JP2002542804A - Improved assays and their reagents - Google Patents

Abstract

The present invention is directed to a method for producing a recombinant virus from a sample of uncharacterized virus or a sample such as a clinical sample, mainly altering the sensitivity of the virus to antiviral drugs. The present invention relates to the use of the viruses thus produced in assays, and reagents for use in such methods and assays, and more particularly to deoxyribonucleic acid (DNA) vector constructs for the purpose of detecting . The assay is adapted to detect resistant viruses more accurately and more sensitively than known assays by taking into account compensatory mutations made in nucleotide sequences other than the nucleotide sequence encoding the target of the antiviral drug. I have.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

DETAILED DESCRIPTION The present invention is directed to a method for producing a recombinant virus from a sample such as a virus sample or clinical sample of unspecified properties, primarily to detect changes in the sensitivity of the virus to antiviral drugs. Thus, the present invention relates to the use of the viruses thus produced in assays, and to reagents for use in such methods and assays, and more particularly to deoxyribonucleic acid (DNA) vector constructs. The assay is adapted to detect resistant viruses more accurately and more sensitively than known assays by taking into account compensatory mutations made in nucleotide sequences other than the nucleotide sequence encoding the target of the antiviral drug. I have.

[0002]

[Prior art]

Human immunodeficiency virus type I (HIV-1) resistance to antiretroviral drugs is a major goal in preventing progression to acquired immunodeficiency syndrome (AIDS) by chemotherapy. The acquisition of resistance by the virus is enhanced by the fast turnover rate of the virus in vivo, coupled with the high error rate of the viral reverse transcriptase (RT) enzyme (Coffin, 1995; Hoe).
t al. , 1995; Mansky and Temin, 1995;
Wei et al. , 1995). Therefore, the ultimate goal of anti-HIV therapy at this stage is to minimize viral replication, delay the emergence of drug-resistant variants, and reduce the normal level of CD4 ⁺ immune cells for as long as possible. To maintain (Vandamme et al., 1998). To this end, recently introduced treatments for HIV infection usually include a combination of three additional antiretroviral drugs.

[0003] Reverse transcriptase (RT) inhibitors (RTI) and viral proteases (Pro or P)
R) Simultaneous use of enzyme inhibitors (protease inhibitors or PIs)
Resulted in the emergence of an HIV-1 virus with a resistance mutation to one or both of the agents targeting Herb. (Hertogs et al., 1998). These mutations alter the structure and / or chemical affinity of the target enzyme such that the ability of the target enzyme to interact with the drug is altered or diminished, and the drug exhibits reduced activity on the mutated virus. . Drug resistance mutations can occur in any other viral drug target molecule. For example, in other viruses, antiviral drugs may act on viral proteases or, if the virus is an RNA virus, on reverse transcriptase, but other targets may be available. Indeed, any viral nucleic acid or protein that is important for the reproduction or infectivity of the virus can be a candidate for a drug target, for example, some anti-herpes nucleoside analog drugs, such as acyclovir, are drug molecules that are activated by the viral thymidine kinase. It acts against HSV-1 or 2, varicella-zoster and other herpes viruses through enzymatic phosphorylation and further processing by host cell enzymes, including incorporation into viral DNA by the DNA polymerase activity of the host. Mutations associated with reduced susceptibility to nucleoside analogs have been demonstrated in the herpesvirus thymidine kinase or DNA polymerase (Kimberlin and Whitley,
1996; Balfour, 1999). Other viral drug targets include DNA maturation factors and DNA polymerases of certain DNA viruses.

[0004] Mutations can occur at sites other than the drug target site itself in the drug-resistant virus, such as a cleavage site (CS) at which the Pro cleaves the precursor protein to form a structural and functional (Producing a viral protein) was recently reported. In HIV-1, the p7 protein is cleaved from the Gag and Gag-Pol precursor polyproteins at the p7 / p1 cleavage site (CS), and the viral proteins p1 and p6 are cleaved from the Gag polyprotein at p1 / p6 CS. Pro, RT and integrase (INT)
Resulting from cleavage of g-Pol. Mutations at the cleavage site are believed to compensate for the impaired polypeptide cleavage activity of the mutant Pro that would otherwise result in loss of virus compatibility, and are referred to herein as compensatory mutations. (Compens
Attention Mutation). Compensatory mutations were p7 / p1 and p1
/ P6 CSs is described in the literature (Doyon et a
l. Maschera et al., 1996; , 1996a, b; Zhan
get et al. , 1997; Carrillo et al. , 1998; M
ammanno et al. , 1998; Zennow et al. , 199
8). Thus, mutations associated with reduced susceptibility to antiretroviral agents currently on the market comprise at least three distinct regions of the HIV-1 genome (R
T, Pro, and CS).

