Non-human mammal model of HSV latent infection

Abstract

The present invention relates to a non-human mammal model of HSV latent infection, which enables detection of HSV in mucosal tissue upon exposure to a stimulus that reactivates latent HSV within the body. The non-human mammal according to the present invention can contribute to elucidating the mechanism of HSV latent infection and reactivation in humans and to developing drugs and the like for HSV.

Description

Non-human mammalian model of latent HSV infection

[0001] The present invention relates to a non-human mammalian model of latently infected with HSV and a method for producing the same, as well as a method for screening drugs and the like and a method for evaluating test substances using the animals.

[0002] Herpes simplex virus type 1 (HHV-1 or HSV-1) and type 2 (HSV-2 or HSV-2) are DNA viruses. HSV-1 primarily infects through the ocular, nasal, and oral mucosa. On the other hand, HSV-2 primarily infects through genital mucosa. After infection through mucosal tissue, HSV particles replicate in mucosal epithelial cells and migrate to ganglia. In ganglia, the virus maintains a latent infection state in which viral particles are not synthesized, allowing it to persist in the body for long periods. The recurrence of herpes, experienced by many people, occurs when the latent virus in ganglia initiates viral replication in response to some kind of stimulus, migrates to the mucosa, and causes inflammation due to viral replication. For example, the well-known phenomenon of "herpes (HSV) recurring and causing bumps around the mouth (herpes simplex)" is one in which the causes of herpes recurrence are empirically recognized phenomena, such as stress and a weakened immune system.

[0003] Numerous studies aimed at elucidating the mechanisms of HSV latent infection and reactivation have been published based on experimental data using rabbits, guinea pigs, and mice. For example, many papers have been published analyzing the reactivation of HSV latently infected in the ganglia of wild-type mice (C57BL / 6 mice) after exposure to ultraviolet light, heat stress, restraint stress, or cold stress (Non-Patent Documents 1-6). Furthermore, there have been published reports analyzing the mechanisms of HSV latent infection and reactivation using experimental systems in which guinea pigs and rabbits, other experimental animals than mice, are infected with HSV (Non-Patent Documents 7-9).

[0004] Kang W et al. Virology, 2003, 312(1):233-44.Clement C et al. Invest Ophtalmol Vis Sci, 2009 50(6):2855-61.Huang W et al. Cell Biochm Biophys, 2011 61(1):115-22.Clement C et al. Invest Ophtalmol Vis Sci, 2011 52(3):1770-9.Yu W et al. Cell Rep., 2018 25(9):2379-2389.e3.Roy S Front Immunol, 2018,9:2922.Wander AH et al. Archives of Virology, 1987,95(3-4):197-209.Bobrowski PJ et al. Int J Dermatol., 1991, 30(1):29-35.Lee S et al. J. Virol., 2015, 89(16):8383-91.

[0005] However, in the studies aimed at elucidating the mechanisms of HSV latent infection and reactivation in Non-Patent Documents 1 to 9, it is difficult to say that the experimental animals reflected the pathological condition in humans. That is, in the experimental system in which mice were infected with HSV, the HSV virus reactivated in the ganglia was not detected in the mucosa. Furthermore, in the experimental system in which guinea pigs were infected with HSV, HSV was unable to maintain a state of latent infection and viral particles were constantly replicating, making it difficult to distinguish between latent and reactivated HSV infection. Furthermore, in the experimental system in which rabbits were infected with HSV, HSV maintained a form of latent infection in the ganglia and viral particles did not replicate.

[0006] A first aspect of the present invention relates to providing a non-human mammalian model of latent HSV infection that is closer to the pathological condition in humans than the above-mentioned experimental systems.

[0007] A second aspect of the present invention relates to making it possible to detect reactivated HSV in mucosal tissues in an experimental system in which the above-mentioned mice are infected with HSV.

