Method for producing cells susceptible to viral infection

Abstract

The purpose of the present invention is to provide a method for producing cells that are susceptible to viral infection, and cells that are susceptible to viral infection. The above problem is solved by providing a method for producing cells that are susceptible to viral infection, said method comprising a step for infecting a host cell with a virus having a long-term presence in cells, or with a vector thereof.

Description

Method for producing virus-susceptible cells

[0001] The present invention relates to a method for producing virus-infectable cells, virus-infectable cells, and methods for using the same.

[0002] Currently, research is underway to develop treatments and vaccines for infectious diseases raging around the world. Infectious diseases are caused by a wide variety of pathogens, including bacteria, viruses, protozoa, and fungi. Viruses are responsible for infectious diseases such as the new influenza virus, the novel coronavirus, Ebola hemorrhagic fever, and severe fever with thrombocytopenia syndrome (SFS), which occurs after tick bites. Viral infections are extremely dangerous. Major infectious diseases worldwide over the past decade include the new influenza virus (2009, over 20 million cases, approximately 250,000 deaths), MERS (2012, approximately 2,500 cases, approximately 900 deaths), Ebola hemorrhagic fever (2014, approximately 29,000 cases, approximately 11,000 deaths), Zika fever (2016, over 4 million cases), and the novel coronavirus (2020, over 600 million cases, over 6 million deaths). All of these are viral infections.

[0003] Viruses are not living organisms because they are made only of DNA or RNA, which contains genetic information, and the proteins that encase it. For this reason, viruses cannot replicate themselves. Viruses require cells to grow. The process of virus growth can be simplified into four steps and explained below.

[0004] The first step is "entry." A virus attaches to receptors on the surface of a host cell and invades the cell through endocytosis or other mechanisms. The second step is "replication." After invading a host cell, the virus releases its genetic information (DNA or RNA). The released DNA or RNA enters the cell nucleus and produces mRNA, which replicates itself (DNA or RNA) and produces viral proteins. A portion of the mRNA then produces viral proteins via human ribosomes and other mechanisms. The third step is "assembly." The proteins that make up the outer layer of the viral coat produced in the endoplasmic reticulum are excised to surround the viral DNA or RNA. This reconstitutes the virus particle. The fourth step is "release." The reconstituted virus particles (multiplied viruses) are released outside the cell through exocytosis or other mechanisms via the Golgi apparatus. This is how viruses multiply in host cells (Figure 1, Non-Patent Document 1).

[0005] Viruses that multiply within a cell invade the next cell. This cycle is repeated, causing the virus to spread. As a result, the cell loses its original functions. However, humans also have immune functions that protect against infection by pathogens. The immune system responds to pathogens such as viruses in two stages, known as innate immunity and adaptive immunity. First, pathogens enter the body, and once they have invaded the cells, innate immunity kicks in.

[0006] Innate immunity, also known as primary immunity, is a mechanism present even in primitive organisms. The main immune cells responsible for primary immunity are myeloid cells such as macrophages and phagocytic cells such as B cells. These cells have various receptors that recognize microorganisms. Examples include Toll-like receptors (TLRs) and C-type lectin receptors (CLRs). To date, 10 types of TLRs and 12 types of CLRs are known in humans, and each receptor specifically recognizes various pathogens or parts of them.

[0007] We will explain the innate immune response to a virus entering the body using the West Nile virus, a (+)-strand RNA virus, as an example. When the West Nile virus infects a cell, viral replication occurs within the cell as described above. During this process, a large amount of double-stranded RNA (dsRNA) is produced as a viral genome replication intermediate. If the infected cell undergoes apoptosis or other such events, the dsRNA within the cell is released. When this released dsRNA is taken up by phagocytes such as macrophages and dendritic cells, it is recognized (bound) and activated by TLR3, a Toll-like receptor (TLR) found in the endoplasmic reticulum and endosomes. The activated receptor activates the transcription factors IRF and NF-κB via intracellular signaling pathways, promoting the production of type I interferon, which has strong antiviral effects. TLRs respond to a variety of pathogens. For example, it has been reported that RNA viruses are recognized by TLR3, TLR7, and TLR8, DNA viruses by TLR9, and viral structural proteins are recognized by TLR2 and TLR4, and that binding activates the cell's defense function (Non-Patent Document 2).

[0008] Type I interferon produced by natural immunity etc. has three main effects: 1) It acts on nearby cells to suppress viral replication, thereby increasing the cells' resistance to viruses. 2) It promotes increased expression of MHC class I molecules in cells that have not yet been infected with viruses, protecting them from NK cells. 3) It activates NK cells, which then eliminate virus-infected cells.

[0009] As mentioned earlier, viruses cannot replicate on their own, so when studying viruses, it is essential to use cells that the virus can infect. Cells that viruses infect or replicate in are called host cells. Four typical examples of the use of host cells are listed below. The first is "virus isolation." To conduct research on the virus itself, it is first necessary to isolate the virus. Researchers utilize the mechanism by which viruses replicate, as described above. Specifically, they collect a sample (such as blood containing the virus) from a patient and mix it with cells in a test tube. By replicating the virus in the cells, they increase the number of viruses they are studying. Finally, they separate the virus from the cells and extract only the virus. Viruses can be stored under liquid nitrogen. Therefore, successfully cultured viruses can be frozen and stored. If necessary, the preserved virus can be used to repeatedly replicate in test tubes using the same cells. Once a virus has been successfully cultured in cells, a stable amount of virus can be obtained.

[0010] The second is "drug evaluation" of antiviral drugs and the like. Suppose researchers have elucidated the mechanism by which the aforementioned virus replicates. They believe that a substance that inhibits this function could be used as an antiviral drug, and use this candidate drug to confirm whether the virus actually multiplies within host cells. For example, in the case of ineffective drug A, the virus multiplies within host cells as usual. On the other hand, in the case of effective drug B, the virus does not multiply within host cells (or is neutralized).

[0011] The third is "vaccine development." Examples of vaccine development using host cells include attenuated vaccines. Specific attenuated vaccines include BCG, polio vaccines, and rotavirus vaccines. Viruses are cultured in various host cells dozens of times, and in some cases, over a hundred times, to produce viruses with reduced toxicity through mutation. For example, in the case of Rotarix (an attenuated rotavirus vaccine, manufactured by GSK), a human rotavirus isolated from a diarrheal patient was passaged 33 times in Vero cells as host cells, and then a strain selected through three limiting dilutions was passaged an additional seven times in Vero cells to obtain an attenuated vaccine strain (Non-Patent Document 3).

[0012] The fourth is "vaccine production." As mentioned above, host cells can propagate viruses and therefore can also be used in vaccine production. An example of a vaccine produced using cells is the aforementioned Rotarix. The aforementioned attenuated vaccine strain is propagated in Vero cells, and the resulting virus solution is purified and additives are added to create an oral liquid formulation for use as a vaccine. It is also used in the production of inactivated vaccines. For example, JEBIC V (Japanese encephalitis inactivated vaccine, Research Institute for Microbial Diseases, Osaka University) propagates the Beijing strain of Japanese encephalitis virus in Vero cells, collects the resulting virus, inactivates it with formalin, treats it with drugs, purifies it, adds stabilizers, fills it, and then freeze-dries it. Cells can also be used in vaccine production in this way (Non-Patent Document 4).

[0013] Thus, host cells that allow viruses to grow are essential for virus research, as well as the research, development, and production of antiviral drugs and vaccines. While there are currently many cultured cells, few function as host cells for viruses. This is because they must be able to grow viruses in sufficient quantities and stably, and the cells themselves must also be able to be supplied in large quantities. In some cases, such excellent host cells are unavailable. For example, there are no such excellent host cells for viruses such as Ebola virus, hepatitis virus, parvovirus, and norovirus. As a result, despite the long-standing desire for vaccines, the development of new drugs and vaccines has been delayed.

[0014] Currently, the most commonly used host cells in virus research are Vero cells derived from the kidneys of African green monkeys. Because these cells were not developed as virus culture cells, the original cells used for their creation are not preserved. Furthermore, because African green monkeys were an endangered species, they were designated a target species of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in the 1970s. Since then, free trade of even cell cultures across borders has been restricted. Despite some issues, Vero cells have been used worldwide for microbiology research, including virus research, due to their ability to propagate many viruses and their high sensitivity to various bacterial toxins. In addition to research, they are also used for pathogen testing in hospitals and, as mentioned above, for the production of virus vaccines.

[0015] One reason why many viruses easily propagate in Vero cells is that Vero cells have a deletion of approximately 9 Mb on chromosome 12. Due to this deletion, Vero cells lose the cyclin-dependent kinase inhibitor genes CDKN2A / 2B, and as a result, they do not produce type I interferon, which should function as a natural immune response during viral infection (Non-Patent Document 5).

