JP7079901B1 - Method of producing extracellular particles - Google Patents
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Abstract
【課題】ブタの感染症ワクチンに利用可能な細胞外粒子の産生方法を提供する。【解決手段】PRRSウイルスが感染し得るブタの細胞に、moi (multiple of infection) 1.0~0.01の濃度でウイルスを感染させると同時にインターフェロン（INF）を添加し、その後洗浄して溶解した細胞を除き、溶解せずに培養器に残った細胞（ウイルス持続感染細胞が多く残った細胞）に培養液を加える。この洗浄と培養液を加える工程を所定回数繰り返し、残ったウイルス持続感染細胞から細胞外粒子を分離精製する。尚、細胞には個体差（個性）があり、インターフェロン（IFN）への感受性も異なる。このため実験条件が同じでも全て持続感染細胞となるわけではない。図2の実験2では、同じ実験条件でもウイルス持続感染細胞とならない例を示している。そのため、ウイルスに感染させた後に細胞が発現するタンパク質を解析し、ワクチン化に最も有望な持続感染細胞を選択しクローン化する必要がある。【選択図】 図１PROBLEM TO BE SOLVED: To provide a method for producing extracellular particles which can be used as a vaccine for infectious diseases of pigs. SOLUTION: The cells of pigs that can be infected with the PRRS virus are infected with the virus at a concentration of moi (multiple of infection) 1.0 to 0.01, and at the same time, interferon (INF) is added, and then washed to remove the lysed cells. , Add the culture solution to the cells that remained in the incubator without lysis (cells in which many persistent virus-infected cells remained). The steps of washing and adding the culture solution are repeated a predetermined number of times to separate and purify the extracellular particles from the remaining virus-persistent infected cells. In addition, cells have individual differences (individuality), and their susceptibility to interferon (IFN) is also different. Therefore, even if the experimental conditions are the same, not all cells become persistently infected cells. Experiment 2 in FIG. 2 shows an example in which the cells do not become persistently infected cells even under the same experimental conditions. Therefore, it is necessary to analyze the proteins expressed by cells after infection with the virus, and to select and clone persistently infected cells that are most promising for vaccination. [Selection diagram] Fig. 1
Description
本発明は、ブタの肺細胞または上皮細胞から感染症免疫ワクチンを製造する出発物質としての細胞外粒子（Extracellular Vesicle；EV）を製造する方法に関する。
この細胞外粒子にはエクソゾーム（Exosome）の他にマイクロベシクル、アポトーシス小体などが含まれるが、性状的に極めて類似しており、２０１６年の細胞外粒子国際会議では、一括して細胞外粒子と呼ぶことが提唱されている。そこで、本明細書では細胞外粒子をエクソゾーム（Exosome）を含んだ意味で用いる。
The present invention relates to a method for producing extracellular particles (EV) as a starting material for producing an infectious disease immune vaccine from porcine lung cells or epithelial cells.
In addition to exosomes, these extracellular particles include microvesicles, apoptotic bodies, etc., but they are very similar in nature. At the 2016 International Conference on Extracellular Particles, extracellular particles were collectively used. It is proposed to call it. Therefore, in the present specification, extracellular particles are used in the sense of including exosomes.
PRRS（豚繁殖・呼吸障害症候群：Porcine Reproductive and Respiratory Syndrome）は、PRRSウイルスを原因とする感染症であり、世界の養豚業に大きな経済的損失を与えている疾病の1つであり、届出伝染病に指定されている。 PRRS (Porcine Reproductive and Respiratory Syndrome) is an infectious disease caused by the PRRS virus, one of the diseases that causes great economic loss to the pig farming industry in the world, and is transmitted by notification. It is designated as a disease.
エクソゾームなどの細胞外粒子は、細胞内に存在する膜小胞が細胞外に分泌された直径が３０～２００ｎｍの粒子で、内部には核酸（マイクロＲＮＡ、ｍＲＮＡ、ＤＮＡなど）の他に細胞内のタンパク質と同じタンパク質を含んでいる Extracellular particles such as exosomes are particles with a diameter of 30 to 200 nm in which membrane vesicles existing inside the cell are secreted outside the cell, and inside the cell, in addition to nucleic acids (microRNA, mRNA, DNA, etc.). Contains the same protein as the protein in
ウイルスに感染した細胞から分泌されるエクソゾームには、ウイルス特有のタンパク質が含まれている。そこで、ウイルスに感染から分泌されたエクソゾームを樹状細胞（dendritic cell）と融合させると、融合した樹状細胞はウイルス特有のタンパク質を分泌するため融合細胞は抗原提示細胞（ＡＰＣ）として機能する。つまり、融合細胞はワクチンとして機能する。 Exosomes secreted by virus-infected cells contain virus-specific proteins. Therefore, when the exosome secreted from the virus infection is fused with the dendritic cell, the fused dendritic cell secretes a protein peculiar to the virus, and the fused cell functions as an antigen-presenting cell (APC). That is, the fused cells function as a vaccine.