[0008] The present invention relates to the following items [1] to

[20] . [1] A non-human mammal model of latently infected with HSV, in which HSV can be detected in mucosal tissues upon exposure to a stimulus that activates latent HSV in the body. [2] The non-human mammal model of latently infected with HSV according to [1], which is a non-human mammal with reduced immune function below normal. [3] The non-human mammal model of latently infected with HSV according to [2], in which the non-human mammal with reduced immune function below normal is a genetically modified non-human mammal deficient in an immunocompetent cell-related gene. [4] The non-human mammal model of latently infected with HSV according to [3], in which the immunocompetent cell-related gene comprises one or more genes selected from the group consisting of a B cell gene, an IFN-γ receptor gene, a CD4 gene, a CD8 gene, an MHC class II gene, and an MHC class I gene. [5] The non-human mammal model of latently infected with HSV according to [3] or [4], in which the genetically modified non-human mammal is a genetically modified mouse. [6] The non-human mammal model of latent HSV infection according to any of [2] to [5], wherein the non-human mammal having a reduced immune function below the normal level is a non-human mammal administered an immunosuppressant. [7] The non-human mammal model of latent HSV infection according to any of [1] to [6], wherein the stimulus that activates latent HSV includes one or more stimuli selected from the group consisting of sleep disturbance stress, ultraviolet light, heat stress, restraint stress, cold stress, and social defeat stress. [8] A method for producing a non-human mammal model of latent HSV infection, comprising the following steps 1 and 2: Step 1: infecting a non-human mammal having a reduced immune function below the normal level with HSV; and Step 2: raising the non-human mammal obtained in Step 1 under the administration of an antiviral drug. [9] The production method according to [8], further comprising the following step 3. Step 3: Exposing the non-human mammal obtained in step 2 to a stimulus that activates latent HSV, and confirming the detection of HSV in mucosal tissue.

[10] The method described in [8] or [9], wherein the non-human mammal with immune function reduced below normal levels is a genetically modified non-human mammal deficient in an immunocompetent cell-related gene.

[11] The method according to

[10] , wherein the immunocompetent cell-related gene comprises one or more genes selected from the group consisting of a B cell gene, an IFN-γ receptor gene, a CD4 gene, a CD8 gene, an MHC class II gene, and an MHC class I gene.

[12] The method according to

[10] or

[11] , wherein the genetically modified non-human mammal is a genetically modified mouse.

[13] The method according to any of [8] to

[12] , wherein the non-human mammal having immune function reduced below normal levels is a non-human mammal administered an immunosuppressant.

[14] The method according to any of [8] to

[13] , wherein the antiviral drug in step 2 comprises one or more antiviral drugs selected from the group consisting of valacyclovir, famciclovir, and acyclovir.

[15] A mouse model of latently infected with HSV-2, in which HSV-2 can be detected in mucosal tissues by administering an immunosuppressant that activates latent HSV-2 in the body.

[16] A mouse in which reactivated HSV can be detected in mucosal tissue.

[17] A method for producing the mouse according to

[16] , comprising the step of administering an immunosuppressant to an HSV-infected mouse.

[18] A method for causing HSV to re-replicate in mucosal tissue of the mouse, comprising the step of administering an immunosuppressant to an HSV-infected mouse.

[19] A method for screening a drug or food or drink that ameliorates and / or prevents HSV infection and / or HSV recurrence in a mammal, using a non-human mammalian model of latent HSV infection according to any of [1] to [7], a mouse model of latent HSV-2 infection according to

[15] , a mouse according to

[16] , or the method according to

[18] .

[20] A method for evaluating a test substance, comprising the step of evaluating the effect of the test substance on HSV infection and / or HSV recurrence in a mammal, using a non-human mammalian model of latent HSV infection according to any of [1] to [7], a mouse model of latent HSV-2 infection according to

[15] , a mouse according to

[16] , or the method according to

[18] .

[0009] According to the first aspect of the present invention, by using a non-human mammalian model of latent HSV infection that is similar to the pathological condition in humans, it is expected that the mechanism of latent HSV infection and reactivation in humans will be elucidated and drugs related to HSV will be developed.

[0010] Furthermore, according to the second aspect of the present invention, in an experimental system in which the above-mentioned mice are infected with HSV, it is possible to detect reactivated HSV in mucosal tissue, which is expected to lead to elucidation of the mechanism of latent infection and reactivation of HSV in humans and the development of drugs and other treatments for HSV.

