JP2002320484A - Hiv-producing cell line and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a T cell line capable of continuously producing CC chemokine receptor 5(CCR5)-directive human immunodeficiency virus(HIV) in a stage of continuous production of HIV or in a stage of latent infection HIV and to separate and to produce the cell line. SOLUTION: (1) Screening of HIV tolerant to a substance having an anti-HIV activity and (2) screening of a substance having an anti-HIV activity, a substance inhibiting the transition of HIV from the latent infection stage into the proliferation stage and a substance capable of eliminating HIV from a T cell line are characterized by using the T cell capable of continuously producing CCR5-directive HIV. This medicine composition contains the substance obtained by the screening (2). Consequently the T cell line capable of continuously producing CCR5-directive HIV is useful for screening a substance having a preventing and treating effect on diseases caused by HIV such as AIDS, etc., or its salt.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to (1) a novel CC
A T cell line (preferably a human T cell line) capable of continuously producing chemokine receptor 5 (CCR5) -directed human immunodeficiency virus (HIV); (2) a method for producing the cell line;
(3) a method for producing i) CCR5-directed HIV, which is characterized by using the cell line, or ii) an HIV resistant to a substance having anti-HIV activity, and (4) using the cell line. I) has anti-HIV activity, or ii) H
A method for screening a substance that suppresses the transition of IV from a latently infected state to a proliferative state, or iii) removes HIV from a T cell line, (5) has i) anti-HIV activity obtained using the screening method Or ii) H
A substance that suppresses the transition of the IV from a latently infected state to a proliferative state, or iii) removes HIV from a T cell line.

[0002]

2. Description of the Related Art Human immunodeficiency virus (HIV) is a retrovirus encoding its own genetic information in ribonucleic acid (RNA), and is known as a causative virus of acquired immunodeficiency syndrome (AIDS). HIV is classified into two types, type 1 and type 2, based on differences in its nucleotide sequence. Among them, human immunodeficiency virus type 1 (HIV-1) has been studied selectively because the number of infected people is much larger than human immunodeficiency virus type 2 (HIV-2), and its life cycle is detailed. Is understood. HIV-1 is CD4 (cluster of differentiation 4)
It infects cells through the chemokine receptor and converts its genetic information from RNA to deoxyribonucleic acid (DNA) using its own reverse transcriptase, which is then integrated into the host's genomic DNA to become a provirus. Proviruses utilize host cell enzymes to transcribe and translate their sequences, ultimately forming large amounts of mature virus particles and budding from the host cells. HIV-1 is responsible for this infection, reverse transcription, integration, particle formation,
Although the life cycle of budding recurs repeatedly, the base sequence of HIV-1 which infects first and the budding offspring virus are often changed due to the low accuracy of the reverse transcription reaction. Changes in the nucleotide sequence have led to the diversity of HIV-1 and, as described below, are the basis for the emergence of HIV-1 ineffective anti-HIV-1 drugs. HIV, especially a substance that inhibits the growth of HIV-1 (anti-
HIV-1 drugs) have been widely screened to prevent the development of AIDS in HIV-1 infected people. To date, reverse transcriptase inhibitors and HIV-1 proteolytic enzymes involved in particle formation have been screened. Inhibitors (protease inhibitors) have actually been clinically administered to HIV-1 infected individuals and have been effective. However, as described above, because of the known diversity of HIV-1, H is ineffective for each anti-HIV-1 drug.
There is a possibility that IV-1 will appear, and many HIV-1 that are resistant to drugs used in clinical practice have been isolated.
With the possibility that HIV-1 in which all existing drugs are ineffective may appear, screening for new anti-HIV-1 drugs is being actively promoted.

[0003] HIV-1 is roughly divided into two groups depending on the type of chemokine receptor used at the time of infection. CCR5 directional (R5) HIV-1 using CD4 and CC chemokine receptor 5 (CCR5) at infection and CXCR4 directional (X4) HIV-1 using CD4 and CXC chemokine receptor 4 (CXCR4) . HIV-1 infection remains asymptomatic for several to more than a dozen years, after which AIDS develops, but R5 HIV-1 is primarily isolated during the asymptomatic phase, and is primarily associated with AIDS. Is known to be X4 HIV-1. It is known that HIV-1 is actively growing even in the asymptomatic period, but it is thought that inhibiting HIV-1 growth during this period can more effectively prevent the development of AIDS So R5 H
Screening for substances that suppress the growth of IV-1 is very widespread. In addition, in order to completely eliminate HIV-1 from infected individuals, it is not enough to only inhibit the growth of HIV-1 and it is necessary to remove the provirus integrated into the host genomic DNA. The anti-HIV-1 drugs are not effective in removing provirus, and it is considered that there is a high need for the development of anti-HIV-1 drugs that have a novel mechanism and can remove provirus.