このようなエクソゾームをがんの免疫療法に用いる提案が、特許文献１及び特許文献２になされている。これら特許文献には、がん細胞から放出されたエクソゾームを樹状細胞やＴ細胞などの免疫細胞に電気穿孔処理（エレクトロポレーション）によって挿入して融合させ、これをワクチンとして患者に投与することが開示されている。 Proposals for using such exosomes for cancer immunotherapy have been made in Patent Documents 1 and 2. In these patent documents, exosomes released from cancer cells are inserted into immune cells such as dendritic cells and T cells by electroporation and fused, and this is administered to patients as a vaccine. Is disclosed.
特許文献３には、ウイルス持続感染細胞を用いてワクチン（ウイルス抗原）を製造すること、ウイルス持続感染細胞を得るためにウイルスレセプターを有する細胞（株化細胞）に直接感染させる方法（cell-free感染法）が開示されている。 Patent Document 3 describes a method (cell-free) of producing a vaccine (virus antigen) using persistent virus-infected cells and directly infecting cells having a virus receptor (strained cells) in order to obtain persistent virus-infected cells. Infection Law) is disclosed.
特許文献４には、エクソゾームにヒートショックを与え、エクソゾーム内に免疫細胞への親和性を高めるヒートショック誘導タンパク質を形成することが開示されている。 Patent Document 4 discloses that an exosome is subjected to heat shock to form a heat shock-inducing protein in the exosome that enhances the affinity for immune cells.
非特許文献１には、ウイルス（SARS-CoV-2 COVID-19ウイルス）がＡＣＥ受容体を介して胚細胞に感染し、ウイルスのタンパク質や核酸がエクソゾームに封入されて細胞外に分泌されること、エクソゾームが免疫細胞応答を誘導すること、エクソゾームを抗ウイルスワクチンとして使用し得ることが記載されている。 Non-Patent Document 1 states that a virus (SARS-CoV-2 COVID-19 virus) infects embryo cells via ACE receptors, and viral proteins and nucleic acids are encapsulated in exosomes and secreted extracellularly. It has been described that exosome induces an immune cell response and that exosome can be used as an antiviral vaccine.
非特許文献２には、エクソゾームを抗ウイルスワクチンとして使用し得ることが記載されている。
更に、非特許文献３には、コロナウイルスが持続感染する症例が挙げられ、非特許文献４及び非特許文献５には、ウイルス感染細胞から細胞外粒子が分泌されることについて記載されている。
Non-Patent Document 2 describes that exosomes can be used as an antiviral vaccine.
Further, Non-Patent Document 3 includes a case of persistent infection with coronavirus, and Non-Patent Document 4 and Non-Patent Document 5 describe that extracellular particles are secreted from virus-infected cells.
上述したように、エクソゾーム（細胞外粒子）はそれが放出された元の細胞に含まれるタンパク質を含んでいる。元の細胞がウイルスに感染していれば当該ウイルスが産生するタンパク質も含まれる。 As mentioned above, exosomes (extracellular particles) contain the proteins contained in the original cell from which they were released. If the original cells are infected with the virus, the proteins produced by the virus are also included.
そこで、ウイルスに感染した細胞から分泌されるエクソゾーム（細胞外粒子）を感染症免疫ワクチンまたはその出発物質として利用することが考えられる。しかしながら、これを感染症の免疫ワクチンとして実現するには、ウイルスが産生するタンパク質を含むエクソゾーム（細胞外粒子）を安定して分泌する細胞が必要となる。 Therefore, it is conceivable to use exosomes (extracellular particles) secreted from virus-infected cells as an infectious disease immune vaccine or a starting material thereof. However, in order to realize this as an immune vaccine for infectious diseases, cells that stably secrete exosomes (extracellular particles) containing proteins produced by the virus are required.
従来技術を組み合わせることにより、エクソゾーム（細胞外粒子）を安定して分泌する細胞としてウイルス持続感染細胞を選定し、この持続感染細胞が分泌するエクソゾーム（細胞該粒子）をワクチンとすること、若しくは樹状細胞と融合することを想定することができる。 By combining conventional techniques, virus persistently infected cells are selected as cells that stably secrete exosomes (extracellular particles), and the exosomes (cells said particles) secreted by these persistently infected cells are used as vaccines, or trees. It can be assumed that it fuses with dendritic cells.