[0011] Figure 1 shows the survival rate of mice in Preparation Example 1. Figure 2 shows the survival rate of mice in Preparation Example 2. Figure 3 shows the survival rate of mice in Preparation Example 1'. Figure 4 shows the results of an analysis of the expression levels of gB and LAT in the cornea (eye) and ganglion (TG). Figure 5 shows the reactivation of HSV-1 by administration of an isotype antibody to B cell-deficient mice latently infected with HSV-1. Figure 6 shows the reactivation of HSV-1 by administration of an anti-CD4 antibody to B cell-deficient mice latently infected with HSV-1. Figure 7 shows the reactivation of HSV-1 by administration of an anti-CD8 antibody to B cell-deficient mice latently infected with HSV-1. Figure 8 shows the reactivation of HSV-1 by administration of an anti-CD4 antibody and an anti-CD8 antibody to B cell-deficient mice latently infected with HSV-1. Figure 9 shows the viral titers 4 and 5 days after administration of the above antibodies. Figure 10 shows the survival rate (%) of mice after administration of the above antibodies. Figure 11 shows the reactivation of HSV-2 by administration of an anti-CD8 antibody to wild-type mice latently infected with HSV-2. Figure 7 shows the reactivation of HSV-2 by administration of anti-CD8 antibodies to wild-type mice latently infected with HSV-2. Figure 7 shows the reactivation of HSV-2 by administration of anti-CD4 and anti-CD8 antibodies to wild-type mice latently infected with HSV-2. Figure 7A shows the results without physiological stress. Figure 7B shows the results with sleep disorder induction. Figure 7B shows the results with sleep disorder induction. Figure 8A shows the results without physiological stress. Figure 8B shows the results with combined UV exposure and sleep disorder stress. Figure 9A shows the results of an experiment administering a JAK inhibitor to wild-type mice latently infected with HSV-1. Figure 9A shows the results with oral administration of DMSO. Figure 9B shows the results with oral administration of a JAK inhibitor.

[0012] The present inventors have investigated the above-mentioned problems and have newly discovered that a non-human mammal in which HSV latently infects nervous tissue, reactivates in the nerves in response to specific stimuli, and HSV replication is detectable in mucosal tissue is closer to the pathological condition in humans. As described above, in conventional experimental animals, there are some in which HSV virus reactivated in ganglia cannot be detected in the mucosa, and some in which viral particles constantly replicate, making it difficult to distinguish between latent infection and reactivation. Therefore, the non-human mammal model of HSV latently infected with HSV of the present invention is a novel experimental animal that has not been seen before (first aspect).

[0013] (First Aspect) The non-human mammal model of HSV latently infected with HSV of Aspect 1 is one in which HSV can be detected in mucosal tissues upon exposure to a stimulus that activates HSV present in the body. Here, the term "non-human mammal model of HSV latently infected" refers to a non-human mammal in which HSV-1 and / or HSV-2 is latently infected in neural tissues such as ganglia. The non-human mammal is not particularly limited as long as it is a mammal other than a human, and examples include mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and monkeys. Preferably, it is an animal classified as a rodent, more preferably a mouse.

[0014] The term "stimuli that activate latent HSV in the body" refers to stimuli that reactivate HSV that has invaded the body and latently infected nervous tissue. Examples of such stimuli include sleep disturbance stress, ultraviolet light, heat stress, restraint stress, cold stress, social defeat stress, and combinations thereof. Sleep disturbance stress is preferred, a combination of sleep disturbance stress and another stimulus is more preferred, and a combination of sleep disturbance stress and ultraviolet light is even more preferred.

[0015] "Detection of HSV in mucosal tissue" can be performed by the method described in the Examples below, etc. A preferred embodiment of the non-human mammal model of HSV latent infection according to Aspect 1 is one in which exposure to sleep disturbance stress results in a detection rate of preferably 10% or more, more preferably 20% or more, and even more preferably 30% or more in mucosal tissue. Another embodiment is one in which exposure to a combination of sleep disturbance stress and ultraviolet light results in a detection rate of preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more. Examples of mucosal tissue include corneal mucosa and genital mucosa, but it is sufficient to be able to detect HSV in any of the mucosal tissues; it is not necessary to be able to detect HSV in all of the mucosal tissues.

[0016] A preferred embodiment of the non-human mammal model of latently infected HSV of Embodiment 1 is a non-human mammal model having immune function reduced below normal levels, which can be prepared by the following method.

[0017] We have recently discovered that administering immunosuppressants to wild-type mice (C57BL / 6 mice) latently infected with HSV leads to reactivation of HSV in neural tissues, and that the reactivated HSV is detected in mucosal tissues, causing inflammation. This finding suggests that reducing the immune function of mice results in reactivated HSV detection in mucosal tissues, similar to human pathology. Meanwhile, in order to generate experimental animals that mimic human pathology, we infected various immunodeficient transgenic mouse models with HSV. Most of these mice died, demonstrating the difficulty of maintaining latent HSV infection in neural tissues. Therefore, to create a condition in which HSV can maintain latent infection in neural tissues in various immunodeficient transgenic mouse models, we administered antivirals such as acyclovir during HSV infection, and successfully induced a condition in which HSV can maintain latent infection in various immunodeficient transgenic mice. The reason why the mortality rate is low and HSV can maintain a state of latent infection is thought to be because antiviral drugs such as acyclovir cannot eliminate latently infected HSV, but can only eliminate replicating HSV.

