JP2002191399A - Method for assaying drug resistance of hiv - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a rapid and simple method for assaying a drug resistance using a cell strain for assaying human immunodeficiency virus-1(HIV-1) infectivity titer and especially the method for assaying the drug resistance by which a multi-sample can be rapidly treated. SOLUTION: This method for assaying the drug resistance against HIV is characterized by bringing an animal cell capable of expressing a secretory reporter protein due to HIV-1 infection into contact with a sample containing HIV-1 in the presence of the test drug and detecting the reporter protein secreted on a culture supernatant due to the HIV-1 infection.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for testing drug resistance of HIV. More specifically, the present invention relates to a method for testing drug resistance of HIV by detecting a reporter protein secreted by a target cell infected with HIV-1 into a culture supernatant in the presence of a test agent.

[0002]

[Prior Art] Human Immunodeficiency Virus type 1
(HIV-1) is a retrovirus belonging to Lentivirus and is a pathogen of acquired immunodeficiency syndrome AIDS. More than a decade after its discovery, it was initially perceived as an inevitable disease that killed the immune system of the infected host and caused most of the infected people to become immunodeficient and die. However, in recent years,
Various inhibitors against reverse transcriptase essential for HIV-1 life cycle and protease related to cleavage of mature protein required for virus particle formation have been developed, and administration of potent anti-HIV-1 drugs by their combination (so-called, HAA
RT therapy (Highly Active AntiRetrovirus Therapy)
As a result, the amount of virus in the infected body can be reduced below the detection limit by a highly sensitive PCR method, and for some patients, HIV-1 infection is no longer considered to be a chronic infection. .

However, when multiple drug administration is stopped, HI
V-1 rapidly proliferates and, due to the high rate of reverse transcriptase mutation, which is a characteristic of this virus, a resistant virus to the administered drug easily appears. It has been often reported that a virus that is resistant to a plurality of drugs based on the mechanism of action is caused, thereby causing difficulty in subsequent treatment.

[0004] In the current situation in which curative therapies are unlikely to be available in the near future, anti-HIV-1 therapeutic strategies will capture how quickly the emergence of resistant viruses and identify the most effective drug against the virus in individual patients. It is thought that the center of gravity shifts in combination in keeping the patient's viral load at a low steady state. Genotyping and Phen as effective drug selection criteria in diversifying HIV treatment using a wide variety of drugs, including newly developed anti-HIV-1 drugs
otyping drug resistance test.

For Genotyping, amplification of the HIV-1 pol gene region by Primer Design and PCR and analysis of its base sequence have already been made into a kit, and have been technically established and spread. However, it has often been pointed out that a discrepancy between drug resistance mutations due to amino acid mutations in a gene encoding reverse transcriptase or protease in the viral genome in a sample and the results regarding biological drug resistance of a patient virus.

[0006] On the other hand, although Phenotyping reflects the actual virus resistance, the method using peripheral blood mononuclear cells (PBMC), which is currently used as a standard method, still has many problems to be solved in its standardization. It has been pointed out that. For example, in a resistance test using human PBMC, it often takes several months to obtain a result.
It is difficult to compare results obtained due to the variation in HIV susceptibility among Donor lots, it is not suitable for processing multiple samples due to the labor required, and HIV-1 gag protein p24 ELI
At present, it is performed only in a limited number of laboratories due to an increase in cost required for a resistance test due to measurement of virus proliferation by SA. However, since the Phenotyping method reflects the biological resistance of the virus, it provides indispensable information as a criterion for drug selection.

In order to overcome these problems, the present inventors have developed a rapid and simple HIV-1 infectious titer cell line MAGIC.
We have reported on drug resistance tests using -5A (Atsuko Hachiya, Saori Aizawa, Mari Tanaka, Yukiko Takahashi, Yoshihiro Hirabayashi,
Setsuko Ida, Masashi Tatsumi, Shinichi Oka, HeLa expressing CCR5
/ CD4-LTR-βGal cells (MAGIC5 clone 1-1
0) Study on anti-HIV drug resistance test using the 13th Japan AIDS Society December 1999 Tokyo, Hachiya, A.,
Aizawa-Matsuoka, S., Tanaka, M., Takahashi, Y., Id
a, S., Gatanaga, H., Hirabayashi, Y., Kojima, A.,
Tatsumi, M. AndOka, S .: Rapid and simple phenotypi
c assay for drug susceptibility of human immunodef
iciency virus type 1 by using CCR5-expressing HeLa
/ CD4 cell clone 1-10 (MAGIC-5) Antimicrob. Agents
Chemother. 45: 495-501, 2001.). This cell line MA
GIC-5A transforms the HIV-1 receptor CD4 and co-receptors CXCR4 and CCR5 so that they can be stably expressed in the human cervical cancer cell line HeLa,
The Tat protein produced by the infected HIV-1 drives β-Galactosidase with the added SV40 nuclear localization signal downstream of the integrated LTR, and the infection titer of HIV-1 in the sample is quickly and easily determined by X-Gal staining. This is an Indicator Cell devised so as to measure. It has been reported that using this cell line, rapid virus isolation from patient sera can be followed by detection of resistant strains against anti-reverse transcription and anti-protease drugs within two weeks.