パンデミックを想定した場合、大量のワクチンを短時間で製造することが要求される。ウイルス持続感染細胞をワクチンの供給源とする場合には、ウイルス持続感染細胞を短時間で大量に産生する必要がある。
しかしながら、従来技術はウイルス持続感染細胞を効率よく短時間で大量に産生する手段については何ら示唆していない。
Assuming a pandemic, it is required to produce a large amount of vaccine in a short time. When virus persistently infected cells are used as a vaccine source, it is necessary to produce a large amount of virus persistently infected cells in a short time.
However, the prior art does not suggest any means for efficiently producing a large amount of persistent virus-infected cells in a short time.
ここで、細胞がウイルスに接すると細胞はインターフェロン（IFN）を発現し、ウイルス感染が阻止され、例外的にインターフェロン（IFN）によるウイルス阻止機能が部分的にしか働かない細胞があり、この細胞がウイルス持続感染細胞となる。即ち、ウイルスに感染してはいるが細胞としての機能を発揮し、細胞外粒子を分泌する細胞である。
しかしながら、インターフェロン（IFN）によるウイルス阻止機能が部分的にしか働かない細胞の出現を待っていたのでは、ワクチンとするための細胞外粒子を短期間で大量に生産することができず、感染症対策に遅れをとることになる。
Here, when a cell comes into contact with a virus, the cell expresses interferon (IFN), and virus infection is blocked. Exceptionally, there are cells in which the virus blocking function by interferon (IFN) works only partially. It becomes a virus persistently infected cell. That is, it is a cell that is infected with a virus but exerts a function as a cell and secretes extracellular particles.
However, waiting for the emergence of cells in which the virus-blocking function of interferon (IFN) works only partially makes it impossible to produce large quantities of extracellular particles for vaccines in a short period of time, resulting in infectious diseases. You will be late for the measures.
細胞には、ウイルス感染した場合にインターフェロン（IFN）が強く発現する細胞と発現力が弱い細胞があり、線維芽細胞（真皮線維芽細胞）などはインターフェロン（IFN）が強く発現され、ウイルス感染が阻止され持続感染系になりにくい。 There are cells that strongly express interferon (IFN) and cells that have weak expression when infected with a virus, and fibroblasts (dermal fibroblasts) etc. strongly express interferon (IFN) and cause viral infection. It is difficult to be blocked and become a persistent infectious system.
一方、上皮細胞（Epithelial cell）や癌腫（Carcinoma）系の細胞はウイルス感染によるインターフェロン（IFN）の発現は弱く、ウイルスが感染すると細胞の溶解が起こり持続感染系になりにくい。 On the other hand, epithelial cells and Carcinoma cells have weak expression of interferon (IFN) due to virus infection, and when the virus is infected, the cells are lysed and it is difficult to become a persistent infection system.
インターフェロン（IFN）に着目してウイルス持続感染細胞を人為的に作成する場合、線維芽細胞（真皮線維芽細胞）などについてはインターフェロン（IFN）の働きを弱めることが考えられる。インターフェロン（IFN）の働きを弱める（阻害する）タンパク質やＲＮＡはあるが、コントロールが極めて困難で、実験では全ての線維芽細胞はウイルス感染してもウイルス抵抗性を示し、ウイルス抗原は極少量かもしくは全く発現していなかった。即ち、線維芽細胞（真皮線維芽細胞）から持続感染細胞を作成するのは困難である。 When artificially creating virus-sustained infected cells focusing on interferon (IFN), it is possible to weaken the action of interferon (IFN) for fibroblasts (dermal fibroblasts) and the like. There are proteins and RNA that weaken (inhibit) the action of interferon (IFN), but they are extremely difficult to control. Or it was not expressed at all. That is, it is difficult to create persistently infected cells from fibroblasts (dermal fibroblasts).
一方、上皮細胞（Epithelial cell）や癌腫（Carcinoma）系の細胞については、インターフェロン（IFN）を強く発現させることで人為的にウイルス持続感染細胞とすることができる可能性がある。インターフェロン（IFN）を強く発現させるにはインターフェロン（IFN）を細胞に供給すればよく、線維芽細胞（真皮線維芽細胞）に比べてコントロールが容易である。 On the other hand, epithelial cells and carcinoma cells may be artificially made into persistent virus-infected cells by strongly expressing interferon (IFN). In order to strongly express interferon (IFN), interferon (IFN) may be supplied to cells, and it is easier to control than fibroblasts (dermal fibroblasts).