[0018] Therefore, one embodiment of the method for producing a non-human mammalian model of latently infected with HSV according to Aspect 1 includes a method for producing a non-human mammalian model of latently infected with HSV, comprising the following steps 1 and 2: Step 1: Infecting a non-human mammalian model having immune function reduced below normal levels with HSV; Step 2: Raising the non-human mammalian model obtained in step 1 under administration of an antiviral drug.

[0019] Step 1 involves infecting a non-human mammal with reduced immune function with HSV. Here, "reduced immune function" refers to a state in which the immune function is normal in the absence of pathogen infection, but becomes vulnerable when infected with a pathogen. Examples of non-human mammals with reduced immune function include genetically modified non-human mammals lacking immunocompetent cell-related genes and genetically modified humanized non-human mammals lacking immunocompetent cell-related genes. Preferred examples include genetically modified non-human mammals lacking immunocompetent cell-related genes, and more preferred examples include genetically modified mice lacking immunocompetent cell-related genes. Furthermore, examples of non-human mammals with reduced immune function include wild-type non-human mammals whose immune function has been reduced by administering an immunosuppressant to the mammal. The immunosuppressant may be any compound capable of delaying or halting immune system activity. Examples of such agents include glucocorticoids such as corticosterone, alkylating agents such as cyclophosphamide, JAK inhibitors such as tofacitinib and baricitinib, anti-CD20 antibodies such as rituximab, and antibodies against T cell surface antigens such as anti-CD4 antibodies and anti-CD8 antibodies. The degree of "immune function reduced below normal levels" is not particularly limited, but in light of the objectives of Embodiment 1, it is a degree to which the survival rate 30 days after HSV infection in the absence of administration of an antiviral drug is preferably 50% or less, more preferably 30% or less. Such non-human mammals with immune functions reduced below normal levels can be obtained from suppliers, or can be produced by known methods using wild-type animals.

[0020] "Immunocompetent cell-related genes" particularly refer to a group of genes related to adaptive immune responses. Examples of immunocompetent cell-related genes include, but are not limited to, B cell genes, IFN-γ receptor genes, CD4 genes, CD8 genes, MHC class II genes, and MHC class I genes. Furthermore, "immunocompetent cell-related gene deficiency" refers to the knockout of one or more immunocompetent cell-related genes, which may be a state in which the entire gene is deleted, or a state in which one or more base sequences of the gene are substituted, deleted, or inserted so as to lose gene function. The method of gene recombination is also not particularly limited, and known methods can be used.

[0021] The HSV to be infected in a non-human mammal with a reduced immune function below normal levels may be either or both of HSV-1 and HSV-2. The infection method may be a known infection route, for example, infection through the corneal mucosa in the case of HSV-1, or infection through the genital mucosa in the case of HSV-2.

[0022] Step 2 is a step of raising the non-human mammal obtained in Step 1 under administration of an antiviral drug. The "antiviral drug" is preferably one that cannot eliminate latently infected HSV but can only eliminate replicating HSV. Note that, as long as it does not completely eliminate latently infected HSV, one or more antiviral drugs can be used. Examples of such antiviral drugs include valacyclovir, famciclovir, and acyclovir, and one or more of these drugs can be used. The dosage and timing of the antiviral drug are not particularly limited. However, after HSV infection, the antiviral drug should be administered in an amount that, in light of the purpose of Mode 1, ensures a survival rate of preferably 60% or more, more preferably 70% or more, 30 days after HSV infection. For example, when valacyclovir is used as the antiviral drug, it can be administered daily at a dose of approximately 100 to 1000 μg / day until the animal reaches a latent infection state. The administration method can be any known method, such as by mixing it with drinking water, feed, etc., or by intraperitoneal or intravenous injection. After 28 to 35 days of rearing, the animals will become latently infected with HSV.

[0023] The above-mentioned preparation embodiment can further include the following step 3. Step 3 below allows confirmation of whether or not a desired non-human mammalian model of latently infected with HSV has been obtained. The stimulus used in step 3 is the same as that described in the above section "Stimulus that activates HSV present in the body." Step 3: A step of exposing the non-human mammalian animal obtained in step 2 to a stimulus that activates HSV, and confirming the detection of HSV in mucosal tissue.