[0008] In addition, since this cell line performs the basic technique of measuring the infectious titer of HIV-1, there is a possibility that it can be applied to many HIV-1 research areas. To date, it has also been applied to drug-resistant Phenotype Assay and anti-HIV drug screening. Although faster, simpler and more economical than the commonly used HIV-1 gag protein p24 based on ELISA or reverse transcriptase activity measurement, the measurement of HIV infectivity with this cell line is microscopic. Based on the count of cell nuclei stained with X-gal below, it was not suitable for multi-sample rapid processing. In the future, the development of anti-HIV-1 drugs will progress, and it is expected that many drugs will be clinically applied. Therefore, a quicker and simpler measurement system has been demanded.

[0009]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art.
That is, an object of the present invention is to provide a rapid and simple drug resistance test method using an HIV-1 infectivity titer cell line. In particular, an object of the present invention is to provide a drug resistance test method capable of rapidly processing multiple samples.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that the MAGIC-5A
In order to evolve the Phenotyping drug resistance test using S.A. more quickly and simply and with high sensitivity, an indicator cell MAGIC-5A was further incorporated with an expression unit incorporating SEAP (secretory alkaline phosphatase) downstream of the LTR, and HIV infection was performed. A high-throughput measurement system was successfully established by detecting alkaline phosphatase secreted in the culture supernatant by chemiluminescence. Furthermore, by drug resistance test using these cells,
It has also been found that HIV drug resistance to various anti-HIV drugs can be evaluated. The present invention has been completed based on these findings.

That is, according to the present invention, an animal cell capable of expressing a secretory reporter protein by HIV-1 infection is brought into contact with a sample containing HIV-1 in the presence of a test agent, and There is provided a method for testing drug resistance of HIV, which comprises detecting a reporter protein secreted into a culture supernatant. Preferably, the animal cell used in the present invention has HIV-1 virus receptor CD4 and HIV-1 virus co-receptor, more preferably has CXCR4 and / or CCR5 as HIV-1 virus co-receptor,
Particularly preferably, it has both CXCR4 and CCR5 as HIV-1 viral co-receptors.

Preferably, the secretory reporter protein is a protein that can be detected by one or more of a colorimetric reaction, fluorescence emission, and chemiluminescence measurement systems. Particularly preferably, the secreted reporter protein is alkaline phosphatase, luciferase or peroxidase.

Preferably, the animal cell used in the present invention is
It is a cell obtained by transforming an expression vector having a gene encoding a secreted reporter protein downstream of the HIV-1 LTR sequence. Preferably, the animal cells used in the present invention are cells derived from mammalian cells, and more preferably cells derived from human HeLa cells or human HOS cells. Particularly preferred animal cells for use in the present invention are accession number FERM P-18.
Animal cells having 078.

[0014] Preferably, the test agent is an anti-HIV agent containing a reverse transcriptase inhibitor or a protease inhibitor,
As the test drug, a combination of two or more drugs can be used. Preferably, the reporter protein secreted into the culture supernatant is detected by one or more of a colorimetric reaction, fluorescence emission, and chemiluminescence measurement system.

[0015]

Embodiments of the present invention will be described below in detail. The method for testing drug resistance according to the present invention comprises:
An animal cell capable of expressing a secreted reporter protein by IV-1 infection is brought into contact with a sample containing HIV-1 in the presence of a test agent, and a reporter protein secreted into a culture supernatant by HIV-1 infection is detected. It is characterized by doing. First, HIV- used in the present invention.
(1) Animal cells capable of expressing a secretory reporter protein by infection will be described.

The type of animal cell used in the present invention is not particularly limited as long as it can be infected with HIV-1. Usually, HIV-1 infection includes HIV-
Since 1 viral receptor CD4 is required,
The animal cell used in the present invention preferably has the HIV-1 virus receptor CD4. In addition, as described hereinabove, in the process of HIV-1 entry into target cells, in addition to the CD4 molecule, which is a major viral receptor on the cell side, T cell-tropic HIV-1 binds CXCR4, Since HIV-1 uses CCR5 as a co-receptor, it is preferable that the animal cells used in the present invention also have these co-receptors.
For the purpose of analyzing a wide range of HIV-1 infectious titers, it is preferable to use animal cells expressing both CXCR4 and CCR5.

These HIV-1 virus receptors C
D4 and the HIV-1 virus co-receptor may be used as they are if they are originally retained and expressed in the starting animal cells. When the starting cells do not express them, a recombinant expression vector in which a gene encoding the receptor or co-receptor is incorporated into a suitable expression vector is introduced into the starting animal cells, whereby the receptor or the receptor is expressed. Coreceptors can be expressed in the animal cells.

The type of starting cells that can be used to establish the animal cells used in the present invention is not particularly limited, but is preferably a mammal (eg, a primate such as a human or monkey, a mouse, a rat, a hamster, or the like). Rodents), and more specifically, human-derived HeLa cells or human-derived HOS cells.

As a technique for selecting cells expressing the receptor or co-receptor of interest, an antibiotic resistance gene is expressed together with the receptor or co-receptor in a recombinant expression vector containing the receptor or co-receptor. By incorporation into such a medium and culturing in a medium containing the antibiotic, cells expressing the desired receptor or co-receptor can be selected. As a technique for cloning a target cell as a single cell derived from one cell, a limiting dilution method can be mentioned.

For example, when a HeLa cell constitutively expressing CXCR4 is used as a starting cell, a mouse retrovirus obtained by adding an antibiotic resistance gene (eg, a neomycin (Neo) resistance gene) to the HeLa cell is used. The vector is transformed to express CD4, and if desired, the LTR-βGal expression unit is transformed together with the hygromycin resistance gene, and viable cells are selected. Furthermore, CD for enhancing CD4 expression
(4) The above-mentioned cells are transformed with an expression vector that confers expression and Puromycin resistance to the transformant, and an expression vector that confers CCR5 expression and blasticidin resistance to the transformant, and the target cells are cloned by limiting dilution. The target cell cloned by this operation is G41
8, total 4 of Hygromycin, Puromycin and Blasticidin
It is resistant to certain selected drugs.