[0004] However, cell lines cultured in the laboratory generally do not express sufficient amounts of CCR5 and do not cause sufficient R5 HIV-1 infection (Lijun Wu et al., Jou
rnalof Experimental Medicine, 185, 1681-1691 (199
7)). For this reason, R5 HIV-1 preparation and various infection experiments are performed using peripheral blood mononuclear cells prepared from blood donated volunteers. The reproducibility of the experiment is not very high, hindering efficient anti-R5 HIV-1 drug screening. In addition, the use of peripheral blood mononuclear cells has
The problem is that the yield is not stable even in the preparation of -1 and the possibility that the repetition of the infection for the preparation causes mutations in the base sequence, resulting in diversity in the virus and reducing the reproducibility of the experimental results, as problems Can be In addition, in order to screen for a substance that removes the R5 HIV-1 provirus, R5 HIV-1 is integrated into genomic DNA, and T5 that is in a state of continuously producing virus particles or in a latent state.
Immortalized HIV-infected T that requires a cell line but can be subcultured
No cell line is known.

[0005]

[Problems to be Solved by the Invention] R5 HIV-1 is genomic DNA
Or immortalized T cell lines capable of sustaining production of virus particles, or subcultured immortalized T cells in which virus particles continue to be produced or virus particles are in a latent state If a strain can be established, it will not only be possible to use it for screening for substances that remove provirus, but also to prepare R5 HIV-1 having the same nucleotide sequence stably. In addition, screening for a substance that suppresses the growth of R5 HIV-1 can be performed with high reproducibility. Reproducible screening increases the likelihood of finding anti-R5 HIV-1 drugs and can provide significant benefits in the pharmaceutical field. Therefore, C
Provided is a T cell line capable of sustaining production of C chemokine receptor 5 (CCR5) -directed human immunodeficiency virus (HIV), which is capable of continuously producing HIV or latently infected with HIV, and isolating the cell line. There is a need to produce and provide a screening method using the T cell line.

[0006]

Means for Solving the Problems In view of the above problems, the present inventors have conducted intensive studies and as a result, have found that the CC chemokine receptor 5 (CCR5) -directed human immunodeficiency virus (HI
V) succeeded for the first time to establish a subcultured immortalized T cell line capable of sustaining the production of HIV and in a state of continuously producing HIV or in which HIV is latently infected, and based on this, completed the present invention. I came to.

[0007] That is, the present invention provides: (1) a T cell line capable of sustaining production of CC chemokine receptor 5 tropic human immunodeficiency virus; and (2) continuous production of CC chemokine receptor 5 tropic human immunodeficiency virus. (3) the T cell line according to the above (1), wherein the T cell line according to the above (1) is in a latently infected state with the CC chemokine receptor 5 tropic human immunodeficiency virus; (1) which is a MOLT-4 cell line
(5) the T cell line according to (1), wherein the CC chemokine receptor 5 tropic human immunodeficiency virus type 1 is CC chemokine receptor 5 tropic human immunodeficiency virus type 1; The T cell line according to (1), wherein the CC chemokine receptor 5 tropic human immunodeficiency virus is an HIV-1 Ba-L strain; (7) persistent infection with the CC chemokine receptor 5 tropic human immunodeficiency virus. A method for producing a T cell line according to the above (1), which comprises infecting a competent T cell line with a human immunodeficiency virus (HIV); A T cell line capable of persistent infection is MOLT-
4 / CCR5 (FERM BP-7060) according to the above (7); (9) culturing a T cell line in a state of continuously producing a human immunodeficiency virus directed against CC chemokine receptor 5; (10) a method of producing a human immunodeficiency virus (HIV); (10) culturing a T cell line in a state of continuously producing a human immunodeficiency virus directed to CC chemokine receptor 5 in the presence of a substance; A method for screening for a human immunodeficiency virus (HIV) having resistance to the substance; (11) a method for screening for a substance having anti-human immunodeficiency virus (HIV) activity, which comprises using the T cell line according to (2). A screening method; (12) using the T cell line according to (3) above, wherein the human immunodeficiency virus (HIV) is in a state from a latently infected state to a proliferated state (13) a method for screening a substance that removes the human immunodeficiency virus (HIV) from a T cell line, which comprises using the T cell line according to (1); (14) a substance obtained by the screening method of (11); (15) a substance obtained by the screening method of (12); (16) a substance obtained by the screening method of (13); A) a pharmaceutical composition comprising the substance according to the above (14), (15) or (16); (18) a pharmaceutical composition according to the above (17), which is an anti-AIDS drug;