As part of infection and disease management, it may be beneficial to monitor the development of drug-resistant viruses. For example, assays for the resistance phenotype of HIV-1 isolates play a major role in disease management by identifying the occurrence of reduced susceptibility to treatment, cross-resistance, and resensitization. Culturing peripheral blood mononuclear cells (PBMC) from infected patients with uninfected donor PBMC is one of the methods that has been used to isolate HIV-1 from patients for phenotypic assays. Method (Japour et al., 1993). Long culture times have been shown to select for minor or low drug resistance variants, including isolating fresh PBMCs (Kusumi et al., 1992; Maye).
rs et al. Co-culture of PBMC is not ideal for large-scale routine applications. Recombinant virus assays (RVA) allow a rapid and reproducible measurement of the phenotypic sensitivity of HIV-1 obtained from plasma (Kellam and Ladder, 1994; Maschera et al.)
al. Hertogs et al., 1995; , 1998) was useful in inducing the selection of agents used in HIV therapy. In RVA, HIV
The RT or Pro sequence was amplified from plasma by reverse transcription-polymerase chain reaction (RT-PCR) and placed in CD4 ⁺ cultured cells (MT4) in a HIV-1 standard laboratory strain named "vector" wild-type. Electroporation with HXB2 RT or Pro deleted "provirus" molecular clones. "Provirus" is a term used to describe a DNA copy of the HIV virus genome that has been integrated into the DNA of a host cell. The RT or PR sequence from plasma was inserted by homologous recombination into the corresponding deletion site of the wild-type vector and the resulting recombinant vector D
NA is inserted into the DNA of the cell to give a full-length infectious provirus.
Gene expression from the provirus creates a population of viruses with a drug-sensitive phenotype that is representative of the patient's plasma virus. The resulting virus strain can be used for drug susceptibility testing for PR or RT inhibitors, depending on which part of the genetic information in the recombinant virus is from the clinical isolate. . RVA has several advantages over PBMC co-culture in addition to its rapidity and reproducibility. The production of viruses with a common central skeleton allows for a more accurate comparison of the drug susceptibility and growth characteristics of the produced viruses, and shorter incubation times minimize the occurrence of minor variants.

[0006] Similarly, an RVA-type assay is based on a DN containing a wild-type (or other standard laboratory strain) virus genome carrying a deletion corresponding to the sequence encoding the drug target.
Application to the detection of drug-resistant variants of viruses other than HIV by recombination of sequences corresponding to antiviral drug targets obtained from patient tissue samples by PCR or RT-PCR using the A vector. it can.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION

According to a first aspect, the present invention relates to a viable set having a resistance profile of a sample of a virus by recombination of a DNA vector with a nucleotide sequence derived from the sample of the virus which is thought to comprise a drug-resistant virus. An assay for detecting a virus that is resistant to an antiviral drug to produce a recombinant virus, wherein the DNA vector comprises a deletion of at least a portion of a sequence encoding a target of the antiviral drug. Assays comprising a wild-type laboratory virus strain genome carrying additional deletions of sequences containing candidate sites for compensatory mutations. Nucleotide sequences derived from a sample of the virus (which may be obtained by PCR, RT-PCR, or related methods) include regions of the viral genome substantially corresponding to sequences deleted from the DNA vector.

[0008] Accordingly, the present invention is an assay for detecting a virus having resistance to an antiviral drug, wherein the recombination of a DNA vector with a nucleotide sequence derived from the sample results in the drug resistance of the virus in the virus sample. Creating a recombinant virus having a profile, wherein said DNA vector has a deletion of at least a portion of a sequence encoding a target of said antiviral drug and further deletion of a sequence comprising a candidate site for a compensatory mutation. An assay comprising the genome of a wild-type laboratory virus strain carrying The assay comprises infecting the cells with the recombinant virus thus produced, incubating the infected cells with an antiviral drug, and incubating the recombinant virus to determine the sensitivity of the recombinant virus to the drug. Detecting the viability of the virus or infected cells.