The animal cell used in the present invention is characterized in that it can express a secretory reporter protein by HIV-1 infection. The animal cell used in the present invention is H
The secretory reporter protein is expressed by the IV-1 infection, and the expressed secretory reporter protein is secreted extracellularly and released into the culture medium. A sample containing HIV-1 is added to the culture system containing the animal cells used in the present invention, the culture is collected, and the reporter protein secreted into the culture is detected or analyzed by an appropriate method.
The infectious titer of HIV-1 in the sample can be measured.

The method for detecting the reporter protein secreted into the culture solution is not particularly limited, and examples thereof include a method for detecting the reporter protein by colorimetric reaction, fluorescence emission or chemiluminescence. The type of secretory reporter protein is not particularly limited, but preferably various enzyme active substrates are easily available, high in measurement sensitivity, and capable of simplifying the measurement procedure.Examples include alkaline phosphatase, luciferase or peroxidase. Of these, alkaline phosphatase is particularly preferred.

One of the preferred methods for producing animal cells expressing a secreted reporter protein upon HIV-1 infection according to the present invention is HIV-1 LTR.
Methods include transforming an animal cell with an expression vector having a gene encoding a secreted reporter protein downstream of the sequence. The expression vector used in the above method,
It preferably contains a drug resistance gene for selection of a desired cell.

For example, G4 as described hereinabove
18, Hygromycin, Puromycin and Blasticidin have resistance to a total of four selected drugs, CXCR4, CD
The animal cells used in the present invention can be prepared by transforming cells expressing 4 and CCR5 with an expression vector having a gene encoding a secreted reporter protein. In this case, the drug resistance gene that can be incorporated into an expression vector having a gene encoding a secretory reporter protein includes the above 4
It may be any other than the drug resistance gene of the species, for example, Zeo
and a cin resistance gene.

More specifically, an expression vector having a gene encoding a secretory reporter protein is used
Transfecting cells having HIV-1 viral receptor CD4 and HIV-1 viral co-receptor;
Surviving colonies can be obtained by selecting with drugs. The cloned cells in which these were grown were plated in duplicate on a microplate, one of which was infected with HIV-1 and the other was used as a non-infected control. Two days after infection, the culture supernatant was collected and kept at 65 ° C for 1 day.
Endogenous alkaline phosphatase and HIV after 5 minutes treatment
Inactivate. SEAP secreted into the culture supernatant can be measured and selected by, for example, a colorimetric reaction using p-Nitrophenol phosphate as a substrate. SEA in non-HIV infection
By selecting a clone having a low P activity and a high SEAP activity due to HIV infection, a target cell can be established. An example of a cell that can be established by the above-mentioned method is accession number FERMP-18078.
And a cell having

The method and conditions for culturing animal cells used in the present invention can be appropriately selected according to the type and properties of the cells. For example, in the case of a cell line derived from HeLa cells, Dulbecco's Modified Minimu containing an antibiotic (eg, Kanamycin) and containing 5% FCS is added.
m 37 in Essential Medium (High glucose; Gibco)
It can be cultured at 5 ° C. with 5% CO ₂ .

The method for testing drug resistance of the present invention comprises the steps of:
And detecting a reporter protein secreted into the culture supernatant by HIV-1 infection. The test drug is preferably an anti-HIV drug,
More specifically, anti-HIV agents containing a reverse transcriptase inhibitor or a protease inhibitor are preferred. As a reverse transcriptase inhibitor, AZT / ZDV (zidovudine: 3'-Azido-3'-
deoxythymidine), d4T (sanilvudine: 2 ', 3'-dideh
ydro-3'-deoxy-thymidine), 3TC (lamivudine: 2'-
deoxy-3'-thiacytidine), COM (ZDV / 3TC combination), ddI (didanosine: 2 ', 3'-dideoxyinosine),
ddIEC (didanosine: 2 ', 3'-dideoxyinosine), d
dC (zalcitabine: 2 ', 3'-dideoxycytidine), ABC
(Abacavir), NVP (nevirapine), EFV (efavir)
enz), DLV (delavirdine) and the like, and protease inhibitors such as RTV (ritonavir), SQ
V (saquinavir), NFV (nelfinavir), IDV (in
dinavir), APV (amprenavir), LPV (ritonavi)
r) and the like. In addition, a combination of two or more drugs can be used as the test drug.