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION As used herein, the term "CC chemokine receptor 5 (CCR5) -directed human immunodeficiency virus" requires CC chemokine receptor 5 as essential when infecting cell lines (directivity). ) Human immunodeficiency virus. A preferred example is human immunodeficiency virus type 1, which essentially infects cell lines with CCR5. Particularly preferably, human immunodeficiency virus type 1 val (HIV-I Ba-
L) strains and the like. As used herein, "a T cell line capable of sustaining production of CC chemokine receptor 5 tropic human immunodeficiency virus" refers to a T cell line in a state of continuously producing CCR5 tropic human immunodeficiency virus and CCR5 tropic. Includes any T cell line that is latently infected by the infectious human immunodeficiency virus. A “latently infected T cell line” refers to a state in which HIV is integrated into the genomic DNA of the cell, but the mature T-cell line does not produce mature virus particles. Preferred examples of T cell lines include the Journal of the National Cancer Institute,
MOL described in Vol. 49, pp. 891-895, 1972
T-4 cell line.

[0009] The T cell line of the present invention having the ability to continuously produce the CCR5-directed human immunodeficiency virus is obtained by adding the CCR5-directed human immunodeficiency virus to a T cell line capable of sustaining infection with the CCR5-directed human immunodeficiency virus. It can be produced by infection. As used herein, “a T cell line capable of persistently infecting CCR5 tropic human immunodeficiency virus” refers to a T cell line that expresses CD4 and CCR5, which are receptors for CCR5 tropic human immunodeficiency virus, in sufficient amounts. Say. C used here
Examples of a source of a T cell line capable of persistently infecting CR5 tropic human immunodeficiency virus include human leukemia T
Immortalized T cell lines obtained by cloning cancerous T cells such as cells. Cloning is performed according to various known methods. Specifically, there is a method of cloning immortalized cells from a human leukemia T cell line, focusing on the fact that cancerous tissue continues to proliferate permanently. Specific examples of the immortalized T cell line thus established include MOLT-4 clone 8 cell line [Journa of Virology (Journa).
l of Virology), March 1986, pp. 1159-1162]. The immortalized T cell line thus obtained is transformed by a method known per se, and expressed by expressing the CC chemokine receptor 5 gene in the cell, thereby obtaining a TCR having the ability to persistently infect CCR5-directed human immunodeficiency virus. Cell lines can be produced. Specifically, Proceedings of the National Academy of Sciences
USA), 1999, Vol. 96, pages 5698-5703, using the plasmid pcDNA3.1-CCR5 to transform the above immortalized T cell line. The transformation is performed by a method known per se, for example, a manual (Sambrook et al., (Molecular Cloning: A Laboratory Ma
nual), Cold Spring Harbor Laboratory Press). Specific examples of the T cells capable of persistently infecting the CCR5-tropic human immunodeficiency virus include MOLT-4 / CCR5 (FERM BP-
7060) strain is a preferred example.

