatitis, skin diseases, encephalitis, meningitis, myelitis, and neurodegenerative diseases that may be caused by HSV-2.

[0059] The HSV-2 vaccine according to one embodiment of the present invention is safer or more effective than a vaccine containing, as an antigen, HSV-2 in which only Us3 has been modified. Furthermore, the HSV-2 vaccine according to one embodiment of the present invention is safer or more effective than a vaccine containing, as an antigen, HSV-2 in which only UL41 has been modified.

[0060] The recombinant live attenuated herpes simplex virus type 2 contained in the HSV-2 vaccine according to one embodiment of the present invention may be one type or two or more types.

[0061] The HSV-2 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.

[0062] Examples of dosage forms of the HSV-2 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-2 vaccine according to one embodiment of the present invention are injections, sprays, and nasal drops.

[0063] The amount of the recombinant live attenuated herpes simplex virus type 2 contained in the HSV-2 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.

[0064] The dose of the recombinant live attenuated herpes simplex virus type 2 contained in the HSV-2 vaccine according to one embodiment of the present invention is 10 ３ pfu ~ 10 ９ pfu, preferably 10 ４ pfu ~ 10 ８ More preferably, 10 pfu ６ pfu ~ 10 ７ More preferably, it is pfu.

[0065] (Administration Method / Administration Route of HSV-2 Vaccine) The administration method (administration route) of the HSV-2 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-2 vaccine according to one embodiment of the present invention is nasal administration or intramuscular administration (intramuscular 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.

[0066] The subjects to which the HSV-2 vaccine according to one embodiment of the present invention is administered are those infected with the HSV-2 virus or at risk of infection with the HSV-2 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.

[0067] The number of administrations and timing of administration of the HSV-2 vaccine according to one embodiment of the present invention can be appropriately determined depending on the type, species, age, weight, condition, etc. of the subject to be administered. From the viewpoint of efficacy and safety, the number of administrations of the HSV-2 vaccine is preferably 1 to 5 times, more preferably 1 to 3 times, and even more preferably 2 times.

[0068] Another aspect of the present invention is a method for treating or preventing an HSV-2 infection, which comprises the step of administering an HSV-2 vaccine according to one aspect of the present invention to a subject.

[0069] 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.

[0070] [Use as a Vaccine Vector] A recombinant live attenuated herpes simplex virus type 2 according to one embodiment of the present invention can also be used as a vaccine vector against pathogens other than HSV-2, such as influenza virus and SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2).

[0071] [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 2 (HSV-2) have been modified, and the modifications of the genes result in a deletion or reduction of gene function.

[0072] 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.

[0073] A virus according to aspect 3 of the present invention may be a virus according to aspect 2 of the present invention, wherein the gene associated with immunogenicity or immune evasion is a gene selected from the group consisting of Us3, UL2, UL3, UL4, UL12, UL12.5, UL13, UL24, UL27, ICP34.5, UL35, UL41, UL43, UL44, UL45, UL46, UL50, UL56, Us2, Us4, Us5, Us8A, Us9, and Us11.

[0074] The virus according to aspect 4 of the present invention may be the virus according to aspect 2 or 3 of the present invention, wherein the genes associated with immunogenicity or immune evasion are two or more genes including Us3.

[0075] A virus according to Aspect 5 of the present invention is the virus according to any one of Aspects 2 to 4 of the present invention, wherein the genes associated with immunogenicity or immune evasion may be two or more genes including UL41.

[0076] A virus according to aspect 6 of the present invention is a virus according to aspect 4 of the present invention, wherein the genes associated with immunogenicity or immune evasion may comprise a combination of genes selected from the group consisting of Us3 and UL2, Us3 and UL3, Us3 and UL4, Us3 and UL13, Us3 and UL27, Us3 and UL35, Us3 and UL41, Us3 and UL44, Us3 and UL45, Us3 and UL46, Us3 and UL50, Us3 and UL56, Us3 and Us4, Us3 and Us5, Us3 and Us8A, Us3 and Us9, and Us3 and Us11.

[0077] A virus according to Aspect 7 of the present invention is the virus of Aspect 5 of the present invention, wherein the genes associated with immunogenicity or immune evasion may include a combination of genes selected from the group consisting of UL41 and UL2, UL41 and UL3, UL41 and UL4, UL41 and UL12, UL41 and UL12.5, UL41 and UL24, UL41 and UL27, UL41 and ICP34.5, UL41 and UL35, UL41 and UL43, UL41 and UL56, UL41 and Us2, UL41 and Us4, UL41 and Us5, UL41 and Us8A, and UL41 and Us9.

[0078] A vaccine according to Aspect 8 of the present invention is a herpes simplex virus type 2 (HSV-2) vaccine comprising the virus according to any one of Aspects 1 to 7 of the present invention as an antigen.