In order to carry out the method of the present invention, first, HI
Inoculating a microplate with animal cells capable of expressing a secreted reporter protein upon V-1 infection,
Samples containing HIV-1 are serially diluted in medium and added to each well. Further, an anti-HIV drug in an appropriate dilution series is added, and appropriate culture conditions (for example, 5% C at 37 ° C.)
After incubating at O ₂ ) for a certain time, the supernatant in each well is collected. Then, the endogenous reporter protein and HI
Heat at 65 ° C. for 15 minutes for V inactivation. Using this supernatant, a reporter protein assay can be performed according to a conventional method. When the reporter protein is alkaline phosphatase, for example, a colorimetric reaction using p-nitrophenol phosphate as a substrate, a fluorescence emission using MUP as a substrate, and a chemiluminescence measurement system using CSPD as a substrate can be used. . The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

[0029]

EXAMPLES Example 1: Establishment of animal cells capable of expressing secreted reporter protein by HIV-1 infection All HeLa cell-derived cell lines were 50 μl as antibiotics.
Dulbecco's Modified Minimum Essential Medium containing g / ml Kanamycin and containing 5% FCS
(High glucose; Gibco) at 37 ° C. with 5% CO ₂ . The CCR5-expressing MAGI cell line MAGIC-5A, which has already been established by the present inventors, is obtained by adding N
CD4 is expressed by a mouse retrovirus vector carrying an eo resistance gene, and an LTR-βGal expression unit is selected by carrying the same together with the Hygromycin resistance gene, and FACS (Fluorescent Activated Cell Sorter) is selected.
In addition to the MAGI cell line selected in
Expression vector pEFBOSpacCD4 (CD4
Expression and Puromycin resistance in Transforma
nt), the expression vector pEFB for CCR5 expression
Transformant OSbsr (CCR5 expression and Blasticidin resistance
Cell line, which was cloned by the limiting dilution method and established by G418, Hygromycin,
It was resistant to a total of four selected drugs, Puromycin and Blasticidin.

PSE downstream of the LTR derived from HIV-1 subtype B
A secretory alkaline phosphatase (SEAP) was incorporated as a reporter gene together with Transcriptional Blocker from AP2-Basic (Clontech), and an expression vector carrying a Zeocin resistance gene as a new selection gene was constructed. After transfection of this expression vector into the MAGIC-5A cell line, selective culture was performed with Zeocin to obtain 165 surviving colonies. These expanded cloned cells were plated in duplicate on a 96-well microplate, one of which was infected with HIV-1 and the other was a non-infected control. Two days after infection, 100 µl of the culture supernatant was collected and incubated at 65 ° C for inactivation of endogenous alkaline phosphatase and HIV.
Treated for 5 minutes and stored at -20 ° C until measurement. Secreted SEAP in the sample was selected and measured by a colorimetric reaction using p-nitrophenol phosphate to which L-Homoarginine was added as an endogenous alkaline phosphatase inhibitor as a substrate (Berger J. et al., Secreted placental alkaline ph).
osphatase: a powerull newquantitative indicator o
f gene expression in eukaryotic cells.Gene 66: 1-
10, 1988).

In the case of non-HIV infection, SEAP activity is low,
Clones showing high SEAP activity due to IV infection were selected and subjected to two consecutive limiting dilution methods in the absence of the selected drug. That is, a total of 162 surviving colonies were obtained by selective culture in the presence of 200 μg / ml Zeocin. However, most of the surviving colonies secreted high SEAP with or without HIV infection. From two very few colonies, HIV-infected non-backgroud S
Only EAP is detected and a large amount of SEAP is caused by HIV infection
Clone MAGIC-5 / which showed stable HIV-1 sensitivity by limiting dilution twice from a cell group secreting
SEAP 3-1-1 was established. MAGIC-5 / S
EAP 3-1-1 is FERM P-18078, dated October 16, 2000, Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, 1-3-1 Higashi, Tsukuba, Ibaraki, Japan
No. (postal code 305-8566).

Example 2: Drug resistance test (method) Subjects and virus isolation 13 patients who visited the AIDS Research and Development Center of the National Medical Center from December 1997 to January 1999
The test was performed on 245 specimens of 7 persons. Blood was collected from patients with consent, and plasma was separated. 5% the day before separation
MAG suspended in Dulbecco's MEM medium supplemented with FCS
IC5A (Masashi Tatsumi, Asato Kojima: Establishment and application of CCR5-expressing HeLa / CD4-LTR-beta Gal cells suitable for measuring HIV-1 infectious titer The 10th Annual Meeting of the Japan AIDS Society, December 1997, Kumamoto. , M. And Kojima,
A .: Establishment of HeLa / CD4-LTR = bGal expressing
CCR5 suitable for infectivity assay of T- and M-tr
opic HIV-1 and its application to clinical isolati
on and cloning of virus.The XIIth International C
ongress of AIDS, Geneve, Switzerland, June, 1998)
20,000 / well in a 48-well microplate
Cells were seeded and allowed to adhere in overnight culture. 1 ml of plasma from the patient on the day of infection was collected at 15,000 rpm
centrifuge for 40 minutes to precipitate the virus, and
μl was removed and 400 μl of InfectionMedium (20
μg / ml DEAE-Dexran 5% FCS-
D'MEM) was added, and the cells were suspended and inoculated into MAGIC-5A cells. After culturing in a 5% CO ₂ incubator at 37 ° C. for 2 hours, the medium was replaced with 5% FCS-D′MEM medium, and observation culture was continued until virus isolation was positive. The separated virus was stored at -80 ° C until use.

1. Drug resistance test Infection titration: MA on 96-well microplate on day before infection
GIC-5A cells were seeded and cultured at 10,000 cells / well. On the day of infection, the virus was thawed and serially diluted from 1- to 1000-fold with Infection Medium.
Each μl was inoculated and cultured for 2 hours. After washing, a fresh culture solution was added and further cultured. After 48 hours, the culture was removed and PB
After washing with S, fixative (1% formaldehyde, 0.2%
100 μl / well of glutaraldehyde in PBS) was added, fixed at room temperature for 5 minutes, washed again with PBS, and stained (4 mM potassium ferrocyanate, 4 mM potassium ferricyanate, 16 mM MgCl ₂ , 400 μg / ml)
X-gal, in PBS), and incubated at 37 ° C. for 2 hours. The number of cells whose cell nuclei were stained blue was counted under a microscope, and HIV-1 in the sample specimen was counted.
The infection titer was used. In the subsequent drug resistance test, 100 to 300 bcc (Blue cell co
unt) was used.