The CCR5-directed human immunodeficiency virus of the present invention is infected by infecting the CCR5-directed human immunodeficiency virus thus obtained with a T cell line capable of persistently infecting the CCR5-directed human immunodeficiency virus. A T cell line capable of sustaining the production of the virus can be produced. Human immunodeficiency virus infection is, for example, T
Medium used for culturing cell lines (eg, RPMI1640
This is performed by culturing a TCR cell line capable of persistently infecting the CCR5-directed human immunodeficiency virus and a CCR5-directed human immunodeficiency virus in a culture medium or the like. The culture temperature is from about 15 ° C to about 55 ° C, preferably about 2 ° C.
0 ° C to about 45 ° C, more preferably about 25 ° C to about 4 ° C.
0 ° C. The culturing time is about 1 hour to about 6 months, preferably about 1.5 hours to about 60 days. The cultivation is preferably performed in an atmosphere of carbon dioxide. The concentration of carbon dioxide is about 3 to 10 (V / V)%.

The establishment of human immunodeficiency virus infection is based on T
It can be detected by measuring an increase in the amount of HIV in the culture supernatant of the cell line. The measurement of the amount of HIV is as follows: 1) H
Method for measuring IV RNA amount by RT-PCR method, 2) p
The amount of 24 antigens can be determined by an enzyme-labeled immunoassay known per se (eg, Retr
o-Tek HIV-1 p24 Antigen ELISA kit, manufactured by Zepto Metrix, USA) or 3) HIV
It is performed by measuring the enzyme activity of reverse transcriptase.

[0012] The CC chemokine receptor 5 (CCR5) -directed using a T cell line capable of continuously producing human immunodeficiency virus of the present invention and a substance or a test substance having an anti-HIV activity. Culturing a T cell line in a state of continuously producing infectious human immunodeficiency virus (HIV) in the presence of a substance having anti-HIV activity, thereby screening for HIV having resistance to the substance having anti-HIV activity, CC chemokine Receptor 5
(CCR5) Screening of a substance having anti-HIV activity using a T cell line in a state of continuously producing tropic human immunodeficiency virus (HIV), CC chemokine receptor 5 (CCR5) tropic human immunodeficiency virus (HI)
V) using a T cell line in a latently infected state.
Screening for a substance that suppresses the transition from a latently infected state to a proliferative state of CC chemokine receptor 5 (CCR)
5) Using a T cell line capable of producing directional human immunodeficiency virus (HIV), a substance capable of removing HIV from the T cell line can be screened.

[0013] Substances having anti-HIV activity include, for example, anti-HIV such as reverse transcriptase inhibitors and protease inhibitors.
Agents and the like. Test substances include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, serum, and the like. It may be a substance produced by a known method or a method analogous thereto.

Hereinafter, each screening method will be described. By culturing a T cell line in a state of continuously producing the CC chemokine receptor 5 (CCR5) -directed human immunodeficiency virus (HIV) in the presence of a substance having anti-HIV activity, H resistant
IV screening. A TCR cell line that continuously produces CCR5-directed HIV or a culture supernatant thereof and cells capable of infecting the HIV (eg, the above-mentioned T cell line capable of persistently infecting CCR5-directed HIV, peripheral blood mononuclear cells, etc.) ) Is cultured in the presence of a substance having anti-HIV activity. Culture conditions are the same as those described above. HIV grown in the culture supernatant is serially diluted, mixed with the HIV-infected cells, and cultured in the same manner. HIV is separated from the culture supernatant and / or infected cells by a method known per se. The isolated cloned HIV is cultured with the cells capable of infecting the HIV under the same conditions as in the presence of the substance having the anti-HIV activity to confirm that the HIV grows. HIV resistant to substances having
Can be screened.

[0015] Screening for a substance having anti-HIV activity using a T cell line in a state of continuously producing human immunodeficiency virus (HIV) directed to CC chemokine receptor 5 (CCR5). A T cell or a culture supernatant thereof in a state of continuously producing CCR5-directed HIV;
Cells capable of infecting IV are cultured in the presence of the test substance under the conditions described above. By measuring the amount of HIV in the culture supernatant by the method described above and selecting a substance that suppresses the increase in the amount of HIV, a substance having anti-HIV activity can be screened.

Screening for a substance that suppresses the transition from a latently infectious to a proliferative state of HIV using a T cell line that is latently infected with CC chemokine receptor 5 (CCR5) -directed human immunodeficiency virus (HIV) . CC
R5 tropic HIV is used in HI
Substances that shift to a state in which V is continuously produced (proliferated state) (eg, LT LT in a latently infected state such as TNF-α)
The cells are cultured under the same culture conditions as described above in the presence of a test substance and a substance that activates the transcription of HIV through an R (Long Terminal Repeat) sequence. Screening for a substance that suppresses the transition from a latently infected state to a proliferated state by measuring the amount of HIV in the culture supernatant in the same manner as described above and selecting a test substance when the amount of HIV is not detected or low. It can be performed.