In a preferred embodiment, the target of the agent is a viral protease, polymerase, or reverse transcriptase. Desirably, the virus is a herpes virus or an HIV virus, preferably an HIV virus, more preferably HIV-1, wherein the DNA vector comprises a gene encoding a drug target,
And the sequence of the HIV provirus carrying the deletion in the site of the compensatory mutation. In one preferred embodiment, the DNA vector carries a deletion in the sequence encoding the viral protease and an additional deletion in the sequence encoding one or more protease cleavage sites. Desirably, the DNA vector carries a deletion of one or both of the p7 / p1 and p1 / p6 protease cleavage sites of HIV-1, in addition to the deletion of the sequence encoding the viral protease or fragment thereof.

In another aspect, the assay employs a DNA vector carrying a deletion of at least a fragment of a sequence encoding a viral reverse transcriptase. In some embodiments, in addition to the deletion of one or more protease cleavage sites, sequences encoding viral reverse transcriptase and viral protease or fragments thereof are deleted. For example, the DN
The A vector lacks one or both of the p7 / p1 and p1 / p6 protease cleavage sites of HIV-1, deletes the entire sequence encoding the viral protease, and deletes the entire sequence encoding the viral reverse transcriptase. Loss and, if necessary, deletion of the sequence encoding the viral polymerase, or deletion of the sequence encoding fragments thereof.

In a second aspect, the invention relates to a wild-type laboratory for carrying at least a portion of a sequence encoding a target of an antiviral drug and a further deletion of a sequence containing a compensatory mutation site. A DNA vector comprising a virus strain genome is provided. In a preferred embodiment,
The target of the drug is a viral protease, polymerase, or reverse transcriptase. Desirably, said virus is a herpes virus or an HIV virus, preferably an HIV virus, more preferably HIV-1, wherein said DNA vector is in a sequence encoding a drug target and Includes the sequence of the HIV provirus carrying the deletion at the site of the mutation. In certain preferred embodiments, the vector carries a deletion in the sequence encoding the viral protease and an additional deletion of the sequence encoding one or more protease cleavage sites. Desirably, the vector carries a deletion of one or both of the p7 / p1 and p1 / p6 protease cleavage sites of HIV-1, in addition to the deletion of the sequence encoding the viral protease or a fragment thereof. In another aspect, the DNA vector carries a deletion of at least a fragment of a sequence encoding a viral reverse transcriptase. In some embodiments, in addition to the deletion of one or more protease cleavage sites, sequences encoding viral reverse transcriptase and viral protease, or fragments thereof, are deleted.
For example, the DNA vector may have a deletion of one or both of the p7 / p1 and p1 / p6 protease cleavage sites of HIV-1, a deletion of the entire sequence encoding the viral protease, and a sequence encoding the reverse transcriptase of the virus. It may carry an entire deletion and, if necessary, a deletion of the sequence encoding the viral polymerase, or a fragment thereof.

In a further aspect, the invention provides a DNA vector as described above for use in an assay according to the first aspect of the invention.

In HIV-1 or other viruses, compensatory mutations may play a role in their functional role (auxiliary proteins) or in the binding, activation, or start site (site) in the RNA genome.
From this it can also be assumed that the RT enzyme can occur in viral proteins associated with the RT enzyme in the first stage of viral replication, which initiates reverse transcription of the genome). Similarly, in viral chemotherapy for certain infectious diseases, the polymerase enzyme of the virus is the target of the drug, and in this context, the resistance mutation may occur in the sequence of the virus encoding the polymerase and cause Alternatively, compensatory mutations may be envisioned in the sequence such as polymerase binding, activation, initiation, etc. of the viral genome. Other possible mechanisms by which the virus can overcome mutations detrimental to drug-induced virus growth include compensatory mutations in regulatory nucleic acid sequences or controlling the level, timing, or pattern of expression Control of the expression of drug targets by mutations affecting the factors of the virus in question. It is not known whether compensatory mutations in control sequences result in amino acid changes in any of the proteins they encode. One of skill in the art would use RVA assays in which the site of such compensatory mutations has been deleted in addition to the deletion of RT, Pro, polymerase, or other drug target sequences (or regions thereof). The teachings of the present invention could be modified to create a DNA vector for Such vectors are also included in the scope of the present invention.