本発明者はウイルス持続感染細胞から分泌される細胞外粒子はワクチンの材料になり得ること、ウイルス持続感染細胞になるか否かにはインターフェロン（IFN）が影響していること、細胞にはウイルスに感染した際にインターフェロン（IFN）の発現が強い種類と弱い種類がある知見に基づき本発明を成したものである。 The present inventor states that extracellular particles secreted from persistently infected cells can be used as a material for vaccines, that interferon (IFN) affects whether or not persistently infected cells are infected with the virus, and that the cells are infected with the virus. The present invention was made based on the finding that there are types in which the expression of interferon (IFN) is strong and types in which the virus is weakly expressed when infected with virus.
本発明は、以下の工程で感染症免疫のワクチンの材料となり得る細胞外粒子を産生する。
工程１：ウイルスとしてPRRS（豚繁殖・呼吸障害症候群：Porcine Reproductive and Respiratory Syndrome）ウイルスを用意する。
工程２：ウイルスが感染し得る細胞としてブタの上皮細胞またはブタの肺細胞を用意する。
工程３：工程２で用意した細胞に、moi (multiple of infection) ０．０１～１．０の割合でウイルスを感染させ、このウイルス感染と同時にインターフェロン（IFN）を添加してウイルス増殖を抑制する。
添加するインターフェロン（IFN）としては、Type1 IFNまたはType 2 IFNをウイルス感染細胞に添加しウイルス増殖を抑制させる。Type1 IFNまたはType 2 IFNの濃度は1ng/ml～50ng/mlが好ましい。
工程４：1～2日経過後に、工程３でウイルス感染させた細胞を洗う。
工程５：工程４で溶解せずに培養器に残った細胞（ウイルス持続感染細胞が多く残っている可能性が高い細胞）に、前記ウイルス由来のタンパク質が含まれているかを確認する。
工程６：前記ウイルス由来のタンパク質が含まれていることが確認できた細胞に培養液を加えて培養する。
工程７：工程６を少なくとも１回以上繰り返して、ウイルス感染持続細胞をクローン化する。
工程８：工程７で得たウイルス感染持続細胞からエクソゾームを含む細胞外粒子を分離精製する。
The present invention produces extracellular particles that can be used as a material for a vaccine for infectious disease immunity in the following steps.
Step 1: Prepare a PRRS (Porcine Reproductive and Respiratory Syndrome) virus as a virus.
Step 2: Pig epithelial cells or pig lung cells are prepared as cells that can be infected by the virus.
Step 3: The cells prepared in Step 2 are infected with a virus at a ratio of moi (multiple of infection) 0.01 to 1.0, and interferon (IFN) is added at the same time as this virus infection to suppress viral growth. ..
As the interferon (IFN) to be added, Type 1 IFN or Type 2 IFN is added to virus-infected cells to suppress virus growth. The concentration of Type 1 IFN or Type 2 IFN is preferably 1 ng / ml to 50 ng / ml.
Step 4: After 1 to 2 days, the virus-infected cells in Step 3 are washed.
Step 5: It is confirmed whether the cells derived from the virus are contained in the cells remaining in the incubator without being lysed in the step 4 (cells in which a large amount of persistent virus-infected cells are likely to remain).
Step 6: A culture solution is added to the cells confirmed to contain the virus-derived protein and cultured.
Step 7: Repeat step 6 at least once to clone the virus-infected persistent cells.
Step 8: Extracellular particles containing exosome are separated and purified from the virus-infected persistent cells obtained in Step 7.
工程３において、感染濃度をmoi ０．０１～１．０としたのは、moi 1.0よりも大きくなると、ウイルス感染拡大が速くなって殆どの細胞がウイルス産生のみを行い溶解するためであり、moi 0.01よりも小さくなると、感染しにくくなるためである。
また、添加するインターフェロン（IFN）濃度を1ng/ml～50ng/mlとするのは、1ng/ml未満ではウイルスにより細胞が溶解しやすく、50ng/mlを超えるとウイルスに感染しにくくなるためである。
In step 3, the infection concentration was set to moi 0.01 to 1.0 because when the infection concentration is higher than moi 1.0, the virus infection spreads faster and most cells only produce the virus and lyse. This is because if it is smaller than 0.01, it becomes difficult to infect.