[0017] Screening for a substance which removes the CC chemokine receptor 5 (CCR5) from a T cell line using a T cell line capable of producing human immunodeficiency virus (HIV). A T cell line capable of producing CCR5-directed HIV is cultured in the presence of a test substance under the same conditions as described above. The proviral DNA content of a T cell line capable of producing CCR5-directed HIV
A substance that removes the above HIV from the T cell line can be screened by selecting a test substance that reduces the amount of proviral DNA by measurement by the CR method or the Southern hybridization method.

In order to measure the amount of proviral DNA by PCR, for example, genomic DNA of a cultured T cell line is extracted by a method known per se, and the extracted genomic DNA is used as a template to quantitatively determine the amount by a known method. Perform a PCR reaction to amplify any region of the proviral DNA,
The amount of genomic DNA is measured using a fluorescently labeled probe, a radioactively labeled probe, or a dye (eg, ethidium bromide) that binds to the amplified fragment. In order to measure the amount of proviral DNA by the Southern hybridization method, for example, genomic DNA of a cultured T cell line is extracted by a method known per se, and the extracted genomic DNA and a probe (eg, a fluorescent-labeled HIV-specific Using a probe, a radiolabeled HIV-specific probe, etc.), Southern hybridization is performed by a method known per se to measure the amount of genomic DNA.

HIV resistant to a substance having anti-HIV activity obtained by the screening method of the present invention
Elucidation of the resistance mechanism using the resistant HIV and the resistance H
It can be used to search for drugs that are effective for IV. The substances obtained by the screening method of the present invention are obtained from the above-mentioned test substances (for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, serum, etc.). Since it is a selected substance and has a preventive / therapeutic effect on a disease caused by HIV (eg, AIDS), it can be used as a safe and low toxic preventive / therapeutic drug for the disease. Furthermore, a substance derived from a substance obtained from the above screening by a method known per se can also be used. These substances or substances derived from these substances may be used alone for treatment or prevention, and may be used in combination with other anti-HIV drugs (eg, nucleoside reverse transcriptase inhibitors such as azidothymidine and dideoxyinosine; nevirapine). And other non-nucleoside reverse transcription inhibitors; HIV protease inhibitors such as indinavir, saquinavir, ritonavir, nelfinavir).

When the substance obtained by the screening method forms a salt, the substance includes the salt.
Salts with physiologically acceptable acids (eg, inorganic acids, organic acids) or bases (eg, alkali metals, organic amines) are used. In particular, physiologically acceptable acid addition salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

The drug containing the substance obtained by the screening method can be manufactured by a known method or a method analogous thereto. The preparation thus obtained is
Since it is safe and has low toxicity, it can be administered to, for example, humans or mammals (eg, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.).

The dosage of the substance obtained by the screening method varies depending on the target disease, the administration subject, the administration method and the like. For example, when the substance or a salt thereof is orally administered for the purpose of treating AIDS, it is generally used. Adult (weight 60kg)
)), About 0.1 mg of the substance per day
To 5 g, preferably about 1.0 mg to 4.5 g, more preferably about 2.0 mg to 4 g. In the case of parenteral administration, the single dose of the substance varies depending on the administration subject, target disease and the like. For example, for the purpose of treating AIDS, the substance is usually administered in the form of an injection to an adult (body weight 60 kg).
), The substance is administered in an amount of about 0,1 per day.
About 01 mg to 35 mg, preferably about 0.1 mg to 20 mg, more preferably about 0.1 mg to 10 mg.
mg injection (eg intravenous, intramuscular, subcutaneous)
It is better to do. In the case of other animals, the human weight is 60k
The amount converted per g can be administered.

Specific examples of the dosage form of the above preparation include tablets (including sugar-coated tablets and film-coated tablets), pills, capsules (including microcapsules),
Granules, fine granules, powders, syrups, emulsions, injections, inhalants, ointments, suppositories and the like are used. These preparations are manufactured according to a known method (for example, a method described in the Japanese Pharmacopoeia). In the preparation, the content of the substance obtained by the above screening method varies depending on the form of the preparation, but is usually about 0.01 to 100% by weight, preferably about 0.1 to 60% by weight, based on the whole preparation. More preferably, it is about 0.5 to 20% by weight.