Reverse transcriptase inhibitor: MAGIC-5A or MAGIC-
5 / SEAP cells were seeded at 10,000 cells / well, cultured, and cells were attached in advance. On the day of infection, 100
３００300 bcc virus was diluted in Infection Medium and added in 100 μl aliquots. Further, 100 μl of the antiviral drug prepared in a 10-fold dilution series was added at 37 ° C., 5
The cells were cultured in a% CO ₂ incubator. 48 hours later,
MAGIC-5A cells are stained as described above,
Count the number of Blue cells, MAGIC-5 / SEAP
In the case of cells (Accession No. FERM P-18078), the culture supernatant is collected and its alkaline phosphatase activity is determined by Repo.
Chemiluminescence was measured with a Luminometer using rterAssay Kit-SEAP- (SAK-101; Toyobo Co. Japan), and at a wavelength of 405 nm in a colorimetric reaction using p-nitrophenol phosphate as a substrate. The value of the well to which no drug was added was taken as 100%, and the concentration at which 50% growth inhibition was obtained was taken as IC50.

Protease inhibitor: 100-300 bc
The virus at a concentration of c was diluted in Infection Medium, and MAG seeded on a 96-well microplate in advance.
100 μl was added to IC-5A cells. Further, 100 μl of the antiviral drug prepared in a 10-fold dilution series was added, and the cells were cultured at 37 ° C. in a 5% CO ₂ incubator. 72 hours later, MAGIC-5A prepared the day before
Then, 100 μl of supernatant and 100 μl of Infection Medium were added to a 96-well plate of MAGIC-5 / SEAP cells and cultured. After 48 hours, the value of the well without the inhibitor was reduced to 10 as described for the reverse transcriptase inhibitor drug.
The concentration at which 50% growth inhibition was obtained was defined as IC50. In all the resistance tests, the infectious molecular clone HIV-1 NL43 was used as a control for the inhibitor-sensitive virus.
Two strains were assayed in parallel and the resistance of the clinical isolate was expressed as multiple times the resistance to the IC50 value of this strain.

Genotype resistance test: RNA was extracted from 100 ml of patient plasma using a high-purity RNA isolation kit (Roche).
The po1 region of HIV-1 was amplified using pRNA PCR Kit (Perkin Elmer). WizadTM PCR Prep DNA Purifi
After concentrating the PCR reaction product using a cation system (Promega) and performing electrophoresis, a gel site containing the target fragment was cut out and purified using SUPREC-01 (Takara Co.). The nucleotide sequence of each sample thus obtained was determined using an Auto sequencer (ABI model 310). The amino acid sequence was deduced from the nucleotide sequence, and the presence or absence of resistance was examined.

(Results) Correlation between MAGIC-5A and viral load Using MAGIC-5A, viruses were isolated from 129 of 210 samples. When the amount of virus in the patient's plasma and the virus isolation rate were examined, the isolation rate was 77% when the virus amount was 4th power or more, and 8% when the viral load was less than 4th power. From this, it was confirmed that if the amount of virus was 4th power or more, virus isolation was possible using this method.

2. Reproducibility in NL432 As a reverse transcriptase inhibitor, AZT (3'-Azido-3'deoxythym
idine), d4T (2 ', 3'-didehydro-3'-deoxy-thymidin
e), and 3TC (2'-deoxy-3'-thiacytidine)
RTV (ritonavir) as a protease inhibitor,
SQV (saquinavir) and NFV (nelfinavir) were examined for reproducibility (triplicate x 5) in a drug resistance test using NL432 as an HIV-1 Wild strain-sensitive virus. As shown in Table 1, good reproducibility was shown. Was done.

[0039]

[Table 1]

3. Phenotype and genotype (gen
otype) Comparison of clinical isolate 1 obtained using this MAGIC-5A
Using 29 specimens and 2 laboratory strains, resistance tests of each drug were performed. Further clinical isolates were AZT46 specimens, d4
T47 sample, 3TC48 sample, RTV41 sample, SQV
Genotypes and analysis results were compared for 41 samples and 43 NFV samples. IC for each drug without treatment
The value of Table. 2 is shown. In spite of no treatment, AZT, RTV, SQV, and NFV were found to be more than four-fold more resistant than NL432.

[0041]

[Table 2]

The results of the resistance test for each drug are described below. AZT (see FIG. 1): The value obtained using NL432 was set to 1 time, and the degree of phenotype resistance to AZT in the untreated group was 0.1 to 5 times (average 1.2 times). there were. On the other hand, when the genotype resistance mutation is a single mutation at amino acid sequence Nos. 41 and 215, or a combination of both mutations is observed, it is 2.6 to 106 times (average 7 times).
(0 times) and the resistance was found to be inclined. Further, as previously reported, it was confirmed that when the mutations at positions 41, 215, and 184 were mutated, they were not resistant but regained sensitivity of 0.53 to 14 times (average 6.7 times). When the resistance mutation at positions 67 and 219 was added to these three combinations, the ratio became 2.6 to 106 times (112 times), indicating that high resistance was acquired again.