[0016] Based on the example of Vero cells and the innate immune mechanism of cells, for example, by blocking the signal that produces type I interferon or by blocking the cellular mechanism that inhibits virus production via the type I interferon receptor, it is possible to produce cells with improved virus production ability and susceptibility. Previously, methods have been reported, such as knocking down genes corresponding to type I interferon production using RNA interference or the like, knocking out (KO) using CRISPR-Cas9 or the like, blocking signals involved in type I interferon production by gene transfer or the like, or temporarily blocking them using compounds or the like. For example, Fusco et al. reported that knocking down the interferon α receptor using siRNA increased the replication efficiency of hepatitis C virus (Non-Patent Document 6).

[0017] Furthermore, G. Wong et al. reported that mice in which type I interferon receptors were knocked out exhibited increased susceptibility, such as higher virus titers in organs or death at a lower viral sensitizing dose, compared to wild-type mice, and therefore have been used in research on many highly pathogenic viruses, including flaviviruses, filoviruses, and bunyaviruses (Non-Patent Document 7).

[0018] Thus, deleting the type I interferon receptor, which blocks the signals produced by type I interferon, is a common method for improving infectivity to microorganisms.

[0019] However, previously reported methods for knocking down genes responsible for type I interferon production and knocking out (KO) genes using CRISPR-Cas9 or similar methods require the steps of preparing components such as the gene to be introduced, introducing them into cells, and verifying the introduction. In other words, creating cells with improved microbial infectivity required a lot of time and effort. Furthermore, methods that use compounds such as siRNA to block signals responsible for type I interferon production only had temporary effects.

[0020] Various viral vectors have been developed to date as a method for introducing genes, etc. For example, retroviral vectors such as lentiviral vectors are commonly used. Furthermore, viral vectors that do not integrate into the genome include adeno-associated viral vectors and Sendai viral vectors. Using these vectors, it is possible to introduce genes related to the suppression or blocking of type I interferon production signals. As a result, it is possible to create cells with reduced responsiveness to type I interferon.

[0021] However, it is known that infection with a specific virus can cause "viral interference," a phenomenon in which the infection and proliferation of other viruses is suppressed (Non-Patent Document 8). Therefore, this method requires the selection of viral vectors used for gene transfer and the evaluation of residual virus. In particular, the creation of cells with improved microbial infectivity using viral vectors in which the virus is present within cells, such as adeno-associated viral vectors and Sendai viral vectors, has not been explored.

[0022] R. Wang-Shick, Molecular Virology of Human Pathogenic Viruses, 2017, 31-45. M. Carty et al., Clin Exp Immunol. 2010 Sep, 161(3), pp. 397-406. R. L. Ward, et al., Clin Infect Dis., 2009 Jan 15, 48(2), pp. 222-8. Dried cell culture Japanese encephalitis vaccine, JEBIC V, drug package insert. N. Osada, et al., DNA Res. 2014 Dec; 21(6): 673-83. D. N. FUSCO, et. al. , GASTROENTEROLOGY 2013;144:1438-1449. G. Wong, et. al. , Zoological Research 39(1):3-14, 2018. C. M. Escobedo-Bonilla, Frontiers in Immunology 12, 674216, 2021.

[0023] The present invention has been made in view of the problems of the prior art, and aims to provide a method for producing virus-susceptible cells, and virus-susceptible cells. Another aim of the present invention is to provide a method for isolating viruses, a method for producing attenuated vaccines, a method for producing vaccines, a method for screening antiviral drugs (drug evaluation), and the like, using the virus-susceptible cells produced by the above method.

[0024] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have found for the first time that viral interference can be avoided and that virus-susceptible cells can be produced by infecting host cells with a long-lasting intracellular virus or its vector. Furthermore, they have found that the virus-susceptible cells produced in this manner can be used for virus isolation from patient samples, the development and production of attenuated vaccines, and the screening of antiviral drugs, and have thus completed the present invention.

[0025] That is, the present invention relates to the following: [1] A method for producing a virus-compromised cell, comprising a step of infecting a host cell with a first virus or a vector thereof, wherein the first virus is a long-lived intracellular virus. [2] The method according to [1], wherein the first virus is present in a location other than the genome of the cell. [3] The method according to [1] or [2], wherein the first virus is present in the cytoplasm. [4] The method according to any one of [1] to [3], wherein the first virus is at least one selected from the group consisting of hepatitis virus, cytomegalovirus, adenovirus, adeno-associated virus, and Sendai virus. [5] The method according to any one of [1] to [4], wherein the host cell is a cell with innate immunity. [6] The method according to any one of [1] to [5], wherein the host cell is a mammalian-derived cell. [7] The method according to any one of [1] to [6], wherein the host cell is at least one selected from the group consisting of immortalized myeloid cells, Vero cells, MDCK cells, Huh7 cells, and HEK293 cells. [8] A method for isolating a virus, comprising the steps of: preparing virus-compromised cells containing a long-lived intracellular virus or a vector thereof; contacting the virus-compromised cells with a sample containing a second virus derived from a subject; propagating the second virus in the virus-compromised cells; and recovering the second virus from the virus-compromised cells after the propagating step. [9] The method according to [8], wherein the step of preparing virus-compromised cells comprises infecting host cells with a long-lived intracellular virus or a vector thereof.

[10] The method according to [8] or [9], wherein the second virus is a different type of virus from the first virus.

[11] A method for screening for an antiviral drug, comprising the steps of: preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof; infecting the virus-susceptible cells with a second virus; contacting the virus-susceptible cells after the infection step with a test substance; and measuring the infection level of the second virus in the virus-susceptible cells.

[12] The method of

[11] , further comprising the steps of:a step of comparing the infection level of the second virus when the test substance is contacted with the virus-susceptible cells with the infection level of the second virus when the test substance is not contacted with the virus-susceptible cells, and determining that the test substance has an antiviral effect if the infection level of the second virus when the test substance is contacted is lower than the infection level of the second virus when the test substance is not contacted with the virus-susceptible cells.

[13] A method for producing a vaccine, comprising the steps of: preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof; infecting the virus-susceptible cells with a second virus; propagating the second virus in the virus-susceptible cells; and recovering the second virus from the virus-susceptible cells after the propagation step, and further comprising a step of recovering the virus obtained after repeating the infection step, propagation step, and recovery step at least five times.

[14] A method for producing a vaccine, comprising the steps of preparing virus-compromised cells containing a long-lived intracellular virus or a vector thereof, infecting the virus-compromised cells with a second virus, propagating the second virus in the virus-compromised cells, and recovering the second virus from the virus-compromised cells after the propagation step.

[15] A method for propagating a virus, comprising the steps of preparing virus-compromised cells containing a long-lived intracellular virus or a vector thereof, and infecting the virus-compromised cells with the second virus.

[16] A virus-compromised cell containing a first virus or a vector thereof, wherein the first virus is a long-lived intracellular virus and the first virus is present in a location other than the genome of the cell.

[17] A kit comprising the virus-compromised cell according to

[16] .

[18] The kit according to

[17] , which is used for virus isolation, drug evaluation, vaccine development, or vaccine production.

[0026] The method of the present invention for producing virus-susceptible cells using a long-lasting intracellular virus or its vector allows virus-susceptible cells to be easily produced from any host cell (e.g., human immortalized myeloid cells, cells previously used as host cells in virus research, etc.). Furthermore, the virus-susceptible cells of the present invention can ensure a stable amount of virus, and therefore can be used in virus research, as well as for virus isolation from patient samples, development of attenuated vaccines, vaccine production, screening of antiviral drugs, etc.

[0027] This is a schematic diagram showing the four steps a virus takes to replicate using host cells. A DNA virus was used as a model for evaluation. The virus recognizes and adsorbs receptors on the cell surface, then "invades" the cell via endocytosis (1). Once inside, the virus sheds its shell and "replicates" the DNA inside (2). The replicated virus then "reconstructs" itself using proteins and other components present in the cell (3). Finally, the reconstructed virus is "released" from the cell (4). Viruses replicate through these four steps. Figure 2(a) shows the results of an evaluation of interferon (IFN-β) production in human induced pluripotent stem cell-derived, immortalized myeloid cells (cMylc-SE2) for novel coronavirus research. Figure 2(b) shows the results of an evaluation of interferon (IFN-γ) production in human induced pluripotent stem cell-derived, immortalized myeloid cells (cMylc-SE2) for novel coronavirus research. Figure 3 shows the results of an evaluation of the exogenous interferon response of human induced pluripotent stem cell-derived, immortalized myeloid cells (cMylc-SE2) for use in novel coronavirus research. Figure 4 is a schematic diagram outlining antiviral signaling in response to endogenous and exogenous interferon stimuli. Figures 5(a) to 5(d) show the results of a dengue virus infection test using human iPS cell-derived, immortalized myeloid cells (iMylc-SE2, cMylc-SE2) and VeroE6 cells. Figure 6 shows the results of an empox virus infection test using human iPS cell-derived, immortalized myeloid cells (iMylc-SE2, cMylc-SE2) and Vero cells. Figure 7 shows the results of a virus isolation test using human iPS cell-derived, immortalized myeloid cells (cMylc-SE2) for use in novel coronavirus research and VeroE6 cells. Figures 8(a) and (b) show the difference in infectivity when infected with dengue virus type 2 single infectious virus particles (D2-SRIPs) in the iMylc-D strain before and after modification to make it susceptible to virus infection, as determined by luciferase assay.Figures 8(c) to (d) show the difference in infectivity of the iMylc-X strain, measured by luciferase assay, when infected with dengue virus type 2 single infectious virus particles (D2-SRIPs) before and after the modification to make it susceptible to virus infection.