The reason why the concentration of interferon (IFN) to be added is 1 ng / ml to 50 ng / ml is that cells are easily lysed by the virus when the concentration is less than 1 ng / ml, and it becomes difficult to infect the virus when the concentration exceeds 50 ng / ml. ..
また、細胞外粒子の分離精製については、一般的には超遠心分離法、またはカラム法を用いて行う。 Further, the separation and purification of extracellular particles are generally carried out by using an ultracentrifugation method or a column method.
本発明に係る細胞外粒子の産生方法によれば、細胞外粒子を分泌するウイルス持続感染細胞から短期間のうちに大量に生産することができる。具体的には、ウイルス持続感染細胞を確立させるまでに３～５日、細胞のクローン化に１～２カ月あれば十分である。 According to the method for producing extracellular particles according to the present invention, a large amount can be produced in a short period of time from a virus-persistent infected cell that secretes extracellular particles. Specifically, 3 to 5 days is sufficient to establish persistent virus-infected cells, and 1 to 2 months is sufficient for cell cloning.
ウイルス持続感染細胞となる前駆体細胞として、上皮細胞（Epithelial cell）や肺細胞（肺がん細胞）を採用したことで以下の効果を発揮する。
（１）線維芽細胞に比べ確立した細胞系は多数あり、細胞外粒子生産に適した細胞を選択するのが容易である。また、ウイルス感染に適合する細胞を選択する可能性が高くなる。
（２）タンパク分解酵素への抵抗性が強く細胞植え継ぎ（継代）は非常に容易である。
（３）すでに浮遊細胞系として確立された系が多く存在し、そのため細胞による物質生産を増やし、その物質を単離するのが容易になる。
（４）接着性の上皮細胞（Epithelial cell）や肺細胞を浮遊細胞へ変えるのが比較的容易である。
（５）細胞が丈夫である故、植え継ぎ時に死滅する細胞が少なく、多数の細胞を培養することが可能であり、細胞外粒子生産系としては線維芽細胞よりはるかに有利である。
The following effects are exhibited by adopting epithelial cells (Epithelial cells) and lung cells (lung cancer cells) as precursor cells that become persistent virus-infected cells.
(1) There are many established cell lines compared to fibroblasts, and it is easy to select cells suitable for extracellular particle production. It also increases the likelihood of selecting cells that are compatible with the viral infection.
(2) Strong resistance to proteolytic enzymes and cell transplantation (passage) is very easy.
(3) There are many systems that have already been established as floating cell lines, which increases the production of substances by cells and facilitates the isolation of the substances.
(4) It is relatively easy to convert adhesive epithelial cells and lung cells into floating cells.
(5) Since the cells are strong, few cells die at the time of subculture, and a large number of cells can be cultured, which is far more advantageous than fibroblasts as an extracellular particle production system.
本発明にかかるウイルス持続感染細胞が分泌した細胞外粒子は、そのままワクチンとして利用できる可能性があるが、ウイルス持続感染細胞が分泌した細胞外粒子を樹状細胞（DC）と融合させて利用することもできる。
この場合は融合樹状細胞を抗原提示細胞（APC）となり、ナイーブＴ細胞を教育し、獲得免疫およびウイルス感染の記憶（memory）を作ることが可能となる。
The extracellular particles secreted by the persistently infected virus cells according to the present invention may be used as they are as a vaccine, but the extracellular particles secreted by the persistently infected virus cells are used by fusing them with dendritic cells (DC). You can also do it.
In this case, the fused dendritic cells become antigen-presenting cells (APCs), which can educate naive T cells and create memory for acquired immunity and viral infection.
細胞外粒子を樹状細胞（DC）と融合させる手段としてはエレクトロポレーション法、ＰＥＧ（ポリエチレングリコール）法などが考えられる。 As a means for fusing extracellular particles with dendritic cells (DC), an electroporation method, a PEG (polyethylene glycol) method, or the like can be considered.
また本発明によれば、ウイルスの種類に左右されずまた変異したウイルスに対しても短期間のうちに大量の細胞外粒子を製造することが可能になる。 Further, according to the present invention, it becomes possible to produce a large amount of extracellular particles in a short period of time regardless of the type of virus and even for a mutated virus.
以下に本発明の実施例を説明する。
図１に示すように、ブタの上皮細胞またはブタの肺細胞に、PRRSウイルスを感染させる。
Examples of the present invention will be described below.
As shown in FIG. 1, pig epithelial cells or pig lung cells are infected with the PRRS virus.