Specifically, the tablet is produced by mixing the substance obtained by the screening method of the present invention as it is, or adding an excipient, a binder, a disintegrant or other appropriate additives and mixing them homogeneously. Into granules according to a conventional method, and then compression-molded by adding a lubricant or the like, or homogeneously mixed with the substance as it is or by adding excipients, binders, disintegrants or other appropriate additives. Then, it can be produced by directly compression-molding, or it can be produced by compression-molding the granules prepared beforehand as they are, or after adding an appropriate additive and mixing them homogeneously. The present agent may contain a coloring agent, a flavoring agent, and the like, if necessary.
Further, the present agent can be provided with an appropriate coating agent. In the method for producing an injection, a certain amount of the substance obtained by the screening method of the present invention is dissolved and suspended in water for injection, physiological saline, Ringer's solution or the like in the case of an aqueous solvent, and usually in vegetable oil or the like in the case of a non-aqueous solvent. It can be made up to a fixed amount by turbidity or emulsification, or can be manufactured by taking a fixed amount of the substance and sealing it in a container for injection (eg, ampoule). Suppositories are substances obtained by the screening method of the present invention and suitable non-irritating excipients, such as cocoa butter and polyethylene glycols, which are solid at normal temperature but become liquid at intestinal temperature and release the substance. And can be manufactured by mixing. As the pharmaceutical carrier for oral use, substances commonly used in the field of pharmaceuticals such as starch, mannitol, crystalline cellulose and sodium carboxymethylcellulose are used. As the carrier for injection, for example, distilled water, physiological saline, glucose solution, oil solution and the like are used. In addition, additives generally used in preparations can be appropriately added as needed.

The MOLT-4 / MOLT-4 /
The CCR5 strain has been deposited with the National Institute of Advanced Industrial Science and Technology, Biotechnology and Industrial Technology Research Institute (NIBH) from February 29, 2000 under the deposit number F.
ERM BP-7060, deposited on January 27, 2000 at the Institute of Fermentation (IFO) under the deposit number IFO
No. 50521 has been deposited.

[0026]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below in detail, but the present invention is not limited thereto. Also,
Unless otherwise noted, individual genetic manipulation techniques are described in the manual of Sombrook et al. ((Molecular Cloning: A
Laboratory Manual), Cold Spring Harbor Laboratory Press).