[0024] (Second Aspect) As described above, in conventional experimental systems in which mice are infected with HSV, HSV reactivated in nervous tissues cannot be detected in mucosal tissues. However, the present inventors have newly discovered that administering an immunosuppressant to wild-type mice (C57BL / 6 mice) latently infected with HSV reactivates HSV in nervous tissues, and the reactivated HSV is detected in mucosal tissues, leading to HSV re-replication and inflammation. This phenomenon mimics the pathology observed in humans with recurrent herpes. An HSV-2 latently infected mouse model, in which HSV-2 can be detected in mucosal tissues by administering an immunosuppressant that activates latent HSV-2 in the body, is also an example of the second aspect of the present invention. Another feature of the present invention is that wild-type mice can be used as the target mice.

[0025] The mouse of Aspect 2 is one in which HSV reactivated in neural tissue can be detected in mucosal tissue. The method for producing the mouse of Aspect 2 includes a step of administering an immunosuppressant to an HSV-infected mouse. The method may also include a step of producing an HSV-infected mouse beforehand. The method for producing an HSV-infected mouse is not particularly limited, and examples include the methods described in the Examples below. Aspect 2 also provides a method for causing HSV to replicate in the mucosal tissue of an HSV-infected mouse, which includes a step of administering an immunosuppressant to the mouse. The HSV-infected mouse may be a wild-type mouse or a mouse of Aspect 1. The terms HSV, neural tissue, mucosal tissue, reactivation, immunosuppressant, and the like in these aspects are the same as those described in Aspect 1.

[0026] In embodiment 2, the immunosuppressant used to reactivate HSV can be any compound capable of delaying or halting immune system activity, including, for example, glucocorticoids such as corticosterone, alkylating agents such as cyclophosphamide, JAK inhibitors such as tofacitinib and baricitinib, and antibodies against T cell surface antigens such as anti-CD20 antibodies such as rituximab, anti-CD4 antibodies, and anti-CD8 antibodies.

[0027] In Embodiment 2, the dosage and timing of the immunosuppressant are not particularly limited. However, after HSV infection, the immunosuppressant may be administered so as to achieve the objectives of Embodiment 2 and induce HSV expression in mucosal tissues. For example, when a JAK inhibitor (tofacitinib) is used as the immunosuppressant, an embodiment in which the immunosuppressant is administered at a dose of approximately 100 to 1000 μg / day daily until HSV expression in mucosal tissues is achieved may be used. The administration method may be any known method, such as administration by mixing with drinking water, feed, or the like, intraperitoneal administration, or intravenous injection. Furthermore, when an anti-CD4 antibody or anti-CD8 antibody is used as the immunosuppressant, an embodiment in which the immunosuppressant is administered at a dose of approximately 300 to 500 μg / day daily to every three days until HSV expression in mucosal tissues is achieved may be used. The administration method may be any known method, such as administration by intraperitoneal administration or intravenous injection.

[0028] The present invention provides a method for screening for a drug or food or drink that ameliorate and / or prevent HSV infection and / or HSV recurrence in a mammal, and a method for screening for a drug or food or drink, using the non-human mammalian model of a latent HSV infection of Aspect 1, the mouse of Aspect 2, and the method for causing HSV re-replication in mucosal tissue of a mouse of Aspect 2. The present invention also provides a method for evaluating a test substance, which evaluates the effect of the test substance on HSV infection and / or HSV recurrence in a mammal, using the non-human mammalian model of a latent HSV infection of Aspect 1, the mouse of Aspect 2, and the method for causing HSV re-replication in mucosal tissue of a mouse of Aspect 2.

[0029] The screening method is not particularly limited, and screening can be easily performed, for example, by administering a candidate drug or food or drink to a non-human mammal infected with HSV and testing whether or not it has the effect of ameliorating and / or preventing HSV infection and / or HSV recurrence in the mammal. The effect of a test substance can also be evaluated using a similar method.

[0030] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples in any way.

[0031] Preparation of a non-human mammalian model of HSV-1 latent infection (Preparation Example 1) HSV-1 (2-5x10) was injected into the corneal mucosa of various gene-deficient mice (IFN-γ receptor-deficient, MHC class II-deficient, B cell-deficient, or MHC class I-deficient) and wild-type mice. 5 pfu / eye). The survival rates of mice after HSV-1 infection are shown in Figure 1. As shown in Figure 1, more than 70% of wild-type mice survived 30 days after HSV-1 infection, whereas the survival rate of most of the gene-deficient mice described above was less than 20% at 30 days after infection.