[0028] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited to this.

[0029] 1. Method for producing virus-susceptible cells In one embodiment of the present invention, there is provided a method for producing virus-susceptible cells, comprising the step of infecting a host cell with a first virus or a vector thereof, wherein the first virus is a long-lived intracellular virus (hereinafter referred to as "the method for producing virus-susceptible cells").

[0030] As used herein, "virus-compromised cells" refers to cells that are susceptible to virus infection and / or in which the infected virus is likely to grow. As described above, in normal cells, virus infection and / or virus growth is suppressed by natural immunity, whereas the virus-compromised cells of the present invention exhibit increased virus infection and / or virus growth compared to cells that are not made susceptible to virus infection. Therefore, the virus-compromised cells of the present invention are capable of producing large amounts of virus and are therefore useful in various fields, including virus research and vaccines.

[0031] In this specification, the term "first virus" is used for convenience to distinguish it from the "second virus" described below.

[0032] (Long-lived intracellular virus and viral vector) In the present method for producing virus-susceptible cells, the first virus is a long-lived intracellular virus. As used herein, "long-lived intracellular virus" refers to a virus that can exist in host cells for a long period of time. By using a long-lived intracellular virus to infect host cells, viral interference can be avoided and susceptible cells can be produced.

[0033] The intracellular long-term residence virus and viral vector may be any virus or viral vector that does not alter the genetic information of the host cell and is attenuated so that the virus or viral vector and the host cell do not mutually kill or eliminate each other. For example, retroviruses such as lentiviruses that act on the genome (genes) of the host cell cannot be used. On the other hand, viruses and their vectors that are not integrated into the genome and can remain dormant or long-term residence in cells, such as adenoviruses, adeno-associated viruses, cytomegaloviruses, and Sendai viruses, can be used.

[0034] Specific examples of long-lasting intracellular viruses are not particularly limited as long as they fall within the above definition, and include, for example, adenovirus vectors (e.g., the adenovirus vectors described in Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (e.g., the adeno-associated virus vectors and Sendai virus vectors described in WO 2010 / 008054), hepatitis viruses (e.g., hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, etc.), and cytomegalovirus.

[0035] The long-lasting intracellular virus is preferably present in a location other than the genome of the cell (for example, in the cytoplasm, in the nucleus, or in an intracellular organelle), and more preferably in the cytoplasm. Examples of long-lasting intracellular viruses present in the cytoplasm include adenovirus vectors, adeno-associated virus vectors, and Sendai virus vectors. Among these, Sendai virus vectors are preferred from the viewpoints of operability and safety.

[0036] Examples of intracellular long-term viruses present in the nucleus include hepatitis viruses (e.g., hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, etc.), cytomegalovirus, etc.

[0037] In one embodiment of the present invention, the long-lived intracellular virus may be at least one selected from the group consisting of hepatitis virus, cytomegalovirus, adenovirus, adeno-associated virus, and Sendai virus.

[0038] (Method for preparing (producing) virus-susceptible cells) The method for producing virus-susceptible cells includes the step of infecting host cells with a long-lasting intracellular virus or a vector thereof.

[0039] As used herein, "host cells" refers to cells that can be infected with a virus and grow. The host cells may be any cells that can be infected with a virus and grow, and may be wild-type cells, established cell lines (immortalized cells), genetic mutants, or cells into which genes have been introduced. The host cells are preferably cells that have innate immunity.

[0040] The host cell is not particularly limited as long as it is a virus within the above definition, and examples thereof include immortalized myeloid cells (e.g., human immortalized myeloid cells), Vero cells, HEK293 cells, MDCK cells, MRC5 cells, BHK-21 cells, Huh7 cells, etc. Furthermore, the host cell may be a gene-transfected or cloned version of these cells, and examples thereof include VeroE6 cells, HEK293T cells, Huh7.5.1 cells, etc.

[0041] The origin of the host cells is not particularly limited, but examples include mammals, such as pet animals such as cats and dogs, livestock animals such as cows, chickens and pigs, and other mammals.

[0042] Virus-susceptible cells can be produced by infecting the host cells with the first virus or its vector. More specifically, for example, host cells can be infected with a long-lasting intracellular Sendai virus vector, and the cells after the infection can be selected with a drug or cloned. In this case, the presence of the long-lasting Sendai virus vector in the cells can be easily confirmed by, for example, introducing a fluorescently detectable gene such as GFP into the Sendai virus vector.

[0043] Hereinafter, one embodiment of the method for producing virus-susceptible cells will be described using specific viruses, host cells, etc. for the sake of convenience, but the present invention is not limited to these embodiments.

[0044] (Method for Producing Virus-Infectable Immortalized Human Myeloid Cells) In one embodiment of the present invention, the method for producing virus-infectable cells relates to a method for producing virus-infectable immortalized human myeloid cells, comprising introducing a first intracellular long-lasting virus or a vector thereof into human immortalized myeloid cells. The human immortalized myeloid cells used in this method may be immortalized monocytes, or may be cells prepared by introducing M-CSF and / or GM-CSF into immortalized monocytes.

[0045] Monocytes can be prepared by collecting them from peripheral blood or umbilical cord blood, by inducing differentiation from pluripotent stem cells, by rendering other somatic cells such as fibroblasts into an undifferentiated state and then inducing their differentiation, or by directly inducing their differentiation through direct reprogramming.

[0046] When monocytes are collected from peripheral blood or umbilical cord blood, they can be obtained as cells expressing CD14 molecules in human blood by known separation and preparation methods. For example, human peripheral blood is gently diluted with an equal volume of physiological saline, phosphate-buffered saline, or Hank's buffer solution, gently layered on Ficoll (registered trademark) (GE Healthcare) in a centrifuge tube, and centrifuged at 15-30°C and 500-1000 xg for 20 minutes. The white band-like layer between the yellowish plasma and the transparent Ficoll can be collected as a peripheral blood mononuclear cell (PBMC) fraction consisting of lymphocytes and monocytes. The collected peripheral blood mononuclear cell fraction may be further washed as necessary. Monocytes can be obtained by further recovering cells expressing CD14 molecules from the recovered PBMC fraction. For example, monocytes can be separated and recovered by contacting PBMCs with a solid phase bound to an anti-CD14 antibody to allow monocytes to bind to the solid phase, and then washing to remove unbound cells. For example, a method using magnetic beads as such a solid phase is known (e.g., Dynabeads (registered trademark) CD14 (Thermo Fisher Scientific Inc.)). Alternatively, monocytes can be obtained by directly contacting peripheral blood with a solid phase to which an anti-CD14 antibody is bound, without separating PBMCs from the peripheral blood.

[0047] When monocytes are obtained by differentiating pluripotent cells, methods for inducing monocytes from pluripotent cells are known. As used herein, "pluripotent stem cells" refer to cells that have pluripotency and the ability to self-renew. As used herein, "pluripotency" is synonymous with multipotency and refers to a cellular state that allows differentiation into cells of multiple lineages. As used herein, "pluripotent stem cells" include stem cells, embryonic stem (ES) cells, cloned embryonic stem cells ("ntES cells") obtained by nuclear transfer, germline stem cells ("GS cells"), embryonic germ cells ("EG cells"), induced pluripotent stem (iPS) cells, and hematopoietic stem cells. Whether a cell is a pluripotent stem cell can be determined, for example, if the test cell forms an embryoid body in an in vitro culture system or differentiates into a desired cell after culture under differentiation-inducing conditions (differentiation treatment).

[0048] Induction of differentiation from pluripotent stem cells to monocytes, for example, macrophage colony-stimulating factor (M-CSF) and interleukin 3 (IL-3) (Fernando O. Martinez et al., Experimental Hematology (2008); 36: 1167-1175 see), IFN-γ or PMA (Annabelle Grolleau et al., J Immunol 1999; 162: 3491-3497 see), or macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) (WO 2012 / 043651, JP 2017-131136 A and JP 2018-171005 A see) can be carried out by contacting pluripotent stem cells. Monocytes may be purified by collecting only cells expressing CD14 according to the above-mentioned method, if necessary.