3TC (see FIG. 2): 3T in untreated group
The degree of phenotype resistance to C is 0.12-
It was 3.86 (1.4 times on average). Genoty in comparison
When the 184th resistance mutation was found in pe, all the samples had at least 120 times higher resistance. Even if the 184th position is a wild type, a low resistance of 0.12 to 31 times (average 9.5 times) was recognized when there was a history of treatment with another reverse transcriptase inhibitor or 3TC.

D4T (see FIG. 3): d4 in the untreated group
The degree of phenotype resistance to T is 0.4 ~
It was 3.2 times (average 1 time). In d4T, many amino acid substitutions related to resistance are unknown, and the correlation with the currently known mutation at position 75 was examined. In this center, a virus that acquired the mutation at position 75 was found. It was not an amino acid substitution reported as a resistance mutation. But like 3TC, 75th place is wild t
Even if ype was used, if it had been treated with other reverse transcriptase inhibitors or 3TC, a low resistance of 0.04 to 8.18 times (2.3 times on average) was observed.

RTV (see FIG. 4): 15 patients without treatment,
In addition, the degree of phenotype-resistant mutation in six patients who had been treated for RTV but had no genotype-resistant mutation at all was 0.33 to 2.53 times (0.8 times on average). The primary mutation at position 82 is a wild type, and the secondary mutation is 0.33 to 1.33 times when there is one (36 or 71) mutation, but 12 to 13 times when two or more mutations are involved. High resistance was obtained from moderate resistance of 106 times (average 72 times). And primarymutation
Is associated with the resistance mutation at position 82,
Irrespective of the number of
Twice) high resistance.

SQV (see FIG. 5): 16 patients without treatment,
The degree of phenotype resistance mutation was 0.1 to 10-fold (average 1.9-fold) for 12 patients who had a history of treatment with SQV but had no genotype resistance mutation at all. If the secondary mutation is one (position 10 or 82), it is 0.34 to 3.2 times.
If a resistance mutation is found at position 90, which is utation, 6.6
High resistance was acquired from moderate resistance of １００100 times (average 60.7 times).

NFV (see FIG. 6): The degree of phenotype resistance mutation was 0.33 for 4 untreated groups and 2 patients who had been treated for NFV but had no genotype resistance mutation at all. It was 10 times. And despite no treatment, secondary mutations 36, 6
A single mutation at position 3,77 or a combined mutation at position 63,77 was considered to be viral polymorphism. Therefore, in the untreated group, secondary mutation
If there is one, 0.3 to 4.6 times (1.2 times on average),
0.3-1.5 times if two are recognized at the same time (1.3 on average)
Both show sensitivity, whereas in the case of one treatment group, 0.6 to 103 times (mean 2)
(1.3 times) 10.6-333 if two are recognized at the same time
High resistance was acquired from moderate resistance (fold 115 times on average). In addition, when a resistance mutation was found at position 30 which was a primary mutation, a high resistance of 53 to 333 (165 times on average) was obtained.

MAGIC-5A and MAGIC-5 / SE
Correlation of drug resistance test using AP cells: Basically, samples measured by Blue Cell Count using MAGIC-5A were simultaneously used for MAGIC-5A and MAGIC-5 / S
The drug resistance was examined in parallel with an infection titer measuring system using EAP cells. 26 samples each of AZT, NVP and NFV, SEAP on the horizontal axis and Blue Cell on the vertical axis
When the concentration showing the IC50 of each drug obtained by Count was plotted, a high correlation was shown in a wide concentration range from a high concentration range to a low concentration range. This correlation was also found in the laboratory strain HIV-
Similarly, a high correlation was shown when plotted as a relative degree of resistance to 1NL432 (see FIGS. 7 and 8). Blue Cell Count using MAGIC-5A cells
And Chemilumines using MAGIC-5 / SEAP cells
Although the labor involved in the culturing process during the cence measurement is almost the same, the Blue Cell Count
Although the labor is reduced by image processing by Computer Soft, it takes less than an hour to read one microplate, whereas microplate compatible
The measurement is completed in tens of seconds using a Luminometer.
When multi-sample processing is required, such labor is large. In terms of detection sensitivity, for example, even when there are more than ten infectious HIV-1 in a sample inoculated in a well of a 96-well microplate, there is an advantage that MAGIC-5A cells can be fixed and determined directly under a microscope. But MAGIC-5 / SEA
P also has the advantage that a significant chemiluminescence value several times that of the HIV-1 non-infected background can be obtained, and that the culture can be continued to obtain a clearer measurement value the next day. However, for the drug resistance test, there is no practical problem since the virus is inoculated with a sufficient infectious titer (100 to 300 bcc / well) at the beginning of the culture. Further, although not shown in the data, the colorimetric reaction using p-nitrophenol phosphate as a substrate was inferior in sensitivity as compared with the chemiluminescence measurement system, but the result was sufficiently practical.

Next, the reproducibility of the drug resistance test for newly approved anti-HIV drugs, abacavia (ABC), nevirapine (NVP) and amprenavia (APV), was examined using HIV-1NL432 as a standard virus strain. did. As a result, good reproducibility was obtained as shown in Table 3. Hereinafter, the results of the resistance test for each drug are described.

[0050]

[Table 3]

Abacavia (ABC) (see FIG. 9): Abacavia (ABC), a reverse transcriptase inhibitor, has 65,
74, 115, 184 resistance mutations are known. The untreated patients tended to be 16.2 times more resistant on average. One more
The addition of 74 resistance mutations to 84 increased the resistance to 26.1-fold.