上記のウイルス感染と同時に細胞にインターフェロンとしてType1 IFNまたはType 2 IFNを添加しウイルス増殖を抑制する。 At the same time as the above virus infection, Type 1 IFN or Type 2 IFN is added to the cells as interferon to suppress the virus growth.
感染させるコロナウイルスの濃度は、moi ０．０１～１．０とした。これは、moi 1.0よりも濃くすると、ウイルス感染拡大が速くなって殆どの細胞がウイルス産生のみを行い溶解するためであり、moi 0.01よりも薄くすると、感染しにくくなるためである。
また、添加するType1 IFNまたはType 2 IFNの濃度は1ng/ml～50ng/mlとした。これは、1ng/mlよりも希釈するとウイルス増殖を抑制できず細胞が溶解し、50ng/mlよりも濃くするとウイルスの感染が阻止される割合が高くなるためである。
The concentration of the coronavirus to be infected was moi 0.01 to 1.0 . This is because if it is thicker than moi 1.0, the virus infection spreads faster and most cells produce only the virus and lyse, and if it is thinner than moi 0.01, it becomes difficult to infect.
The concentration of Type 1 IFN or Type 2 IFN to be added was 1 ng / ml to 50 ng / ml. This is because if it is diluted more than 1 ng / ml, the virus growth cannot be suppressed and the cells are lysed, and if it is more than 50 ng / ml, the virus infection is blocked at a high rate.
感染させた細胞を培養器内で室温（25℃）で２４～４８時間培養し、培養器を洗浄する。
この洗浄で溶解した細胞は除去され、感染していない細胞とウイルス持続感染細胞が培養器内に残る。残った培養器に再び培養液を加え、再度培養する。
以上の洗浄と培養の操作を繰り返すことで、培養器内の細胞は殆どがウイルス持続感染細胞となる。繰り返し回数は任意であるが数回行えば十分である。
The infected cells are cultured in an incubator at room temperature (25 ° C.) for 24-48 hours, and the incubator is washed.
The cells lysed by this wash are removed, leaving uninfected cells and persistently infected cells in the incubator. Add the culture solution to the remaining incubator again and incubate again.
By repeating the above washing and culturing operations, most of the cells in the incubator become virus-persistent infected cells. The number of repetitions is arbitrary, but it is sufficient to repeat it several times.
上記によって得た培養器内の細胞（ウイルス持続感染細胞）にPRRSウイルス特異タンパク質が含まれているかは、蛍光免疫染色法で確認する。 Whether or not the cells in the incubator obtained by the above (virus persistently infected cells) contain the PRRS virus-specific protein is confirmed by fluorescent immunostaining.
図２は、PRRS（豚繁殖・呼吸障害症候群：Porcine Reproductive and Respiratory Syndrome）に対して本発明を適用した例である。実験方法は、以下の通りである。
（1）ブタの肺細胞に、PRRSウイルスをmoi 0.1で感染させ、同時に濃度10ng/ml のType1 IFNを添加し、37℃インキュベータに保存した。
（2）1日ごとに浮遊してきた細胞をピペットで取り除き、培養液（ダルベッコMEM 5％牛胎児血清含む）で洗浄した。
（3）（2）の作業を2～3日ごとに繰り返した。
（4）その後、5日ほどで浮遊細胞の出現はほとんど無くなり、細胞はシャーレに付着していた。
（5）感染細胞を抗PRRSウイルス、ポリクローン抗体で直接染色し、顕微鏡で観察した。
FIG. 2 shows an example in which the present invention is applied to PRRS (Porcine Reproductive and Respiratory Syndrome). The experimental method is as follows.
(1) Pig lung cells were infected with PRRS virus with moi 0.1, and at the same time, Type 1 IFN at a concentration of 10 ng / ml was added and stored in a 37 ° C incubator.
(2) The floating cells were removed with a pipette every day and washed with a culture medium (including Dulbecco MEM 5% fetal bovine serum).
(3) The work of (2) was repeated every 2 to 3 days.
(4) After that, the appearance of floating cells almost disappeared in about 5 days, and the cells were attached to the petri dish.
(5) Infected cells were directly stained with anti-PRRS virus and polyclone antibody and observed under a microscope.
感染後10日の感染細胞にはウイルス由来のタンパク質の発現がみられ、その後ウイルス由来のタンパク質の発現は徐々に失われたが、感染後60日経過した感染細胞からは安定してタンパク質が発現された。更に感染後90日まで観察したが、細胞形態、免疫染色図に変化は見られず、PRRSウイルスによる持続感染系が完成したと思われる。 Virus-derived protein was expressed in infected cells 10 days after infection, and then the expression of virus-derived protein was gradually lost, but the protein was stably expressed in infected cells 60 days after infection. Was done. Further observation was performed up to 90 days after infection, but no changes were observed in the cell morphology and immunostaining diagram, suggesting that the persistent infection system by PRRS virus was completed.