[0049] Monocyte immortalization can be achieved by introducing at least one gene selected from the BMI1 gene, EZH2 gene, MDM2 gene, MDM4 gene, HIF1A gene, BCL2 gene, and LYL1 gene, and the cMYC gene into the monocytes obtained as described above, thereby imparting proliferation ability while maintaining monocyte function. Preferably, a combination of the BMI1 gene, BCL2 gene, and cMYC gene is used, and one or more of the EZH2 gene, MDM2 gene, MDM4 gene, HIF1A gene, and LYL1 gene may be additionally introduced. Introduction of these genes into monocytes can be performed with reference to the descriptions in WO 2012 / 043651 and JP 2017-131136 A. Gene introduction can be performed using the gene introduction method described below.

[0050] The proliferation ability of immortalized monocytes can be maintained by further introducing M-CSF and GM-CSF. Thus, the method of this embodiment may further include introducing M-CSF and GM-CSF into immortalized myeloid cells or immortalized monocytes. Alternatively, cells expressing M-CSF and GM-CSF may be used as the immortalized myeloid cells or immortalized monocytes. Introduction of these genes into immortalized myeloid cells or immortalized monocytes can be performed by referring to the description in JP 2018-171005 A.

[0051] Virus-infectable human immortalized myeloid cells can be prepared by infecting the human immortalized myeloid cells (including human immortalized monocytes) obtained as described above with the aforementioned viruses or viral vectors. More specifically, a long-lasting intracellular virus or its vector is introduced into the human immortalized myeloid cells. Infection with the viral vector may be performed before, after, or simultaneously with the production of human immortalized monocytes.

[0052] (Method for introducing a long-lived intracellular virus or its vector into a host cell) A method for introducing a long-lived intracellular virus or its vector into a host cell can be carried out using a known method. Furthermore, the method for introducing a long-lived intracellular virus or its vector into a host cell is not particularly limited as long as the desired effect is achieved. In one embodiment of the present invention, a method for introducing a long-lived intracellular virus vector into a human immortalized myeloid cell involves, for example, placing 0.5 to 1.0 x 10 human immortalized myeloid cells in a 24-well plate. ７ Sendai virus vectors are added under culture conditions at a concentration of 1000 cells / well, and after cell proliferation, drug selection is performed to obtain immortalized myeloid cells harboring Sendai virus vectors (viral-susceptible human immortalized myeloid cells).

[0053] (Evaluation of interferon signaling in modified cells susceptible to virus infection) Methods for evaluating whether cells modified to be susceptible to virus infection are more susceptible to virus infection than unmodified host cells include direct evaluation by infection, such as evaluation of the virus concentration added or the amount of progeny virus, and indirect evaluation methods based on type I interferon, an antiviral substance in natural immunity. Indirect evaluation methods include two types: endogenous and exogenous. An example of an endogenous evaluation method is a method for evaluating the amount of type I interferon produced during virus infection as endogenous interferon. An example of an exogenous evaluation method is a method for evaluating signal activation from interferon receptors under interferon addition / production conditions.

[0054] (Endogenous evaluation: evaluation of interferon production in prepared cells) As a method for evaluating whether cells that have been processed to be susceptible to virus infection have become more susceptible to virus infection than cells before the processing, an endogenous evaluation method is, for example, a method for evaluating the amount of type I interferon produced upon virus infection. For example, SARS-CoV2 virus can be added to human immortalized myeloid cells that have been processed to be susceptible to virus infection, and the amount of type I interferon in the culture supernatant after 3 days can be measured by RT-PCR.

[0055] (Exogenous evaluation: evaluation of suppression of interferon signal activation in prepared cells) As an evaluation method for determining whether cells that have been processed to be susceptible to virus infection have become more susceptible to virus infection than cells before processing, an exogenous evaluation method is, for example, an evaluation method in which interferon α is added externally and signal activation from the interferon receptor is examined. As such an evaluation method, for example, a method for evaluating downstream genes that respond when type I interferon receptor signal activation is reported by Zhang et al. (Virology Journal, (2022), p. 19: 28). In one embodiment, cells that have been processed to be susceptible to virus infection are plated, and then interferon α is added. After culturing for about 1 to 2 days, the cells are recovered and the mRNA and protein levels of molecules (MxA, OAS, SOCS1, SOCS3, USP18) and ISGs (ISG15, ISG20) that respond to type I interferon and are involved in antiviral activity signals are measured, thereby evaluating the activation of antiviral signals by type I interferon.

[0056] (Evaluation of Viral Infection Effect Using Pseudovirus) In addition to viral infection evaluation, a method for evaluating whether cells modified to be susceptible to virus infection exhibit greater susceptibility to virus infection than existing cells can also be performed using pseudoviruses such as single-time infectious virus particles (SRIPs). For example, the single-time infectious virus particles (DENV-SRIPs) of dengue virus described by Yamanaka et al. (mSphere. 2021, Aug. 25; 6(4): e0033921) are added to cells. When cells are infected with DENV-SRIPs, DENV-SRIPs mRNA is amplified within the cells. Because the mRNA contains a luciferase system, evaluating luciferase activity after 24 hours allows for quantitative evaluation of whether the cells modified to be susceptible to virus infection exhibit increased infectivity compared to existing cells.

[0057] 2. Virus-susceptible cells In one embodiment of the present invention, there is provided a virus-susceptible cell comprising a first virus or a vector thereof, wherein the first virus is a long-lived intracellular virus and is present in a location other than the genome of the cell (hereinafter referred to as "the virus-susceptible cell").

[0058] The virus-susceptible cells contain the long-lived intracellular virus within the cells, thereby avoiding viral interference and, as a result, becoming susceptible to viral infection. Furthermore, in the virus-susceptible cells, the long-lived intracellular virus is present in a location other than the genome of the cells, so that the long-lived intracellular virus does not act on the genome (genes) of the host cell, allowing the cells to coexist with the virus.

[0059] The virus-infectible cells are used in various applications that require securing a certain amount of virus, such as virus isolation, drug evaluation, vaccine development, and vaccine production.

[0060] 3. Virus Isolation Method In one embodiment of the present invention, there is provided a virus isolation method (hereinafter referred to as "this isolation method") comprising the following steps: - a step of preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof (hereinafter referred to as "preparation step"); - a step of contacting the virus-susceptible cells with a sample containing a second virus derived from a subject (hereinafter referred to as "contact step 1"); - a step of propagating the second virus in the virus-susceptible cells (hereinafter referred to as "propagation step"); and - a step of recovering the second virus from the virus-susceptible cells after the propagation step (hereinafter referred to as "recovery step 1").

[0061] (Preparation Step) In this step, virus-susceptible cells containing a long-lasting intracellular virus or its vector, to be used in the present isolation method, are prepared.

[0062] To prepare virus-susceptible cells containing a long-lasting intracellular virus or its vector, separately produced virus-susceptible cells may be purchased, or the virus-susceptible cells may be produced within this process.

[0063] In one embodiment of the present invention, this step may include a step of infecting a host cell with a long-lived intracellular virus or a vector thereof.

[0064] Virus-compromised cells containing a long-lasting intracellular virus or a vector thereof can be produced by the method described in the section [1. Method for producing virus-compromised cells].

[0065] (Contacting Step 1) In this step, the virus-compromised cells prepared in the preparing step are brought into contact with a sample containing a second virus derived from a subject (hereinafter referred to as a "subject sample").

[0066] The specimen sample is not particularly limited, but examples thereof include tissue, blood, plasma, serum, lymph, urine, feces, serous fluid, cerebrospinal fluid, synovial fluid, saliva, throat swab, or fractions or processed products thereof. The specimen sample is appropriately selected depending on the purpose. For example, saliva, throat swab, etc. are used as specimen samples for isolating the novel coronavirus (SARS-CoV2 virus).

[0067] The method for contacting the virus-susceptible cells with the test sample is not particularly limited, but for example, the test sample can be added to a culture medium in which the virus-susceptible cells are cultured.

[0068] (Proliferation Step) In this step, the second virus that has infected the virus-susceptible cells is propagated.

[0069] The second virus is propagated by culturing the virus-susceptible cells under appropriate conditions. Therefore, the propagation step can also be described as a step of culturing the virus-susceptible cells.

[0070] The culture conditions in this step are not particularly limited as long as they allow the second virus to grow, and can be appropriately set depending on the type of virus-susceptible cells, the type of the second virus, the desired degree of growth, etc.

[0071] (Recovery step 1) In this step, the second virus grown in the growth step is recovered. The second virus can be recovered from the culture supernatant or from the lysate after cell lysis. The recovered second virus is appropriately purified as needed before use. Examples of purification methods include density gradient centrifugation, ultrafiltration, and affinity chromatography.

[0072] (Others) In the present isolation method, the second virus is preferably a virus of a different type from the first virus. By using the second virus of a different type from the first virus, virus isolation can be performed appropriately.