[0079] The vaccine according to Aspect 9 of the present invention is superior in safety or efficacy to the vaccine according to Aspect 8 of the present invention, which contains, as an antigen, HSV-2 in which only Us3 has been modified.

[0080] The vaccine according to Aspect 10 of the present invention is superior in safety or efficacy to the vaccine according to Aspect 8 or 9 of the present invention, which contains, as an antigen, HSV-2 in which only UL41 has been modified.

[0081] The vaccine according to Aspect 11 of the present invention may be for intramuscular administration or nasal administration in any one of Aspects 8 to 10 of the present invention.

[0082] The vaccine according to aspect 12 of the present invention may be administered twice to a subject in any of aspects 8 to 11 of the present invention.

[0083] A vaccine according to aspect 13 of the present invention is a vaccine according to any one of aspects 8 to 12 of the present invention, wherein the dose of the virus is 10 ６ pfu ~ 10 ７ It may be pfu.

[0084] A use according to a fourteenth aspect of the present invention is the use of the virus according to any one of aspects 1 to 7 of the present invention as a vaccine vector against a pathogen other than herpes simplex virus type 2 (HSV-2).

[0085] The use according to aspect 15 of the present invention may be the use according to aspect 14 of the present invention, wherein the pathogen is influenza virus or SARS-CoV-2.

[0086] 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.

[0087] 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 wild-type HSV-2 virus strain 186. 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.

[0088] 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.

[0089] [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 4°C and 5,000 rpm 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 20°C and 70,000 rpm.

[0090] 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.

[0091] [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

[0092] 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-2 vaccine" (hereinafter referred to as "HSV-2 vaccine") was produced using the obtained virus as an antigen and used in experiments.

[0093] [Example 4] Primary infection protection test in mice. ４ pfu of HSV-2 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-2 strain 186 was administered subcutaneously. ３ 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.

[0094] A schematic diagram of the mouse primary infection protection test is shown in Figure 1. The HSV-2 vaccine containing the double mutant virus as an antigen suppressed virus shedding more than the group vaccinated with ΔTk, a conventional vaccine prototype live attenuated virus of the same origin (Figures 2 to 32). In the figure, "Us3KM" refers to a virus in which the 220th lysine of Us3 has been converted to methionine, "UL13KM" refers to a virus in which the 173rd lysine of UL13 has been converted to methionine, "UL27YA" refers to a virus in which the 889th tyrosine of UL27 has been converted to alanine, "UL35TA" refers to a virus in which the 111th threonine of UL35 has been converted to alanine, "UL41DN" refers to a virus in which the 215th aspartic acid of UL41 has been converted to asparagine, "UL50DA" refers to a virus in which the 97th aspartic acid of UL50 has been converted to alanine, and "UL12YF" refers to a virus in which the 376th tyrosine of UL12 has been converted to phenylalanine. Furthermore, notations with a Δ, such as "ΔUL2," refer to viruses in which the gene is deleted and the protein encoded by the gene is not expressed. *Double mutant virus: Us3 / UL2, Us3 / UL3, Us3 / UL4, Us3 / UL13, Us3 / UL27, Us3 / UL35, Us3 / UL41, Us3 / UL44, Us3 / U L45, Us3 / UL46, Us3 / UL50, Us3 / UL56, Us3 / Us4, Us3 / Us8A, Us3 / Us9, Us3 / Us11, UL41 / UL2, UL41 / UL3, UL41 / UL4, UL41 / UL12, UL41 / UL12.5, UL41 / UL24, UL41 / UL27, UL41 / ICP34.5, U L41 / UL35, UL41 / UL43, UL41 / UL56, UL41 / Us2, UL41 / Us4, UL41 / Us5, UL41 / Us8A, UL41 / Us9

[0095] The HSV-2 vaccines containing the following double mutant viruses as antigens significantly reduced vaginal lesion symptoms compared to the ΔTk-vaccinated group, demonstrating a good protective effect against the onset of the disease (Figures 33 to 61). *Double mutant viruses: Us3 / UL2, Us3 / UL3, Us3 / UL4, Us3 / UL13, Us3 / UL27, Us3 / UL35, Us3 / UL41, Us3 / UL45, Us3 / UL50, U3 / UL56, U3 / Us4, Us3 / Us8A, Us3 / Us9, Us3 / Us11, UL41 / UL2 , UL41 / UL3, UL41 / UL4, UL41 / UL12, UL41 / UL12.5, UL41 / UL24, UL41 / UL27, UL41 / ICP 34.5, UL41 / UL35, UL41 / UL43, UL41 / UL56, UL41 / Us2, UL41 / Us4, UL41 / Us5, UL41 / Us9

[0096] [Example 5] Mouse intracerebral infection test 1 × 10 ４ pfu of HSV-2 vaccine was administered intracerebrally to the mice, and the survival rate was monitored for two weeks.