Nevirapine (NVP) (see FIG. 10):
Nevirapine (NVP), a non-diffusible reverse transcriptase inhibitor
Are known to have 103, 106, 108, 181, 190 resistance mutations. These are all primar for NVP
y mutation. The number of untreated patients was 1.05 times, and even in patients who have used reverse transcriptase inhibitors, 1.
The sensitivity range was 51 times. That is, cross-resistance was not recognized between the reverse transcriptase inhibitor and nevirapine. With the exception of 27.6-fold of the 181 mutation, all the other (106, 103, 190) resistance levels were over 100-fold and the values were far out, indicating that high resistance was obtained. Most of the resistance mutations of the non-diffusible reverse transcriptase inhibitors are recognized as cross-resistance, but only the NVP 106 resistance mutation has no cross-resistance to EFV (Efavirenz). It was found that the sample in which 106 resistance mutations measured this time were highly resistant to NVP by 131 times or more, but did not acquire 5.7 times resistance to EFV.

Amprenavia (APV) (see FIG. 11): 1.47-fold in untreated patients, and 1.04-fold more sensitive in untreated, but polymorphic, mutants with a mutation of 10. Range. However, the same 10 mutations were observed, and it was found that those treated showed an increase in resistance of 19.6-fold. Furthermore, when the secondary mutation of amprenavia, 10,46,84, was prepared, the resistance increased 232 times on average. Amprenabia, a protease inhibitor, was known as a drug with low cross-resistance because 50, the primary mutation, was not a resistance mutation to other protease inhibitors.
It was found that the presence of a plurality of tations alone led to a tendency toward resistance like other proteases. Previously, Parkin et al. Reported that the N88S mutation found in 20% of patients who had used IDL or NFV in the past also returned sensitivity to APV (Journal of Viorology, 2000, 4414-441).
9) had. In the present assay, the group in which 46 resistance mutations were observed was 24.2-fold resistant,
The presence of N88S in 46 returned the sensitivity to 5.5-fold, indicating that these patients had previously been treated with NFV.

(Discussion) At present, in order to detect a drug-resistant mutant virus, (1) a genotype (genotype method) for examining a mutation in the nucleotide sequence of a drug action region and (2) direct HIV culture in the presence of a drug And a phenotype (phen) for evaluating and evaluating concentrations (IC50, IC90) that can suppress proliferation.
otype method), and (3) recombinant DNA
Recombinant virus assay using the technology to create infectious recombinant HIV from patients and perform the phenotype method.
One is. Of these, in a short time, excellent operability,
The genotype method, which can obtain results relatively easily, is the mainstream, but on the other hand, it is difficult to make comprehensive judgments in the case of unreported resistance mutations or accumulation of multiple mutations . In contrast, the detection method using the phenotype method, which can judge the degree of resistance comprehensively, is rapidly improving, but it still takes time, cost,
It is considered labor-intensive and is not generally done. Also, regarding the recombinant virus assay, the technology for incorporating the drug-active region of the patient-derived virus into the HIV vector plasmid requires extremely skill, and the obtained recombinant virus often does not grow. Regions other than the region are known to be involved in HIV-1 particle formation, and it is not known whether the obtained recombinant virus reflects the actual properties of the virus in the patient. Like, it is not generally done.

Thus, the present inventors have now proposed MAGIC-5.
A and phenotype by MAGIC-5 / SEAP cell line
The aim of this study was to solve these problems using the method and to conduct a susceptibility test directly from clinical isolates. In the conventional phenotype method using PBMC, it takes several months to obtain a result, and it takes p
Although it is costly to measure 24, the use of these cells can provide results in only two weeks from virus isolation to drug susceptibility test, and infection by cell staining or measurement of enzyme activity in the culture supernatant. Could be quickly determined.

From the results of this example, it was found that the average degree of resistance of untreated patients was about 1-fold as compared with NL432 as a control, but some samples were 10-fold more. These viruses were found to have acquired low-level resistance by the phenotype method, despite no resistance mutations being detected by the genotype method. Data. Further genotype method and phenotype
When the methods were compared, a good correlation was obtained for drugs other than d4T, for which no resistance mutation at amino acid sequence No. 75 could be detected. However, with respect to 3TC and d4T, low-level resistance was observed even in the case of a wild type, which had been treated with a reverse transcriptase inhibitor.

AZT was also calculated for 41,184,21
When the amino acid mutation at the 5-position was simultaneously observed, it was also confirmed using this cell that the sensitivity was restored.
However, how the change in sensitivity caused by such amino acid mutations occurs in terms of the reaction and interaction between the enzyme and the substrate has not been clarified by current technology and knowledge. Furthermore, when the primary mutation was observed, the protease inhibitor showed about 100-fold higher resistance, indicating that the results obtained by the genotype method and the phenotype method were consistent. It is known that the HIV-1 virus acquires a secondary mutation to adapt under drug administration. It was reported that patients who had been treated with other protease inhibitors had a response to NFV of only about 50%, but in this case, even if no primary mutation for NFV was found, the secondary mutation did not. found. this
The secondary mutation shows susceptibility in untreated patients, but shows resistance in patients who have been treated with protease inhibitors, phenotyp
Suggested by the e method. From these results, MAGIC-
The phenotype method using 5A clarified the degree of resistance that could not be determined by the genotype method, and was considered to be useful as one of the determinations for selecting drugs to be used in future multidrug combination therapy.