[0073] In another embodiment of the present invention, there is provided a method for isolating a second virus from blood derived from an infectious patient, the method comprising the steps of contacting virus-susceptible cells with the patient-derived blood, culturing the infected virus-susceptible cells, and detecting and / or identifying the second virus in the cultured cells to identify the infectious virus.

[0074] Hereinafter, one embodiment of the virus isolation method will be described using specific viruses, host cells, etc. for the sake of convenience, but the present invention is not limited to these embodiments.

[0075] (Virus infection and culture using cells that have been modified to be susceptible to virus infection) Virus infection using cells that have been modified to be susceptible to virus infection can be carried out by contacting the cells with the virus. The culture conditions of the original host cells are used as a reference for the medium used during infection and / or culture after infection. In one embodiment, when infecting human immortalized myeloid cells that have been modified to be susceptible to virus infection with dengue virus, the evaluation conditions used for the cells before processing are used as a reference. For example, if the evaluation conditions before processing are α-MEM (10% fetal bovine serum) medium supplemented with non-essential amino acids, or RPMI 1640 medium (SIGMA) containing hypoxanthine and HEPES (SIGMA), 10% fetal bovine serum, sodium bicarbonate, 100 units / ml penicillin, and 100 μg / ml streptomycin, the same conditions are used. The evaluation time can be set appropriately depending on the type of cells, virus, and medium, and can be, for example, 24 to 96 hours, 36 to 84 hours, or 72 hours. Other culture conditions can be determined in accordance with general culture conditions for human cells, for example, 37°C, 5% CO ２ It can be under conditions.

[0076] (Virus isolation using cells processed to be susceptible to virus infection) Isolating a virus from a patient allows the type of infecting virus to be identified. The amount of virus varies depending on the patient's symptoms and the time elapsed since infection. Virus isolation requires cells that are susceptible to infection and proliferation. Therefore, cells processed to be susceptible to virus infection are useful. Virus isolation using cells processed to be susceptible to virus infection can be performed by contacting a patient sample (throat swab, blood, saliva, etc.) with cells processed to be susceptible to virus infection and culturing the cells for a certain period of time. The culture medium may be, for example, a culture medium used for infection assessment. In one embodiment, human immortalized myeloid cells processed to be susceptible to virus infection are contacted with a throat swab or saliva sample from a patient infected with COVID-19, and after culturing for about one week, the amount of virus amplified in the culture supernatant is measured by RT-PCR. The supernatant from the group in which viral growth was confirmed by RT-PCR can be collected and added again to host cells to produce a viral isolate, or the virus can be identified by analyzing the genome using a next-generation sequencer (NGS).

[0077] 4. Method for screening antiviral drugs In one embodiment of the present invention, there is provided a method for screening antiviral drugs (hereinafter referred to as "the screening method") comprising the following steps: a step of preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof (the "preparation step"); a step of infecting the virus-susceptible cells with a second virus (hereinafter referred to as the "infection step"); a step of contacting the virus-susceptible cells after the infection step with a test substance (hereinafter referred to as the "contact step 2"); and a step of measuring the infection level of the second virus in the virus-susceptible cells (hereinafter referred to as the "measurement step").

[0078] Furthermore, it is preferable that this screening method further comprises the following steps: - A step of comparing the infection level of the second virus when the test substance is brought into contact with virus-susceptible cells with the infection level of the second virus when the test substance is not brought into contact with virus-susceptible cells, and determining that the test substance is a substance having antiviral activity if the infection level of the second virus when the test substance is brought into contact with the virus-susceptible cells is lower than the infection level of the second virus when the test substance is not brought into contact with the virus (hereinafter referred to as the "determination step").

[0079] In this screening method, the "preparatory step" is as described in the section [3. Virus isolation method].

[0080] (Infection step) In this step, virus-susceptible cells are infected with a second virus. This step can be carried out by the method described in the section "1. Method for producing virus-susceptible cells."

[0081] (Contacting Step 2) Any substance can be used as the test substance. The type of test substance is not particularly limited, and examples of the test substance that can be used include compounds present in extracts of natural products, low molecular weight synthetic compounds, synthetic peptides, antibodies, antisense RNA, siRNA, miRNA, and aptamers.

[0082] In one embodiment of the present invention, the test substance may be a compound contained in a chemical library, a phage display library, a combinatorial library, or the like.

[0083] In one embodiment of the present invention, the test substance is preferably a low molecular weight compound, and more preferably a compound library of low molecular weight compounds. The compound library may be a commercially available compound library or a compound library constructed by a conventional method in the art.

[0084] In one embodiment of the present invention, the antibodies, antisense RNAs, siRNAs, miRNAs, and aptamers used as test substances may be commercially available or may be constructed by conventional methods in the art. Those skilled in the art can prepare antibodies, antisense RNAs, siRNAs, miRNAs, and aptamers based on conventional methods in the art.

[0085] In this process, "contact" means that two or more substances are close enough to be in physical contact with each other.

[0086] In one embodiment of the present invention, contact between the virus-susceptible cells and the test substance may be carried out by adding the test substance to a container in which the virus-susceptible cells are cultured, or by adding the cells to a solution containing the test substance. Contact between the virus-susceptible cells and the test substance is preferably carried out by adding the test substance to a container in which the virus-susceptible cells are cultured.

[0087] In this step, various conditions such as the contact time between virus-susceptible cells and the test substance, the concentration of the test substance, etc. can be appropriately determined by those skilled in the art.

[0088] (Measurement step) The infection level of the second virus can be measured by measuring the infectious titer of the second virus contained in the supernatant after the completion of the culture using cell-ELISA, immunostaining, plaque formation, or PCR. Alternatively, the infection level can be measured by fixing the virus-infectible cells after the completion of the culture and performing immunostaining or cell ELISA.

[0089] When the infection level of virus-susceptible cells with a second virus is measured by Cell-ELISA or immunostaining, the screening method may include immobilizing the infected virus-susceptible cells and contacting the immobilized cells with an antibody specific to the second virus. For example, in this specification, the Cell-ELISA method may include contacting the immobilized cells with an antibody (primary antibody) that specifically binds to the virus, removing (by washing) the unbound antibody, contacting the immobilized cells after contact with the primary antibody with a labeled antibody (secondary antibody) that can bind to the primary antibody, removing (by washing) the unbound antibody, measuring the labeling level (intensity, area, number, etc.) of the secondary antibody bound to the fixed cells, and calculating the infection level of the virus that has infected the cells from the measured labeling level. Alternatively, the immunostaining method herein may include contacting a labeled antibody capable of specifically binding to the virus with immobilized cells, removing (by washing) unbound antibody, measuring the labeling level (intensity, area, number, etc.) of the antibody bound to the immobilized cells, and calculating the infection level of the virus that has infected the cells from the measured labeling level. The label used for the labeled antibody may be a detectable label such as a radioactive label, an enzyme label, a fluorescent label, a bioluminescent label, a chemiluminescent label, a metal label, or a near-infrared label.

[0090] When the infection level of a second virus in virus-susceptible cells is measured by a plaque formation method, the method may include mixing a virus-containing solution recovered from the culture supernatant or cell lysate of the infected virus-susceptible cells with a cell line known to be infected with the virus (e.g., K562 cell line, Vero cell line, or BHK cells), adding the mixed cell / virus solution to a medium, and culturing for 12 hours to 10 days, immobilizing the infected cells after culturing, and contacting the immobilized cells with an antibody specific to the second virus. Specifically, contacting the immobilized cells with the virus-specific antibody can be carried out by the Cell-ELISA method or immunostaining method described above.

[0091] When the infection level of a second virus in virus-susceptible cells is measured by PCR, for example, real-time PCR can be performed using a virus-containing liquid recovered from the culture supernatant or cell lysate of the virus-susceptible cells after infection as a sample.

[0092] (Determination step) This step is a step of comparing the infection level of the second virus when the test substance is brought into contact with virus-susceptible cells with the infection level of the second virus when the test substance is not brought into contact with virus-susceptible cells, and determining that the test substance is a substance having antiviral activity if the infection level of the second virus when the test substance is brought into contact with the virus-susceptible cells is lower than the infection level of the second virus when the test substance is not brought into contact with the virus.

[0093] The "infection level" is not particularly limited as long as it is a numerical value that reflects the number of infected viruses, and for example, PFU, PFU / mL, the area of ​​the infected area, the amount of virus (units), or the virus concentration (units / mL) can be used.

[0094] (Others) In the present screening method, the second virus is preferably a virus of a different type from the first virus. By using the second virus of a different type from the first virus, screening can be carried out appropriately.

[0095] In another embodiment of the present invention, there is provided a method for screening antiviral drugs, comprising the following steps: infecting virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof with a second virus in the presence of a test substance; measuring the infection level of the second virus in the virus-susceptible cells after the infection step; and determining whether or not the test substance has an antiviral effect based on the measured infection level of the second virus.

[0096] Hereinafter, one embodiment of the present screening method will be described using specific viruses, host cells, etc. for the sake of convenience, but the present invention is not limited to these embodiments.