[0032] Preparation of a mouse model of latent HSV-2 infection (Preparation Example 1') In addition, HSV-2 (0.5-1.0 x10 4 pfu / genital tissues). The survival rates of mice after HSV-2 infection are shown in Figure 3. As shown in Figure 3, wild-type mice that were not administered antiviral drugs had a survival rate of approximately 50% 10 days after HSV-2 infection, and most of them had a survival rate of less than 10% 15 days after infection. These gene-deficient mice and wild-type mice were obtained from The Jackson Laboratory or CLEA Japan, Inc.

[0033] Preparation of a non-human mammalian model of latent HSV-1 infection (Preparation Example 2) HSV-1 (2-5x10) was inoculated into the corneal mucosa of various gene-deficient mice (IFN-γ receptor-deficient, CD4-deficient, MHC class II-deficient, B cell-deficient, or MHC class I-deficient) and wild-type mice. 5Mice were infected with 1000 μL of HIV-1 virus (pfu / eye). On day 4 after infection, an antiviral drug (valacyclovir hydrochloride) was diluted in PBS to 0.5 mg / ml and administered intraperitoneally at a concentration of 1000 μL. Additionally, from days 4 to 12, the antiviral drug (valacyclovir hydrochloride) was diluted to 0.5 mg / ml in drinking water and placed in a drinking bottle in the cage. The survival rates of the mice were then analyzed (Figure 2). As shown in Figure 2, wild-type mice and most gene-deficient mice had a survival rate of nearly 100% at 30 days after infection. Furthermore, the survival rate of B cell-deficient mice was over 70%. Therefore, by administering the antiviral drug at the appropriate time, it is possible to prevent lethality and maintain the survival of various gene-deficient mice. IFN-γ receptor-deficient, MHC class II-deficient, B cell-deficient, MHC class I-deficient, or CD4-deficient mice were obtained from The Jackson Laboratory.

[0034] Preparation of a mouse model of latently infected with HSV-2 (Preparation Example 2') Regarding HSV-2 infection of the genital mucosal tissue, HSV-2 (0.5-1.0 x10 4 Mice were infected with 1000 μL of 0.5 mg / ml of valacyclovir hydrochloride (VHHCl) diluted in PBS and administered intraperitoneally on day 4 of infection. Additionally, from day 4 to day 12, the antiviral drug (VHHCl) was diluted to 0.5 mg / ml in drinking water and placed in a drinking bottle in the cage. The survival rate of the mice was then analyzed (Figure 3). As shown in Figure 3, wild-type mice treated with the antiviral drug maintained a nearly 100% survival rate even 80 days after infection.

[0035] <Confirmation of Latent Infection> To determine whether HSV-1 had established a latent infection in the living body of the mice obtained in Example 2 that had been rendered viable by antiviral drug administration, the corneas and ganglia of the B cell-deficient mice obtained in Example 2 were isolated and the expression of HSV-1-derived genes was analyzed (Figure 4). Note that the control mice in Figure 4 were B cell-deficient mice uninfected with HSV-1. It is known that when HSV-1 is latently infected in ganglia, no expression of genes necessary for viral replication, such as glycoprotein B (gB), is detected, and only LAT (latency associated transcript) is expressed. Therefore, we analyzed the expression levels of gB and LAT in the cornea (eye) and ganglia (TG). Neither gB nor LAT was expressed in the cornea. On the other hand, gB expression was not detected in the ganglia, but LAT was highly expressed. In other words, it was revealed that the B cell-deficient mice obtained in Example 2, which were able to survive after administration of an antiviral drug, had latently infected ganglia with HSV-1. The expression level of each gene was determined by extracting total RNA from corneal or ganglion cells and performing real-time PCR on the reverse transcription product. The vertical axis of the graph in Figure 4 represents the relative value calculated from the Ct value.