[0097] A schematic diagram of the mouse intracerebral infection test is shown in Figure 62. The survival rate after administration of HSV-2 vaccines using Us3 / UL3, Us3 / UL4, Us3 / UL27, Us3 / UL35, Us3 / UL44, Us3 / UL46, Us3 / UL56, Us3 / Us4, Us3 / Us5, and Us3 / Us8A as antigens was higher than that of the group inoculated with the Us3 single mutant attenuated live virus, confirming that the pathogenicity was attenuated (Figures 63 to 72).

[0098] [Example 6] Mouse nasal infection test 1 × 10 ５ The mice were intranasally inoculated with pfu of HSV-2 vaccine, and the survival rate was observed for two weeks.

[0099] A schematic diagram of the mouse nasal infection test is shown in Figure 73. The survival rate after administration of HSV-2 vaccines using Us3 / UL3, Us3 / UL56, Us3 / Us4, UL41 / UL4, and UL41 / Us5 as antigens was higher than that of the groups inoculated with the Us3 single mutant attenuated live virus or the UL41 single mutant attenuated live virus, confirming that the pathogenicity was attenuated (Figures 74 to 78).

[0100] [Example 7] Guinea pig recurrence suppression test 1 × 10 ５ Two weeks later, 1 × 10 pfu of HSV-2 wild-type virus MS strain was administered intravaginally. ６ pfu or 1 x 10 ７ pfu of HSV-2 vaccine was administered intranasally or intramuscularly (prime inoculation). Two weeks later, the same amount of HSV-2 vaccine was administered intranasally or intramuscularly (boost inoculation). The presence or absence of blisters in the vagina was observed for up to 7 weeks after prime inoculation. If new blisters appeared in a different location, it was considered a recurrence (recurrent onset).

[0101] A schematic diagram of the guinea pig relapse prevention test is shown in Figure 79. By administering two doses (prime and boost), HSV-2 vaccines using Us3 / UL27, Us3 / UL35, Us3 / Us4, and UL41 / Us5 as antigens showed a 40% or higher preventive effect on relapse, and Us3 / UL35 showed an 80% or higher preventive effect on relapse (Figures 80 to 83).

[0102] The HSV-2 vaccine of the present invention, in which mutations have been introduced into two types of genes of herpes simplex virus type 2, is expected to be industrially useful as a vaccine that is both highly safe and effective.

Claims

1. Two or more genes of herpes simplex virus type 2 (HSV-2) are modified, The aforementioned gene modification is a deficiency or reduction of gene function, The modification of the two or more genes described above is a modification of two or more genes selected from genes related to immunogenicity or immune evasion. A multiple mutant virus in which the genes associated with immunogenicity or immune evasion include combinations of genes selected from the group consisting of Us3 and UL2, Us3 and UL3, Us3 and UL4, Us3 and UL13, Us3 and UL27, Us3 and UL35, Us3 and UL41, Us3 and UL44, Us3 and UL45, Us3 and UL46, Us3 and UL50, Us3 and Us4, Us3 and Us5, Us3 and Us8A, Us3 and Us9, and Us3 and Us11.

2. Two or more genes of herpes simplex virus type 2 (HSV-2) are modified, The aforementioned gene modification is a deficiency or reduction of gene function, The modification of the two or more genes described above is a modification of two or more genes selected from genes related to immunogenicity or immune evasion. A multiple mutant virus in which the genes associated with immunogenicity or immune evasion include combinations of genes selected from the group consisting of UL41 and UL2, UL41 and UL3, UL41 and UL4, UL41 and UL12, UL41 and UL12.5, UL41 and UL24, UL41 and UL27, UL41 and ICP34.5, UL41 and UL35, UL41 and UL43, UL41 and UL56, UL41 and Us2, UL41 and Us4, UL41 and Us5, UL41 and Us8A, and UL41 and Us9.

3. A herpes simplex virus type 2 (HSV-2) vaccine comprising the virus described in claim 1 as an antigen.

4. A herpes simplex virus type 2 (HSV-2) vaccine comprising the virus described in Claim 2 as an antigen.

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

6. The vaccine according to claim 4, which has superior safety or efficacy compared to a vaccine containing HSV-2 modified only in UL41 as an antigen.

7. The vaccine according to claim 3 or 4, which is for intramuscular or intranasal administration.

8. The vaccine according to claim 3 or 4, which is administered to the target in two doses.

9. The dose of the aforementioned virus is 10 ６ pfu~10 ７ The vaccine according to claim 3 or 4, wherein it is a pneumococcal fungicide (PFU).

10. Use of the virus described in claim 1 or 2 as a vaccine vector against pathogens other than herpes simplex virus type 2 (HSV-2).

11. The use as a vaccine vector according to claim 10, wherein the pathogen is influenza virus or SARS-CoV-2.