[0097] (Evaluation of immune-evading viruses using immortalized myeloid cells engineered to be susceptible to virus infection) In another embodiment of the present invention, immortalized myeloid cells engineered to be susceptible to virus infection are particularly susceptible to infection by a second virus, and the virus is more likely to undergo immune evasion due to immune cells. Therefore, it is possible to prepare a virus that has undergone immune evasion and / or evaluate drugs in the presence of the virus. In one embodiment of the present invention, human immortalized myeloid cells engineered to be susceptible to virus infection are contacted with a novel coronavirus, and after three days, the virus in the culture supernatant is recovered and diluted, and then contacted with new human immortalized myeloid cells engineered to be susceptible to virus infection. After repeating this process, for example, five times, the amplified virus is analyzed using a next-generation sequencer (NGS) to identify the immune-evading virus strain. By using immune-evading viruses and adding candidate drugs under culture conditions with host cells, it is possible to select drugs that are effective against immune-evading viruses.

[0098] 5. Method for producing a vaccine In one embodiment of the present invention, there is provided a method for producing a vaccine (hereinafter referred to as "the method for producing the attenuated vaccine") comprising the following steps: a step of preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof (the "preparation step"); a step of infecting the virus-susceptible cells with a second virus (the "infection step"); a step of propagating the second virus in the virus-susceptible cells (the "propagation step"); a step of recovering the second virus from the virus-susceptible cells after the propagation step (the "recovery step 1"); and a step of recovering the virus obtained after repeating the infection step, the propagation step, and the recovery step 1 at least five times (hereinafter referred to as "recovery step 2").

[0099] In the method for producing this attenuated vaccine, the "preparation step," "multiplication step," and "recovery step 1" are described by reference to the descriptions in Section 3. Virus isolation method. In addition, in the method for producing this attenuated vaccine, the "infection step" is described by reference to the descriptions in Section 4. Antiviral drug screening method.

[0100] (Recovery Step 2) In the recovery step 2, the infection step, the growth step, and the recovery step 1 are repeated at least five times, thereby obtaining a virus strain with attenuated pathogenicity.

[0101] The number of repetitions is not particularly limited as long as it is 5 or more, and may be, for example, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more. The number of repetitions can be appropriately set based on the type of the second virus, the conditions of each step, etc., so as to obtain the desired virus strain.

[0102] The recovery method in recovery step 2 can be the same as that described in recovery step 1.

[0103] The virus recovered in the recovery step 2 is a strain with attenuated pathogenicity and can therefore be used as a live vaccine.

[0104] In another embodiment of the present invention, there is provided a method for producing a vaccine (hereinafter referred to as "the method for producing the vaccine") comprising the following steps: - a step of preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof ("preparation step"); - a step of infecting the virus-susceptible cells with a second virus ("infection step"); - a step of propagating the second virus in the virus-susceptible cells ("propagation step"); and - a step of recovering the second virus from the virus-susceptible cells after the propagation step (recovery step 1).

[0105] In the method for producing this vaccine, the "preparation step," "multiplication step," and "recovery step 1" are described by reference to the descriptions in Section 3. Virus isolation method. In addition, in the method for producing this vaccine, the "infection step" is described by reference to the descriptions in Section 4. Antiviral drug screening method.

[0106] The virus obtained by the present vaccine production method can be made into an inactivated vaccine (including a toxoid). When used as an inactivated vaccine, the present vaccine production method may further include decomposing the recovered virus and, if necessary, recovering an antigenic portion from the decomposition product, or extracting and inactivating a toxin in the virus.

[0107] (Others) In the method for producing the attenuated vaccine and the method for producing the vaccine, the second virus is preferably a virus of a different type from the first virus. By using the second virus of a different type from the first virus, the vaccine can be produced appropriately.

[0108] 6. Method for Propagating a Virus In one embodiment of the present invention, there is provided a method for propagating a virus (hereinafter referred to as "the present propagation method") comprising the following steps: preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof (the "preparation step"), and infecting the virus-susceptible cells with a second virus (the "infection step").

[0109] In this propagation method, the "preparation step" is described in Section 3. Virus isolation method. In addition, the "infection step" is described in Section 4. Antiviral drug screening method.

[0110] In this propagation method, cells susceptible to infection with the virus are used, and therefore the infected virus can be propagated efficiently and in large quantities.

[0111] (Others) In the present propagation method, the second virus is preferably a virus of a different type from the first virus.

[0112] 7. Kit In one embodiment of the present invention, a kit (hereinafter referred to as "the kit") containing cells susceptible to infection with the virus is provided.

[0113] As used herein, the term "kit" refers to a package that includes containers (e.g., vials, plates, tubes, dishes, etc.) that contain specific materials.

[0114] In addition to the virus-susceptible cells, the kit may also include, for example, a medium for culturing the cells, instructions for use, and the like.

[0115] This kit can be used for various purposes that require securing a certain amount of virus, such as virus isolation, drug evaluation, vaccine development, and vaccine production.

[0116] The present invention will be described in more detail below using examples, but these are not intended to limit the scope of the present invention. Note that all references cited throughout this specification are incorporated herein by reference in their entirety.

[0117] (Example 1) Preparation of infection-compromised MDCK cells (MDCK-SE) Compromised MDCK cells were prepared using commercially available MDCK cells. ６ Cells were seeded at 1000 cells / well and cultured in MEM medium containing 10% FBS and 1% NEAA, supplemented with a long-lasting intracellular Sendai virus vector carrying a puromycin resistance gene. Two weeks after the start of viral vector transduction, puromycin was used to select only vector-transduced cells. These cells were then used as MDCK-SE cells. Transduced cells were confirmed to be 85% or more by visual inspection using a fluorescent microscope. MDCK-SE cells were expanded under the same culture conditions and then cryopreserved under liquid nitrogen using a commercially available cell freezing solution such as Bambanker.

[0118] (Example 2) Preparation of Compromised Huh-7 Cells (Huh-7-SE) Compromised Huh-7 cells were prepared using Huh-7 cells provided by a distributing organization. Specifically, after thawing the Huh-7 cells, they were subcultured for two passages and then plated at 5 × 10 cells per well on a 6-well plate. ６Cells were seeded at 1000 cells / well, and a long-lasting intracellular Sendai virus vector containing a puromycin resistance gene was added. Culture was performed in DMEM medium containing 10% FBS and 1% NEAA. Two weeks after the start of viral vector transduction, only transfected cells were selected using puromycin to produce Huh-7-SE cells. These cells were expanded and then cryopreserved under liquid nitrogen using a commercially available cell freezing solution (Bambanker).

[0119] (Example 3) Preparation of infection-compromised immortalized monocytic cells (aMylc-SE) derived from human peripheral blood (Preparation of immortalized monocytic cells derived from human peripheral blood) Human immortalized monocytic cells derived from human peripheral blood were prepared with reference to previous reports (WO 2012 / 043651 and JP 2017-131136 A). Specifically, a CD14-positive fraction was extracted from human peripheral blood, and c-Myc, BMI-1, and genes reported in the human immortalized monocytic cell report were introduced into a lentiviral vector, which was then used to introduce the vector into CD14-positive cells to produce the cells. Human peripheral blood-derived immortalized monocytic cells were cultured in α-MEM medium containing 20% ​​FBS, 50 ng / ml M-CSF, and 50 ng / ml GM-CSF. Proliferative cells were harvested 2 to 5 weeks after the start of culture and stored under liquid nitrogen using a commercially available cell preservation solution.

[0120] (Confirmation of prepared cells) To confirm that the prepared cells were monocyte-like cells, morphological observations such as evaluation of color by concentration and measurement of surface markers were performed before infection evaluation. Evaluation of color by concentration was performed by visually observing the color of the cells when they were collected in a centrifuge tube. Surface marker measurement was performed using a CD14 antibody (both Biolegend) and a BD flow cytometer, Accuri.

[0121] (Preparation of human peripheral blood-derived immortalized monocytic cells (aMylc-SE)) The prepared human peripheral blood-derived immortalized monocytic cells were seeded at 5 x 10E5 cells / mL in a 24-well plate, and a long-lasting intracellular Sendai virus vector containing a drug resistance gene was added. The cells were then cultured in α-MEM medium containing 20% ​​FBS, 50 ng / mL M-CSF, and 50 ng / mL GM-CSF. One week after the start of viral vector transduction, only transfected cells were selected using a drug, and these cells were designated aMylc-SE cells. After expansion, they were cryopreserved under liquid nitrogen using a commercially available cell freezing solution.