[0036] Anti-CD4 and CD8 Antibody Administration Experiments: Anti-CD4 and / or anti-CD8 antibodies were administered to B cell-deficient mice latently infected with HSV-1 obtained in Example 2, and the detection of reactivated HSV-1 in the ganglia was examined in the cornea. An isotype control antibody (also referred to as an isotype antibody) was used as a control. CD4+ and CD8+ T cell depletion was induced in B cell-deficient mice obtained in Example 2, after confirming latently infected mice (Figures 5A–F). Anti-CD4 or anti-CD8 antibodies, or anti-CD4 and anti-CD8 antibodies (0.3 mg / mouse) were intravenously injected (0, 3, 6, and 9 days later) (Figures 5A–F). After antibody administration, 10 μL of PBS was pipetted 10 times onto the ocular surface of one eye every day. The same 10 μL was pipetted 10 times into the other eye. The collected fluid from both eyes was diluted with 190 mL of PBS and stored at -80°C (viral wash solution). Viral titers in the corneas were measured by plaque-forming unit (pfu) / ml. Anti-CD4 and anti-CD8 antibody treatments demonstrated HSV-1 virus particles in 87.5% of mice. Significant increases in viral titers were observed on days 4 and 5 after antibody administration (Figure 5E). Furthermore, the survival rate was below 40% at 20 days after administration in mice treated with anti-CD4 and anti-CD8 antibodies (Figure 5F). In contrast, isotype antibody treatments, which were used as a control, maintained viral titers below the detection limit in all mice from days 1 to 12 after administration (Figure 5A). Anti-CD4 antibody treatments also demonstrated an increase in viral titers in 11.1% of mice (Figure 5B). Furthermore, when anti-CD8 antibody was administered, an increase in viral titer was observed in 25.0% of cases (Fig. 5C).

[0037] In addition, the mice obtained in Preparation Example 2', i.e., wild-type mice, were infected with HSV-2 (0.5-1.0 x 10 4After establishing a latent infection state by infecting mice with 100 pfu / genital tissues, anti-CD4 and / or anti-CD8 antibodies (0.3 mg / mouse) were injected intravenously (days 0, 3, 6, and 9). Each day after antibody administration, 50 μL of PBS was pipetted 10 times into the genital mucosa tissue. The collected solution was diluted with 150 mL of PBS and stored at -80°C (viral wash solution). Viral titers in the genital mucosa were measured by plaque-forming unit (pfu) / ml. HSV-2 virus particles were detected in 75.0% of mice treated with anti-CD4 and anti-CD8 antibodies (Fig. 6C). In contrast, viral titers in all mice treated with anti-CD8 antibodies were below the detection limit from day 0 to day 12 (Fig. 6A). In addition, administration of anti-CD4 antibody resulted in an increase in viral titer in 20.0% of cases (Figure 6B). Isotype antibodies, anti-CD4 antibody, and anti-CD8 antibody were obtained from BioXcell.

[0038] <Sleep Disorder Stress Test> B cell-deficient mice latently infected with HSV-1 were subjected to sleep disorder stress to examine whether reactivated HSV-1 in the ganglia could be detected in the cornea. Sleep disorders were induced in the B cell-deficient mice obtained in Example 2, which had been confirmed to be latently infected. First, the mice were acclimatized to the running wheel in an acclimation wheel (SW-15S, MELQUEST) for one week. Then, the mice were transferred to a sleep disorder mouse model generation device (SW-15-SD, MELQUEST) to induce sleep rhythm disruption. After transfer to the cage, 10 μL of PBS was pipetted 10 times onto the ocular surface of one eye. The same 10 μL was pipetted 10 times into the other eye. The liquid collected after pipetting in both eyes was diluted with 190 mL of PBS and stored at -80°C (virus wash solution). We then measured viral titers in the corneas using the plaque-forming unit (pfu) / ml assay, and found that HSV-1 viral particles were detected in 37.5% of the mice (Fig. 7B). In contrast, no virus was detected in the corneas of mice not exposed to UV light or sleep deprivation stress (Fig. 7A).

[0039] <Stress test of UV exposure + sleep disorder> We further tested the combination of UV exposure and sleep disorder stress exposure. The B cell-deficient mice obtained in Example 2 were acclimatized to the running wheel in an acclimation running cage (SW-15S, MELQUEST). After one week of acclimation, they were irradiated with 500 mJ / cm using a TM20 Chromato-Vu transilluminator. 2The eyes were irradiated with UV-B (302 nm) light. UV-B was irradiated to both eyes. The mice were then transferred to a sleep disorder model mouse production device (SW-15-SD, MELQUEST) to induce sleep rhythm disruption. After being placed in this cage, the virus washout solution was obtained daily by pipetting as described above and stored at -80°C. The virus titer in the cornea was then measured using the same method as above. HSV-1 virus particles were detected in nearly 100% of the mice (Fig. 8B). In contrast, no virus was detected in the corneas of mice not exposed to UV light or the stress of sleep disorder (Fig. 8A).