When the assay of the invention is applied to HIV, it is obtained from an infected person, together with a vector from any previously characterized HIV strain, to produce a viable recombinant virus. The (RT-) PCR product of the sample may be co-transfected; for example, strains other than the standard HXB2 experimental strain may be used. In this way, the assay can be easily adapted to create variants in different "vector" strains, which may reduce the effect of the vector on fitness, drug resistance, or virulence. It may be useful to examine. The strain selected to provide the vector sequence is preferably one in which the growth in the recombinant virus produced and the variation in susceptibility to the antiviral agent resulted from the vector strain, or a sample of the virus (eg patient). Should be well-characterized so that it can be ascertained from sequences derived from
Wild-type strains are generally appropriate because they do not contain existing resistance mutations. In addition, it is necessary to select the vector strain with respect to the cell culture conditions used for producing the recombinant virus and the subsequent drug resistance. One of skill in the art will be able to select a vector strain that exhibits sufficiently strong replication characteristics in the cell culture of interest. Thus, throughout this specification and the following claims, the notation "laboratory strain,""experimental virus strain,""wild-typestrain," or "wild-type experimental virus strain" Should be understood to mean any virus strain that has been characterized so far.

In a further aspect, the present invention provides a kit for performing an assay according to the first aspect of the invention, comprising a DNA vector construct according to the second aspect of the invention. Also provided is the use of a DNA vector construct according to the present invention in an assay for detecting a virus having resistance to an antiviral drug.

The present invention is further described and illustrated in the following non-limiting examples and with reference to the accompanying drawings.

EXAMPLES In these studies, we used existing RT or Pro deleted RVA constructs (Kel
lam and Ladder, 1994; Maschera et al. ,
1995; Goulden), extending 1) p7 / p1 and p1 / p
A new plasmid was created with a continuous deletion of 6 CS and the entire Pro gene, 2) CS, Pro and RT (up to codon 232), or 3) CS, Pro and RT (up to codon 483). These constructs allow for a rapid method for assessing the drug-susceptible phenotype of plasma viruses in patients receiving any currently available anti-HIV drugs. In addition, by performing competitive RVA with vector / PCR fragment combinations that contain CS changes or where CS changes have been omitted, we have demonstrated the importance of including CS in RVA.

Example 1 Construction of a CS-Deficient HIV-1 Provirus Clone for Use in RVA To perform a phenotypic analysis of HIV-1 obtained from patients receiving Pro and RT inhibitors, we RVA with deletions in Pro, RT, p7 / p1 and p1 / p6 CS by extending the deletion in an existing Pro-deleted HIV-1 provirus clone (Maschera et al., 1995). The plasmid was constructed. After extending the Pro deletion (FIG. 1A) to include CS (FIG. 1C), the CS and Pro deletion constructs were modified using the current RT deletion construct to delete the deletion in RT. Was introduced (FIGS. 1D and 1E).

The CS is located in a 200 bp region in pHXBΔPro between the ApaI site in gag and the BstEII site located in the deletion. CS was deleted by removing this fragment. The provirus clone pHXBΔPro is pIB
Pro-deleted wild-type HIV-1 virus H cloned into H.120 vector
XB2. As is evident from FIG. 1A, the provirus contained A in gag.
In addition to the paI site, Apa was added to the cloning region of the pIB120 vector.
Has an I site. Before the 200 bp ApaI-BstEII fragment containing CS could be removed, this additional ApaI site had to be removed first. Plasmid pHXBΔPro was digested with MluI and XbaI and ApaI
The 29 bp fragment containing the site was removed and T4 DNA polymerase (New Eng)
(Land Biolabs). The blunt end was ligated to an XbaI linker (Stratagene) to obtain plasmid pHXBΔProA. Ligation was performed using Roche Diagnostics' rapid DNA ligation kit. The ligated DNA was transformed into E. coli strain XL-1 Blue supercompetent bacteria (Stratagene) and positive colonies were retransformed into strain JM109 (Stratagene) to maximize the yield of preparation. Digestion with ApaI and BstEII and custom-made HpaI linker (
Ligation with Applied Biosystems (ApaI and BstEII)
The site was restored and the HpaI site was introduced) to remove CS from pHXBΔProA, yielding pHXBΔCSPPro (FIG. 1B). The sequence of the HpaI linker is ApaI HpaI BstEII 5′-CGCGTTAACGCG-3 ′ 3′-CCGGGGCGCAATTGCCGCCACTG-5 ′.