[0122] Example 4: Preparation of Immortalized Myeloid Cells (iMylc-SE) Derived from Human Induced Pluripotent Stem Cells (Preparation of Immortalized Myeloid Cells Derived from Human Induced Pluripotent Stem Cells) Immortalized myeloid cells derived from human induced pluripotent stem (iPS) cells were prepared by reference to the previously published method for preparing immortalized human induced pluripotent stem (iPS) cells (iPS-ML) (WO 2012 / 043651 and JP 2017-131136 A). Specifically, undifferentiated iPS cells were seeded onto a basement membrane pre-coated with Matrigel, such as laminin 511, and differentiation-inducing culture was initiated using α-MEM medium (containing 20% ​​FBS). Culture was continued thereafter, with the α-MEM medium (containing 20% ​​FBS) replaced every three days. Eighteen days after the start of differentiation induction, cells were treated with trypsin-EDTA (ethylenediaminetetraacetic acid)-collagenase solution (37°C, 60 minutes) to dissociate and recover, and a cell suspension was prepared by pipetting. Subsequently, cells from one 10 cm diameter dish were suspended in 10 ml of α-MEM medium (containing 10% FBS) and seeded onto two 10 cm diameter dishes without feeder cells or gelatin coating, and allowed to stand. After 3 hours, cells that had not adhered to the dishes were recovered and passed through a 100-micrometer mesh (BD Falcon Cell Strainer) to obtain a cell suspension from which aggregated cell clumps had been removed.

[0123] The cell suspension obtained above was suspended in α-MEM medium (containing 20% ​​FBS, 50 ng / ml M-CSF, and 100 ng / ml GM-CSF) and cultured. After approximately 3 to 9 days, floating or weakly adherent cells appeared, and a gradual increase in cell number was observed. These floating cells (iPS-MCs) were collected and the expression of the leukocyte marker CD45 and the myeloid cell markers CD11b and CD33 was confirmed using a flow cytometer.

[0124] The suspension cells (iPS-MCs) obtained above were transfected with c-Myc, BMI-1, and / or genes reported in the human immortalized monocytic cells report via lentivirus, and these lentiviral vectors were used to generate immortalized myeloid cells derived from human induced pluripotent stem cells. These cell lines were cultured in α-MEM medium containing 20% ​​FBS, 50 ng / ml M-CSF, and 50 ng / ml GM-CSF. After 2 to 5 weeks of culture, proliferative cells were obtained and stored under liquid nitrogen using a commercially available cell preservation solution.

[0125] (Confirmation of Prepared Cells) To confirm that the prepared iPS cell-derived immortalized myeloid cells (iMylc) were myeloid cells, morphological observations such as color evaluation by concentration and measurement of surface markers were performed before infection evaluation. Color evaluation by concentration was performed by visually observing the color of the cells when they were collected in a centrifuge tube. Surface marker measurement was performed using a BD flow cytometer, Accuri, using a CD14 antibody (both Biolegend).

[0126] (Preparation of iPS cell-derived, infection-resistant, immortalized human myeloid cells (iMylc-SE)) The prepared iPS cell-derived immortalized myeloid cells were seeded at 5 x 10E5 cells / mL in a 24-well plate, and a long-lasting intracellular Sendai virus vector containing a drug resistance gene was added. The cells were then cultured in α-MEM medium containing 20% ​​FBS, 50 ng / mL M-CSF, and 50 ng / mL GM-CSF. One week after the start of viral vector transduction, only transfected cells were selected using a drug, and these cells were designated aMylc-SE cells. After expansion, they were cryopreserved under liquid nitrogen using a commercially available cell freezing solution.

[0127] Example 5: Preparation of iPS cell-derived, susceptible, immortalized human myeloid cells (cMylc-SE) for novel coronavirus research. Following the method of Shimizu et al. (Scientific Reports 11(1):23713), iPS cell-derived, immortalized human myeloid cells for novel coronavirus research can be prepared by introducing the ACE2 and TMPRSS2 genes into iPS cell-derived immortalized myeloid cells. These cells were seeded at 5 x 10E5 cells / mL in a 24-well plate, supplemented with a long-lasting intracellular Sendai virus vector containing a drug resistance gene, and cultured in α-MEM medium containing 20% ​​FBS and growth factors. One week after the start of viral vector transduction, only transfected cells were selected using a drug. These cells were designated cMylc-SE cells, expanded, and then cryopreserved under liquid nitrogen using a commercially available cell freezing solution.

[0128] Example 6: Preparation of Compromised Immortalized Myeloid Cells (iMylc-SE2) Derived from Human Induced Pluripotent Stem Cells iPS-MCs were prepared by reference to previously reported methods for preparing immortalized human induced pluripotent stem (iPS) cell-derived cells (iPS-ML) (International Publication No. WO 2012 / 043651 and JP 2017-131136 A). Subsequently, long-lasting Sendai virus was introduced with c-Myc, BMI-1, and / or genes and / or growth factors (e.g., GM-CSF) reported for human immortalized monocytic cells. Using this Sendai virus vector, compromised immortalized myeloid cell lines (cMylc-SE2) derived from human induced pluripotent stem cells were prepared. This cell line was cultured in α-MEM medium containing 20% ​​FBS, 50 ng / ml M-CSF, and 50 ng / ml GM-CSF. After 2 to 5 weeks of culture, the cells were harvested as proliferative cells and stored under liquid nitrogen using a commercially available cell preservation solution.

[0129] Example 7: Preparation of Immortalized Myeloid Cells (cMylc-SE2) Derived from Human Induced Pluripotent Stem Cells for Novel Coronavirus Research. Immortalized myeloid cells (cMylc-SE2) derived from human induced pluripotent stem cells were prepared for novel coronavirus research by introducing the ACE2 and TMPRSS2 genes into immortalized myeloid cells (iMylc-SE2) derived from human induced pluripotent stem cells according to the method of Shimizu et al. (Scientific Reports 11(1):23713). These cells were cultured in α-MEM medium containing 20% ​​FBS, 50 ng / ml M-CSF, and 50 ng / ml GM-CSF. After 2 to 5 weeks of culture, the cells were harvested as proliferative cells and stored under liquid nitrogen using a commercially available cell preservation solution.

[0130] Example 8 Evaluation of interferon production by human induced pluripotent stem cell-derived immortalized myeloid cells (cMylc-SE2) for novel coronavirus research. The prepared human induced pluripotent stem cell-derived immortalized myeloid cells (cMylc-SE2) for novel coronavirus research were plated at 2 x 10 ５The cells were seeded at 1000 cells / well, and the novel coronavirus (SEARS-CoV2, Omicron strain BA5) was added and cultured for 3 days. VEROE6 / TMPRSS2 cells were also used as controls. After 3 days, the culture supernatant was collected and IFN-β and λ in the supernatant were measured by ELISA.

[0131] The results are shown in Figure 2. The novel coronavirus (Omicron strain, BA5) was contacted with cMylc-SE2 cells (cMylc-SE2-PhF, cMylc-SE2-D05) prepared from two types of iPS cells and Vero cells, and cultured for three days. The amounts of IFN-β and λ in the culture supernatant were measured by ELISA. The Y-axis represents the OD value obtained by ELISA. As a result, Vero cells did not produce interferon-β or λ upon infection with the novel coronavirus. On the other hand, cMylc-SE2 cells (cMylc-SE2-PhF, cMylc-SE2-D05) prepared from two types of iPS cells significantly produced interferon-β and λ. For example, with cMylc-SE2-PhF, the amount of interferon-β produced was 1.6 (OD value), and the amount of interferon-λ produced was 0.3 (OD value).

[0132] Example 9: Evaluation of exogenous interferon response of human induced pluripotent stem cell-derived immortalized myeloid cells (cMylc-SE2) for novel coronavirus research Two types of Mylc cells (cMylc-Kα) before processing for infection and cMylc-SE2 cells (cMylc-SE2-D05) after processing for infection were cultured at a final cell count of 3 × 10 ７ The cells were seeded in a 10-cm dish or a T25 flask at a density of 10 cells / well. Each well was filled with α-MEM medium containing 10% or 20% FBS supplemented with type I interferon (interferon α: final concentrations of 0, 100, and 1000 U / mL), or with α-MEM medium alone, and incubated at 37°C in 5% CO. ２ The cells were cultured in an incubator. 24 hours after the start of interferon α addition, RNA extracted from the cells was used as a template to analyze the mRNA expression level of the downstream gene of type I interferon receptor (Interferon Stimulated Gene 15: ISG15) by real-time PCR.

[0133] The results are shown in Figure 3. In Mylc cells (cMylc-Kα) before the compromised cell line, interferon α treatment increased the expression level of ISG15 mRNA by more than 1,000-fold compared to untreated cells. On the other hand, no change in the expression level of ISG15 mRNA was observed in cMylc-SE2 cells (cMylc-SE2-D05) after the compromised cell line.

[0134] The results of Examples 8 and 9 show the effect of infection-compromising processing based on the report by H. Xia et al. (Cell Rep, 2020 Oct. 6, 33(1):108234) (Figure 4). The effect of the cell-dwelling viral vector is to block the antiviral mechanism stimulated by interferon, such as ISG15, in response to external (exogenous) addition, rather than interferon production.