Example 1 Establishment of MOLT-4 / CCR5 Cell Line (1) Determination of Selective Agent (Geneticin) Concentration MOLT-4 cells were cultured under the conditions of 37 ° C. and 5% carbon dioxide.
RP containing 0% fetal bovine serum (FBS, manufactured by ICN (ICN), USA), 100 U / ml penicillin G (manufactured by Life Technology, USA), 100 μg / ml streptomycin (manufactured by Life Technology, USA)
MI1640 medium (Life Technology, USA)
(Hereinafter referred to as MOLT-4 basic medium). 100, 200, 300, in MOLT-4 basal medium
400, 500, 600, 700, 800, 900, 1
Geneticin (Geneticin, manufactured by Life Technology, USA) was added at 2,000 μg / ml, and 2 × 10 ³ cells / ml was added.
After culturing for a week, the growth of the cells was observed under a microscope, and the minimum concentration that completely suppressed the growth was determined. (2) Transfection 15 μl of cellfectin (Cellfectin, manufactured by Life Technology, USA) in 250 μl of RPMI1640 medium without FBS, penicillin G and streptomycin
Was added as a lipid solution and added to the wells of a 24-well cell culture plate. RPMI 164 also without additives
3.2 μg of pcDNA3.1-CCR5 (Baba et al., Proceedings National Academic
Science U.S.A. (Proceedings of the N)
ational Academy of Sciences, USA), 1999, 9
6, 5698-5703) to give a DNA solution. This DNA solution is mixed with the lipid solution, and
After standing for 0 min, 250 μl of DNA from A1 well
-The lipid mixture was removed. MOL suspended in RPMI1640 medium without addition to 8 × 10 ⁶ cells / ml
T-4 cells were added in an amount of 50 μl and cultured at 37 ° C. for 4 hours under 5% carbon dioxide. In addition, 15% FBS, 100U /
600 μl of RPMI1640 medium containing 100 ml / ml of penicillin G and 100 μg / ml of streptomycin was added and cultured for 2 days. The total volume of this culture was 1 mg / ml Genetic
1 containing 10 ml of MOLT-4 basic medium containing in
The cells were transferred to a 00 mm culture dish, and the culture was continued under the conditions of 37 ° C. and 5% carbon dioxide, the culture solution was replaced at appropriate times, and Geneticin-resistant cells were grown. (3) Preparation of feeder cells MOLT-4 cells were suspended in a phosphate buffer (PBS (manufactured by Life Technology, USA)) at 1 × 10 ⁷ cells / ml, and 200 μl was dispensed into a sample tube. . 20 μl of 0.5 mg / ml mitomycin C (manufactured by Wako Pure Chemical Industries, Japan) solution was added thereto, and reacted at 37 ° C. for 30 minutes. After washing 5 times with MOLT-4 basic medium, 1 mg / ml Geneti was adjusted to 1 × 10 ⁵ cells / ml.
The cells were suspended in cin-containing MOLT-4 basal medium. (4) Single cell cloning 100 μl of the above feeder cells were dispensed into a 96-well cell culture plate. Proliferate Geneticin-resistant cells by 3cel
MOLT containing 1mg / ml Geneticin to be ls / ml
-4 The suspension was suspended in a basal medium, and dispensed into a 96-well cell culture plate to which feeder cells had been previously added. The growth of the cells was observed under a microscope, the proliferating cells were separated, and the culture scale was increased in a timely manner. (5) Examination of CCR5 expression The Geneticin-resistant clone obtained in the above (4) was used to prepare a reaction mixture [2% (V / V) FBS, 0.1% (W / V) sodium azide]
(Wako Pure Chemical Industries, Japan) / phosphate buffer], and suspended in 40 μl of the same reaction solution. 10
μl of fluorescein isothioanaate [(fluores
cein isothiocyanate (FITC)]-labeled anti-CCR5 antibody clone 2D7 (Farmingen, USA) was added and reacted at 4 ° C. for 45 minutes. Separate the cells and add 1
After washing three times with the above reaction solution (3 ml), the suspension was suspended in 300 μl of the reaction solution, and the binding of the antibody was analyzed using a Flow Cytometer (JASCO CytoACE-300, manufactured by JASCO Corporation, Japan). The results are shown in FIG. 1 and FIG. MOLT4 / CC
In the Flow Cytometer chart of the R5 cell line (Fig. 2), the peak of the anti-CCR5 antibody (broken line) is clearly on the right (higher fluorescence intensity side) as compared with the flow Cytometer chart of the MOLT4 cell line as the raw material (Fig. 1). ) And CCR
5 was confirmed.

Example 2 MOLT-4 / CCR5 / Ba-L
Establishment of Cell Line (1) Infection with HIV-1 Ba-L The MOLT-4 / CCR5 cell line obtained in Example 1 was incubated at 37 ° C.
Using MOLT-4 basic medium under the condition of 5% carbon dioxide
Cultured. 1 × 10 in MOLT-4 basal medium⁶cells / ml
MOLT-4 / CCR5 cells prepared to
HIV-1 Ba-L corresponding to 10 ng as 24 quantities
, And cultured for 2 hours at 37 ° C and 5% carbon dioxide.
Was. The cells are washed with MOLT-4 basal medium and non-adsorbed
1 × 10 after removing the loose ^FiveRe-adjust to cells / ml
And cultured under conditions of 37 ° C. and 5% carbon dioxide. (2) Establishment of persistently infected cell line M infected with HIV-1 Ba-L obtained in (1) above
Collect culture supernatant of OLT-4 / CCR5 cell line every 4 days
At the same time, count the number of viable cells by trypan blue staining.
1 × 10^FiveMOLT-4 basic medium to be cells / ml
And continue culturing under conditions of 37 ° C and 5% carbon dioxide.
did. The amount of p24 antigen in the collected culture supernatant was measured using Retro-Tek HI
V-1 p24 Antigen ELISA kit (Zeptometrics (Zepto
 Metrix), USA). The result
The results are shown in [Table 1]. Thereafter, when the culture is continued, HIV-1
Most MOLT-4 / CCR infected with Ba-L
5 cells died, but continued culture for about one month
Some cells have survived and proliferated. P24 in culture supernatant
As a result of measuring the amount of antigen, production of p24 antigen was
Sequentially infected cells (MOLT-4 / CCR5 / Ba-L) were established. Persistent infection
Cloning by limiting dilution of cells resulted in p24 production
A clone was obtained that had been lost. 10 ng /
When cultured for 3 days in a medium containing ml TNF-α, p24 was found in the supernatant.
The antigen was detected and the establishment of the latently infected cell line was confirmed.