PRRSウイルスの濃度をmoi 0.1に固定し、添加するType1 IFNの濃度を1ng/ml、5ng/ml、25ng/ml、50ng/mlとし、他の条件を上記実験例と同様にして行った。
結果は、PRRSウイルスによる持続感染系が確認できた。
The concentration of PRRS virus was fixed at moi 0.1, the concentration of Type 1 IFN to be added was 1 ng / ml, 5 ng / ml, 25 ng / ml, 50 ng / ml, and other conditions were the same as in the above experimental example.
As a result, it was confirmed that the persistent infection system was caused by the PRRS virus.
Type1 IFNの濃度を10ng/mlに固定し、PRRSウイルスの濃度をmoi 0.01、moi 0.05、moi0.25、moi1.0とし、他の条件を上記実験例と同様にして行った。
結果は、PRRSウイルスによる持続感染系が確認できた。
The concentration of Type1 IFN was fixed at 10 ng / ml, the concentration of PRRS virus was moi 0.01, moi 0.05, moi0.25, and moi1.0, and other conditions were the same as in the above experimental example.
As a result, it was confirmed that the persistent infection system was caused by the PRRS virus.
ブタの肺細胞の代わりにブタの上皮細胞を用いて、上記と同様の検証を行ったところ、同じ結果が得られた。またIFNに関してはType1 IFNとType2 IFNに変化はなかった。 The same verification as above was performed using pig epithelial cells instead of pig lung cells, and the same results were obtained. Regarding IFN, there was no change between Type 1 IFN and Type 2 IFN.
上記の安定して発現しているタンパク質の中にワクチンとして使用できるタンパク質が存在しているかを検証し、これらのタンパク質を発現している感染持続細胞をクローン化（細胞集団の作製）する。 It is verified whether proteins that can be used as a vaccine exist among the above-mentioned stably expressed proteins, and persistent infected cells expressing these proteins are cloned (preparation of cell population).
細胞には個体差（個性）があり、インターフェロン（IFN）への感受性も異なる。このため実験条件が同じでも全て持続感染細胞となるわけではない。図2の実験2では、同じ実験条件でもウイルス持続感染細胞とならない例を示している。
そのため、ウイルスに感染させた後に細胞が発現するタンパク質を解析し、ワクチン化に最も有望な持続感染細胞を選択しクローン化する必要がある。
There are individual differences (individuality) in cells, and their susceptibility to interferon (IFN) is also different. Therefore, even if the experimental conditions are the same, not all cells become persistently infected cells. Experiment 2 in FIG. 2 shows an example in which the cells do not become persistently infected cells even under the same experimental conditions.
Therefore, it is necessary to analyze the proteins expressed by cells after infection with the virus, and to select and clone persistently infected cells that are most promising for vaccination.
Claims (1)
工程１：ウイルスとしてPRRS（豚繁殖・呼吸障害症候群：Porcine Reproductive and Respiratory Syndrome）ウイルスを用意する。
工程２：ウイルスが感染し得る細胞としてブタの上皮細胞またはブタの肺細胞を用意する。
工程３：工程２で用意した細胞に、moi (multiple of infection) ０．０１～１．０の割合でウイルスを感染させ、同時に1ng／ml～50ng／mlの濃度のインターフェロン（IFN）を添加する。
工程４：1～2日経過後に、工程３でウイルス感染させた細胞を洗う。
工程５：工程４で溶解せずに培養器に残った細胞（ウイルス持続感染細胞が多く残っている可能性が高い細胞）に、前記ウイルス由来のタンパク質が含まれているかを確認する。
工程６：前記ウイルス由来のタンパク質が含まれていることが確認できた細胞に培養液を加えて培養する。
工程７：工程６を少なくとも１回以上繰り返して、ウイルス感染持続細胞をクローン化する。
工程８：工程７で得たウイルス感染持続細胞からエクソゾームを含む細胞外粒子を分離精製する。 A method for producing extracellular particles, which comprises the following steps.
Step 1: Prepare a PRRS (Porcine Reproductive and Respiratory Syndrome) virus as a virus.
Step 2: Pig epithelial cells or pig lung cells are prepared as cells that can be infected by the virus.