The plasmid pHXBΔCSPRTA (FIG. 1D) contains CS, Pro and codon 2
It contains up to 32 RT deletions and is 5.9 kbp from pHXBΔCSPro
Of the HpaI / BamHI fragment of 5.2 kb from plasmid pHIVΔRTBst11071 (Goulden) lacking RT from codons 39 to 232.
It was constructed by replacing the Bst11071 / BamHI fragment of p. Plasmid pHXBΔCSPRTC (FIG. 1E) contains CS, Pro and codon 483.
Up to 5.9 kbp B from pHXBΔCSPro
The stEII / BamHI fragment was ligated with the plasmid pHIVΔBstEII (Kellam and Ladder, 19) with the RT deleted from codons 41-483.
94) was replaced by a 4.4 kbp BstEII / BamHI fragment. Plasmids were analyzed by restriction endonuclease analysis and Perki
n Elmer ABI Prism 377 DNA Sequencer and Big
The entire modified region was confirmed by sequencing using the Dye Terminator cycle sequencing method. The primer used for sequencing was CS1 (Table 1).
, FIG. 2). Sequences were analyzed by Factura feature identification software and
quence Navigator (Perkin Elmer Applier
d Biosystems).

Experiment 2 Detection of Drug Resistance For co-transfection experiments, three new RVA constructs are used to produce a virus with a drug-sensitive phenotype consistent with the introduced PCR product and the deleted region in the plasmid. This ensured that they could detect drug resistant viruses in the sample. 3 created by site-directed mutagenesis
Amprenavir resistant mutants with two Pro mutations (M46I, I47V, 150V) (see below), a single M in RT created by site-directed mutagenesis
A lamivudine-resistant mutant having the 184V mutation (Tisdale et al.,
1993) and a PCR product derived from HXB2 WT virus. The effect of introducing or excluding CS mutations from the two cloned CS variants (see below) was also examined. Although these experiments are similar to the plasma virus RVA, it is important to note that the template used to obtain the PCR products is clonally compared to the heterogeneous population of viruses found in clinical samples. Due to the differences, the possibility that more compatible viruses outperforming the mutants could occur in the heterogeneous plasma virus population was ruled out.

One of the cloned CS variants was a patient designated as Patient A (was receiving indinavir therapy, but viral load data indicated treatment failure)
Was from. By sequencing of plasma virus obtained from patient A, p7 /
A to V mutation at the P2 position (AP2V) of p1 CS (mutation previously observed in patients receiving indinavir therapy (Zhang et al., 1).
997)). Clone A1 has the AP2V p7 / p1 CS mutation and has consensus subtype B Pro sequences with I15V, I54V, R
57K, I62V, L63P, H69Y, A71T, I72E, V82A and I
85V was also different. Amplenavir therapy did not work, p1 / p6 CS P
A clone was also isolated from a patient (patient B) that acquired an L to F mutation at the I 'position (LP1'F). This mutation is associated with the protease inhibitor BILA 1906 BS
Or resistance selection in vitro using BILA 2185 BS (Doyon
et al. , 1996), in vitro resistance selection during indinavir or saquinavir therapy (Zhang et al., 1997; Mammano et a.
l. , 1998), and also after in vitro resistance selection using ABT378 in vitro (Carrillo et al., 1998). Clone B1 contains the consensus subtype B Pro sequence in addition to the LP1'F p1 / p6 CS mutation and the amino acids I15V, E34G, M36I, S37E
, I50V and L63P were different.

As already mentioned (Zoller et al., 1982; Kunkel, 19
85), using a single synthetic oligonucleotide, the M13 clone mpRT1 /
H (Larder et al., 1989) followed by electroporation into MT4 cells with the cloned RT-deleted HXB2 provirus to give amprenavir-resistant M46I / I47V / I50V Pro.
Mutants were created.

Using infected MT4 cell DNA as a template, amprenavir resistant M46I
/ I47V / I50V Pro mutant, lamivudine resistant M184V RT mutant and WT HXB2 PCR product were obtained. The DNA of the cells was incubated with
5 mM Tris pH 7.5, 5 mM EDTA, 0.5% (w / v) SDS
, And 50 μg / mL proteinase K, followed by phenol / chloroform extraction, and purification from infected MT4 cell pellets by chloroform extraction and ethanol precipitation. Plasma viral RNA is Ro
Using the Che Amplicor HIV-1 Monitor test kit,
Prepared according to manufacturer's instructions (Mulder et al., 1994).