From the comparison of the results using MAGIC-5A and MAGIC-5 / SEAP cells in a drug resistance test, a high correlation was obtained between the two. This is MAG
This was also predicted from the fact that IC-5 / SEAP cells were modified to secrete SEAP extracellularly by HIV-1 infection using MAGIC-5A cells as a parent strain. Moreover
As a reporter, the system using SEAP was remarkably superior to β-galactosidase in terms of stability after addition of a chemiluminescent reagent and quick and simple operation. SEAP was also superior in terms of detection sensitivity,
Measurement was possible even if the amount of virus to inoculate was small.
In the case of the Assay system using MAGIC-5 / SEAP, an actually measured value can be obtained only by inoculating cells, inoculating a virus at the same time as a drug, collecting a culture supernatant, adding an enzyme substrate solution, and applying the same to a measuring instrument. , P24 EL currently used
The operating procedure is much simpler than that of ISA or RT Assay, and it is considered that conversion to robotization by Work Station is also possible. MAGIC-5A
Virus isolation from patient samples by cell line and MAGIC
-5 / Drug resistance by SEAP Phenotyping Assay reveals that drug resistance is found in a short period of about two weeks,
It is useful for early detection of resistant virus and selection of susceptible drugs, and also measures drugs that are newly approved in the future.As a combination therapy, a high-throughput drug resistance test system that can respond to various combinations of drugs It is expected to be an indispensable measurement system for construction.

In addition, it has been found that the present test method can efficiently and sensitively detect resistant viruses of newly approved anti-HIV drugs ABC, NVP and APV. This means that this test has the flexibility to cope with various anti-HIV drugs that are further developed and expected to have clinical applications.

[0060]

Industrial Applicability According to the present invention, it is possible to provide a quick and simple drug resistance test method using an HIV-1 infectious titer cell line, and in particular, a drug resistance test method capable of rapidly processing multiple samples. became.

[Brief description of the drawings]

FIG. 1 is a diagram showing the correlation between AZT resistance in the phenotype and genotype.

FIG. 2 is a diagram showing a correlation between 3TC resistance in a phenotype and a genotype.

FIG. 3 is a diagram showing a correlation between d4T resistance in a phenotype and a genotype.

FIG. 4 is a diagram showing the correlation between phenotypic RTV resistance and genotype.

FIG. 5 is a diagram showing a correlation between SQV resistance in a phenotype and a genotype.

FIG. 6 is a diagram showing the correlation between phenotypic NFV resistance and genotype.

FIG. 7 shows chemiluminescence assay using MAGIC-5 / SEAP cells and Bl using MAGIC-5A cells.
It is a figure which shows correlation with anti-HIV-1 agent density | concentration in ue Cell Count.

FIG. 8 shows chemiluminescence assay using MAGIC-5 / SEAP cells and Bl using MAGIC-5A cells.
It is a figure which shows correlation with anti-HIV-1 agent density | concentration in ue Cell Count.

FIG. 9 is a diagram showing the correlation between ABC resistance and genotype in the phenotype.

FIG. 10 is a diagram showing the correlation between NVP resistance in phenotype and genotype.

FIG. 11 shows a correlation between phenotypic APV resistance and genotype.

Continued on the front page (72) Inventor Shinichi Oka 2-13-7-502 Yayoi Bunyo-ku, Tokyo F-term (reference) 4B024 AA11 AA20 DA03 EA04 GA11 4B063 QA01 QA18 QR02 QR13 QR77 QR80 QX02 4B065 AA93X AA93Y AA97X AC15 BA02 CA46

Claims (13)

[Claims] 1. An animal cell capable of expressing a secreted reporter protein upon HIV-1 infection is brought into contact with a sample containing HIV-1 in the presence of a test agent,
A test method for drug resistance of HIV, comprising detecting a reporter protein secreted into a culture supernatant by IV-1 infection. 2. The test method according to claim 1, wherein the animal cell has the HIV-1 virus receptor CD4 and the HIV-1 virus coreceptor. 3. The test method according to claim 1, wherein the animal cell has CXCR4 and / or CCR5 as an HIV-1 virus coreceptor. 4. The test method according to claim 1, wherein the animal cell has both CXCR4 and CCR5 as HIV-1 virus co-receptors. 5. The test method according to claim 1, wherein the secretory reporter protein is a protein that can be detected by at least one of a colorimetric reaction, fluorescence emission, and chemiluminescence measurement system. . 6. The test method according to claim 1, wherein the secretory reporter protein is alkaline phosphatase, luciferase or peroxidase. 7. The animal cell according to claim 1, wherein the animal cell is obtained by transforming an expression vector having a gene encoding a secreted reporter protein downstream of the HIV-1 LTR sequence. Test method described in 8. The test method according to claim 1, wherein the animal cell is a cell derived from a mammalian cell. 9. The test method according to claim 1, wherein the animal cell is a cell derived from a human HeLa cell or a human HOS cell. 10. The method according to claim 10, wherein the animal cell has an accession number of FERM P-.
The test method according to any one of claims 1 to 9, which is an animal cell having 18078. 11. The test agent is an anti-HIV agent containing a reverse transcriptase inhibitor or a protease inhibitor.
11. The test method according to any one of items 1 to 10. 12. The test method according to claim 1, wherein a combination of two or more drugs is used as the test drug. 13. The reporter protein secreted in the culture supernatant is subjected to any one of colorimetric reaction, fluorescence emission and chemiluminescence.
The test method according to any one of claims 1 to 12, wherein the detection is performed using at least one kind of measurement system.