Step 3: The cells prepared in Step 2 are infected with the virus at a ratio of moi (multiple of infection) 0.01 to 1.0 , and at the same time, interferon (IFN) at a concentration of 1 ng / ml to 50 ng / ml is added. ..
Step 4: After 1 to 2 days, the virus-infected cells in Step 3 are washed.
Step 5: It is confirmed whether the cells derived from the virus are contained in the cells remaining in the incubator without being lysed in the step 4 (cells in which a large amount of persistent virus-infected cells are likely to remain).
Step 6: A culture solution is added to the cells confirmed to contain the virus-derived protein and cultured.
Step 7: Repeat step 6 at least once to clone the virus-infected persistent cells.
Step 8: Extracellular particles containing exosome are separated and purified from the virus-infected persistent cells obtained in Step 7.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001097885A (en) * | 1999-10-01 | 2001-04-10 | Kyoritsu Shoji Kk | Feline retrovirus vaccine and method for preparing the same |
| JP2001299336A (en) * | 2000-04-28 | 2001-10-30 | Eiji Konishi | Preparation of flaviviruslike particle stable as vaccine and diagnostic antigen without toxicity |
| JP2006061057A (en) * | 2004-08-26 | 2006-03-09 | Kyoritsu Seiyaku Kk | Established cell for producing vaccine, and virus |
| JP2006230397A (en) * | 2005-02-24 | 2006-09-07 | Buddhist Tzu Chi General Hospital | Method for screening anti-flavivirus infectious compound by persistent flavivirus-infected cell line |
| JP2013523824A (en) * | 2010-04-06 | 2013-06-17 | ジョン ダブリュ． ホラデイ、 | How to treat cancer |
| JP2016082925A (en) * | 2014-10-27 | 2016-05-19 | 学校法人 岩手医科大学 | Murine norovirus persistent infection cells |
| JP6635637B1 (en) * | 2018-11-22 | 2020-01-29 | 株式会社細胞治療技術研究所 | Method for producing immunotherapy vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001097885A (en) * | 1999-10-01 | 2001-04-10 | Kyoritsu Shoji Kk | Feline retrovirus vaccine and method for preparing the same |
| JP2001299336A (en) * | 2000-04-28 | 2001-10-30 | Eiji Konishi | Preparation of flaviviruslike particle stable as vaccine and diagnostic antigen without toxicity |
| JP2006061057A (en) * | 2004-08-26 | 2006-03-09 | Kyoritsu Seiyaku Kk | Established cell for producing vaccine, and virus |
| JP2006230397A (en) * | 2005-02-24 | 2006-09-07 | Buddhist Tzu Chi General Hospital | Method for screening anti-flavivirus infectious compound by persistent flavivirus-infected cell line |
| JP2013523824A (en) * | 2010-04-06 | 2013-06-17 | ジョン ダブリュ． ホラデイ、 | How to treat cancer |
| JP2016082925A (en) * | 2014-10-27 | 2016-05-19 | 学校法人 岩手医科大学 | Murine norovirus persistent infection cells |
| JP6635637B1 (en) * | 2018-11-22 | 2020-01-29 | 株式会社細胞治療技術研究所 | Method for producing immunotherapy vaccine |
| JP2020090476A (en) * | 2018-11-22 | 2020-06-11 | 株式会社細胞治療技術研究所 | Method for manufacturing immunotherapy vaccine |
Non-Patent Citations (10)
| Title |
|---|
| BIOTECHNOLOGY JOURNAL, vol. 13, JPN6022008919, 2018, pages 1 - 7, ISSN: 0004776526 * |
| Biotechnology Journal，13，1，2018年，1-7，article number 1700443 |
| EUROPEAN JOURNAL OF CASE REPORTS IN INTERNAL MEDICINE, vol. 7, JPN6022008922, November 2020 (2020-11-01), ISSN: 0004776524 * |
| European Journal of Case Reports in Internal Medicine，7，2020年11月，doi: 10,12890/2020_001707 |
| INFECTION, GENETICS AND EVOLUTION, vol. 85, JPN6022008924, June 2020 (2020-06-01), pages 1 - 12, ISSN: 0004776522 * |
| Infection, Genetics and Evolution，85，2020年06月，1-12，article number 104422 |
| THE OPEN VIROLOGY JOURNAL, vol. 12, JPN6022008921, 2018, pages 134 - 148, ISSN: 0004776525 * |
| The Open Virology Journal，12，2018年，134-148 |
| VIROLOGY, vol. 362, JPN6022008923, 2007, pages 26 - 37, ISSN: 0004776523 * |
| Virology，362，2007年，26-37 |
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