[Table 1]

[0029]

According to the present invention, the CC chemokine receptor 5 (CC
R5) T cell lines capable of sustained production of tropic human immunodeficiency virus (HIV) include, for example, HI such as AIDS.
It is useful for screening a substance having a prophylactic or therapeutic effect on a disease caused by V or a salt thereof.

[0030]

[Brief description of the drawings]

FIG. 1 shows a Flow Cytometer chart of a MOLT4 cell line, in which a solid line indicates a control antibody and a dashed line indicates an anti-CCR5 antibody.

FIG. 2 Flow Cytometer of MOLT4 / CCR5 cell line
In the figure, the solid line represents the control antibody, and the broken line represents the anti-C
3 shows the CR5 antibody.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI theme coat ゛ (reference) C12Q 1/02 C12N 5/00 BF term (reference) 4B024 AA01 BA32 CA04 DA03 EA02 GA11 4B063 QA01 QA18 QQ08 QQ10 QR41 QR68 QS40 4B065 AA94X AA97X AA97Y AB01 AC14 BA02 BA06 CA24 CA44 4C084 AA16 ZB33 ZC55

Claims (18)

[Claims] 1. A T cell line capable of sustainably producing human immunodeficiency virus directed against CC chemokine receptor 5. 2. The T-cell according to claim 1, which is in a state of continuously producing a human immunodeficiency virus directed against CC chemokine receptor 5.
Cell line. 3. The T cell line according to claim 1, wherein the human immunodeficiency virus directed against CC chemokine receptor 5 is in a latently infected state. 4. The T cell line according to claim 1, wherein the T cell line is a MOLT-4 cell line. 5. The T cell line according to claim 1, wherein the CC chemokine receptor 5 tropic human immunodeficiency virus is CC chemokine receptor 5 tropic human immunodeficiency virus type 1. 6. The human immunodeficiency virus directed against CC chemokine receptor 5 is the HIV-1 Ba-L strain.
The described T cell line. 7. The T cell line according to claim 1, wherein the T cell line having the ability to persistently infect the human immunodeficiency virus directed to CC chemokine receptor 5 is infected with human immunodeficiency virus (HIV). Manufacturing method. 8. A T cell line capable of persistently infecting CC immunochemokine receptor 5 directed human immunodeficiency virus is an MO cell line.
The method according to claim 7, which is LT-4 / CCR5 (FERM BP-7060). 9. A method for producing a human immunodeficiency virus (HIV), which comprises culturing a T cell line that is in a state of continuously producing a human immunodeficiency virus directed against CC chemokine receptor 5. 10. A human immunodeficiency virus resistant to said substance, characterized by culturing in the presence of a substance a T cell line in a state of continuously producing a human immunodeficiency virus directed to CC chemokine receptor 5 ( HIV) screening method. 11. A method for screening a substance having anti-human immunodeficiency virus (HIV) activity, comprising using the T cell line according to claim 2. 12. A method for screening a substance that suppresses the transition of a human immunodeficiency virus (HIV) from a latently infectious state to a proliferative state, comprising using the T cell line according to claim 3. 13. A method for screening a substance for removing the human immunodeficiency virus (HIV) from a T cell line, comprising using the T cell line according to claim 1. 14. A substance obtained by the screening method according to claim 11. 15. A substance obtained by the screening method according to claim 12. 16. A substance obtained by the screening method according to claim 13. 17. A pharmaceutical composition comprising the substance according to claim 14, 15, or 16. 18. The pharmaceutical composition according to claim 17, which is an anti-AIDS drug.