[0135] (Example 10) Dengue virus infection test of human iPS cell-derived immortalized myeloid cells (iMylc-SE2, cMylc-SE2) and VeroE6 cells. 3 × 10 iMylc-SE2 cells (iMylc-SE2-XF) and cMylc-SE2 cells (cMylc-SE2-D05) and VeroE6 cells after infection were infected. ４ Dengue virus (types 1 to 4) was added to the culture supernatant and allowed to infect for 2 hours. After that, medium was added and the cells were incubated at 37°C and 5% CO ２ The cells were cultured in an incubator. During this time, the infected dengue virus multiplied within the cells, and progeny particles were released into the culture supernatant. After 72 hours, the amount of dengue virus RNA was quantified by real-time PCR using RNA extracted from the culture supernatant as a template.

[0136] The results are shown in Figure 5. Compared with VeroE6 cells, which have traditionally been used as host cells for dengue virus, iMylc-SE2 cells (iMylc-SE2-XF) and cMylc-SE2 cells (cMylc-SE2-D05) had higher amounts of viral RNA in the culture supernatant, demonstrating their suitability as virus-infected cells.

[0137] Example 11: Empox virus infection test of human iPS cell-derived immortalized myeloid cells (iMylc-SE2, cMylc-SE2) and Vero cells. 5 × 10 iMylc-SE2 cells (iMylc-SE2-XF) and cMylc-SE2 cells (cMylc-SE2-D05) and Vero cells were infected with the virus. ４ The cells were seeded in a 96-well plate at 100 cells / well. Empox virus was added to the culture supernatant and allowed to infect for 1 hour. After that, the virus solution was removed, and a compound-containing medium was added. The cells were then incubated at 37°C, 5% CO ２ The cells were cultured in an incubator. During this time, the infected empoxvirus proliferated within the cells, producing progeny particles. 24 hours later, the amount of empoxvirus DNA was quantified by real-time PCR using DNA extracted from the cells as a template.

[0138] The results are shown in Figure 6. Compared to Vero cells, which have traditionally been used as host cells for empox viruses, iMylc-SE2 cells (iMylc-SE2-XF) and cMylc-SE2 cells (cMylc-SE2-D05) had the same or higher intracellular viral DNA levels and were highly sensitive to antiviral agents, demonstrating their suitability as virus-infected cells.

[0139] Example 12 Virus isolation test using human iPS cell-derived immortalized myeloid cells (cMylc-SE2) for novel coronavirus research and VeroE6 cells. cMylc cells (cMylc-Kα), immortalized myeloid cells for novel coronavirus research derived from human iPS cells before processing to make them susceptible to infection, cMylc-SE2 cells (cMylc-SE2-D05), immortalized myeloid cells for novel coronavirus research derived from human iPS cells after processing to make them susceptible to infection, and VeroE6 / TMPRSS2 cells were used to isolate 3 × 10 ４ Throat swab or saliva samples collected from COVID-19 patients were added to three wells each and incubated at 37°C, 5% CO ２The cells were cultured in an incubator. After one week, the amount of novel coronavirus RNA was quantified by real-time PCR using RNA extracted from the culture supernatant of each well as a template.

[0140] The results are shown in Figure 7. Wells with qPCR quantification and virus levels of 10E3 Copies / µL or more are marked with a +. The results demonstrated that, although some samples were able to be isolated using unprocessed cMylc cells, the infection-compromised cMylc-SE2 cells were significantly more effective at isolating viruses. Furthermore, the results demonstrated a similar success rate of isolation to that of VeroE6 / TMPRSS2 cells, which are conventionally used for virus isolation.

[0141] Example 13: Test of infection efficacy of human iPS cell-derived new type immortalized myeloid cells (iMylc) and human iPS cell-derived new type immortalized myeloid cells (iMylc) that have been made susceptible to infection Two strains of human iPS cell-derived immortalized myeloid cells, iMylc (strain D (denoted as "iMylc-D") and strain X (denoted as "iMylc-X")), were seeded at 5 x 10E5 cells / mL in a 24-well plate, and a long-lasting intracellular Sendai virus vector containing a drug resistance gene was added, followed by culture in α-MEM medium containing 20% ​​FBS and growth factors. One week after the start of viral vector transduction, only the transfected cells were selected using a drug, and cells of the cell line that had been made susceptible to infection were prepared.

[0142] Infection evaluation using single-infectious dengue virus type 2 virus particles (D2-SRIPs) was performed as described below. D2-SRIPs were prepared based on the literature of Yamanaka et al. (J Virol Methods. 2023 Jan;311:114641). D2-SRIPs were diluted in 10% FBS-containing α-MEM medium at six 2-fold serial dilutions from 20% to 0.625%. 50 μL of the prepared D2-SRIP diluted solutions were added to two wells of a 96-well plate at each concentration. Next, each cell was prepared at 4 x 10E5 cells / mL, and 50 μL was added to each well. The 96-well plate containing D2-SRIPs and iMylc cells before and after processing was incubated at 37°C and 5% CO. ２The plate was placed in an incubator and cultured for 24 hours, after which the plate was removed, the culture supernatant was removed, and a cell lysate was prepared, to which a luminoassay reagent was added and evaluation was carried out.

[0143] The results are shown in Figure 8. The X axis represents the dilution concentration of D2-SRIPs, and the Y axis represents the amount of luminescence detected by a luminometer, expressed in RLU. The results showed that the infectivity of iMylc cells after processing was enhanced compared to that of iMylc cells before processing for compromised infection.

[0144] The present invention can be widely used in various fields, for example, in fields related to medicine, vaccines, virus research, etc. This application is based on Japanese Patent Application No. 2024-057807 (filing date: March 29, 2024), the contents of which are incorporated in full herein.

Claims

1. A method for producing a virus-susceptible cell, comprising the step of infecting a host cell with a first virus or a vector thereof, wherein the first virus is a long-lived intracellular virus.

2. The method of claim 1, wherein the first virus is present in a cell at a location other than the genome.

3. The method of claim 1 or 2, wherein the first virus is present in the cytoplasm.

4. The method according to claim 1 or 2, wherein the first virus is at least one selected from the group consisting of hepatitis virus, cytomegalovirus, adenovirus, adeno-associated virus and Sendai virus.

5. The method of claim 1 or 2, wherein the host cells are cells with innate immunity.

6. The method of claim 1 or 2, wherein the host cell is a cell of mammalian origin.

7. The method of claim 1 or 2, wherein the host cell is at least one selected from the group consisting of immortalized myeloid cells, Vero cells, MDCK cells, Huh7 cells, and HEK293 cells.

8. A method for isolating a virus, comprising the steps of: preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof; contacting the virus-susceptible cells with a sample containing a second virus derived from a subject; growing the second virus in the virus-susceptible cells; and recovering the second virus from the virus-susceptible cells after the growth step.

9. The method according to claim 8, wherein the step of preparing a virus-susceptible cell comprises a step of infecting a host cell with a long-lasting intracellular virus or a vector thereof.

10. The method of claim 8 or 9, wherein the second virus is a virus of a different species than the first virus.

11. A method for screening an antiviral drug, comprising the steps of: preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof; infecting the virus-susceptible cells with a second virus; contacting the virus-susceptible cells after the infection step with a test substance; and measuring the infection level of the second virus in the virus-susceptible cells.

12. The method according to claim 11, further comprising the following step: comparing the infection level of the second virus when the test substance is contacted with the virus-susceptible cells with the infection level of the second virus when the test substance is not contacted with the virus-susceptible cells, and determining that the test substance has antiviral activity if the infection level of the second virus when the test substance is contacted is lower than the infection level of the second virus when the test substance is not contacted.

13. A method for producing a vaccine, comprising the steps of: preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof; infecting the virus-susceptible cells with a second virus; propagating the second virus in the virus-susceptible cells; and recovering the second virus from the virus-susceptible cells after the propagation step; and further comprising the step of recovering the virus obtained after repeating the infection step, propagation step, and recovery step at least five times.

14. A method for producing a vaccine, comprising the steps of: preparing virus-susceptible cells containing a long-lasting intracellular virus or a vector thereof; infecting the virus-susceptible cells with a second virus; propagating the second virus in the virus-susceptible cells; and recovering the second virus from the virus-susceptible cells after the propagation step.

15. A method for propagating a virus, comprising the steps of: preparing a virus-susceptible cell containing a long-lasting intracellular virus or a vector thereof; and infecting the virus-susceptible cell with a second virus.

16. A virus-susceptible cell containing a first virus or a vector thereof, wherein the first virus is a long-lived intracellular virus, and the first virus is present in a location other than the genome of the cell.

17. A kit comprising the virus-susceptible cells of claim 16.

18. The kit of claim 17 for use in virus isolation, drug evaluation, vaccine development or vaccine production.

Images