[0040] <Administration experiment of JAK inhibitors to wild-type mice latently infected with HSV-1> HSV-1 (2-5x10) was administered to the corneal mucosa of wild-type mice (C57BL / 6 mice). 5 pfu / eye). In this study, no antiviral drug (valacyclovir hydrochloride) was administered. Six to seven weeks after infection, the JAK inhibitor (tofacitinib) was suspended in corn oil at 4 mg / ml and orally administered (0.4 mg / mouse) for 7 days. After administration, the virus wash was obtained by pipetting as described above and stored at -80°C. The virus titer in the cornea was measured using the same method as above. HSV-1 virus particles were detected in approximately 60% of the corneas (Fig. 9B). In a control group, corn oil suspended in DMSO was orally administered, but no HSV-1 virus particles were detected in the cornea (Fig. 9A).

[0041] This invention is expected to lead to elucidation of the mechanism of latent infection and reactivation of HSV in humans and to the development of drugs and other treatments for HSV.

Claims

1. A non-human mammalian model of latent HSV infection that allows detection of HSV in mucosal tissues upon exposure to stimuli that activate latent HSV in the body.

2. A non-human mammalian model of latently infected with HSV according to claim 1, which is a non-human mammalian animal with immune function reduced below normal levels.

3. A non-human mammalian model of latently infected with HSV according to claim 2, wherein the non-human mammalian animal with immune function reduced below normal levels is a genetically modified non-human mammalian animal lacking genes related to immunocompetent cells.

4. A non-human mammalian model of latently infected with HSV according to claim 3, wherein the immunocompetent cell-related genes include one or more genes selected from the group consisting of B cell genes, IFN-γ receptor genes, CD4 genes, CD8 genes, MHC class II genes, and MHC class I genes.

5. A non-human mammalian model of latently infected with HSV according to claim 3, wherein the transgenic non-human mammalian animal is a transgenic mouse.

6. A non-human mammalian model of latently infected with HSV according to claim 2, wherein the non-human mammalian animal with immune function reduced below normal levels is a non-human mammalian animal administered an immunosuppressant.

7. A non-human mammalian model of latent HSV infection according to claim 1, wherein the stimulus that activates latent HSV comprises one or more stimuli selected from the group consisting of sleep deprivation stress, ultraviolet light, heat stress, restraint stress, cold stress, and social defeat stress.

8. A method for producing a non-human mammalian model latently infected with HSV, comprising the following steps 1 and 2: Step 1: infecting a non-human mammalian animal whose immune function is reduced below normal levels with HSV; and Step 2: raising the non-human mammalian animal obtained in step 1 under the administration of an antiviral drug.

9. The method according to claim 8, further comprising the following step 3: exposing the non-human mammal obtained in step 2 to a stimulus that activates latent HSV, and confirming the detection of HSV in mucosal tissue.

10. The method according to claim 8, wherein the non-human mammal with immune function reduced below normal levels is a genetically modified non-human mammal deficient in immunocompetent cell-related genes.

11. The method of claim 10, wherein the immunocompetent cell-related genes include one or more genes selected from the group consisting of B cell genes, IFN-γ receptor genes, CD4 genes, CD8 genes, MHC class II genes, and MHC class I genes.

12. The method of claim 10, wherein the genetically modified non-human mammal is a genetically modified mouse.

13. The method according to claim 8, wherein the non-human mammal with immune function reduced below normal levels is a non-human mammal administered an immunosuppressant.

14. The method of claim 8, wherein the antiviral drug in step 2 comprises one or more antiviral drugs selected from the group consisting of valacyclovir, famciclovir, and acyclovir.

15. A mouse model of latently infected with HSV-2 in which HSV-2 can be detected in mucosal tissues by administering an immunosuppressant that activates latent HSV-2 in the body.

16. Mice in which reactivated HSV can be detected in mucosal tissues.

17. A method for producing the mouse according to claim 16, comprising the step of administering an immunosuppressant to an HSV-infected mouse.

18. A method for causing HSV to replicate again in the mucosal tissue of an HSV-infected mouse, comprising the step of administering an immunosuppressant to the mouse.

19. A method for screening drugs or foods and beverages, using a non-human mammalian model of latent HSV infection according to any one of claims 1 to 7, a mouse model of latent HSV-2 infection according to claim 15, a mouse according to claim 16, or the method according to claim 18, to screen for drugs or foods and beverages that ameliorate and / or prevent HSV infection and / or HSV recurrence in mammals.

20. A method for evaluating a test substance, which uses a non-human mammalian model of latent HSV infection according to any one of claims 1 to 7, a mouse model of latent HSV-2 infection according to claim 15, a mouse according to claim 16, or the method according to claim 18, to evaluate the effect of the test substance on HSV infection and / or HSV recurrence in a mammal.

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