The primers used to generate the RT-PCR product for cloning were:
In the first round, RVA5 'and RVA3' are nested reactions (nes
In ted reaction, they were CS1 and dRTC3 ′ (Table 1 and FIG. 2).
B). The product was cloned using the TOPO TA cloning kit (Invitrogen). For co-transfection with pHXBΔCSPro, primers CS2 and CP2 were used to generate PCR products covering the CS and Pro genes (Table 1 and FIG. 2B). Pr from pHXBΔCSPro
In order to create a virus having the same mutation as the o mutation but not having the CS mutation, pHXBΔPro and a PCR product obtained from a clone A1 or B1 that covers the Pro gene but does not cover CS (−CS [-CS [ RVA using the [outside] and CP2). P for RVA with pHXBΔCSPRTA
Primers for generating the CR product were CS2 and dRTA3 '. pH
CS2 and IN3 ′ were used as primers for XBΔCSPRTC.

[0026]

[Table 1]

[0027] RT-PCR of RNA obtained from plasma was performed as follows. The first round of PCR consists of two layers of reagent (Amp) in a single tube separated by a wax layer.
liwax PCR Gem 100, Perkin Elmer). The upper layer contains the reagent for reverse transcription in a total volume of 50 μL and contains 50 mM Tris-H.
Cl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 10 mM DT
T, 5% (v / v) DMSO, 500 μM of each dNTP, 20 μg / mL BS
A, 250 ng 3 'primer, 40 units of Rnasin ribonuclease inhibitor (Promega), 200 units of Superscript II
RT (Gibco BRQ, and 25 μL of RNA template.
1 mM Tris (pH 8.0), 0.1 mM ED (in a total volume of 50 μL)
TA, 5% (v / v) DMSO, 250 ng 5 'primer and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer). A second round of PCR reaction and amplification of DNA or plasmid clones from infected MT4 cells was performed (in a total volume of 100 μL) with 20 mM Tris (pH 8.0).
8), 25 mM KCl, 1.5 mM MgCl ₂ , 5% (v / v) DMSO,
200 μM of each dNTP, 250 ng of each primer, and 5 μL of template DNA
Consisted of Thermal cycling is available at Perkin Elmer GeneA
The following cycles were performed in the mp PCR system 9600. 45 ° C for 45 minutes (first round only); 95 ° C for 20 seconds, 55 ° C for 10 seconds, 72 ° C
For 60 seconds (5 cycles); 90 ° C. for 10 seconds, 55 ° C. for 10 seconds, 72 ° C. for 6 seconds.
0 seconds + 5 additional seconds for each successive cycle (30 cycles).

Prior to transfection, the RVA plasmid was linearized by digestion with a restriction enzyme that cuts the deletion site. Plasmid pHXBΔPro, pHXBΔ
CSPPro and pHXBΔCSPRTC were cut with BstEII to give pHXBΔ
CSPRTA was cut with ApaI. Transfection of MT4 cells
Based on the method described by Ellam and Larder (1994). Briefly, 10 μg of the linearized plasmid was added to QIAquick.
MT4 cells were electroporated with about 5 μg of the PCR product purified by Spin PCR Purification Kit (Qiagen). The medium was changed every few days, and when extensive cytopathic effect (CPE) was evident, 11-
Collected 14 days later. Drug sensitivity is described in Pauwels et al. (1988
3-)-(4,5-dimethylthiazol-2-yl) -2,
5-Diphenyltetrazolium bromide (MTT)-determined by a reduced colorimetric cell viability assay. A two-fold dilution series of the drug was prepared in a 96-well tissue culture plate, and 4 × 10 ⁴ MT4 cells infected with 4 viruses of 4 TCID units were added to each well. After 5 days incubation at 37 ° C., M
After adding TT and incubating at 37 ° C. for 2 hours, the amount converted to blue formazan product was evaluated by reading the absorbance at 540 nm. The drug concentration required to increase absorbance to a level of 50% of the absorbance of uninfected control cells (IC
50) was calculated by regression analysis of a plot of absorbance versus drug concentration. Resistance was expressed as fold increase in IC50 compared to wild-type HXB2 control (fold resistance, FR).

While the virus from the M184V RT mutant showed an APV sensitivity comparable to HXB2, plasmids pHXBΔCSPR and pHXBΔCSPRTA
APV resistant virus was produced when transfected with PCR product from M46I / I47V / I50V PR mutant (25FR). Therefore,
The construct appears to produce a virus with the expected PR phenotype from the introduced PCR product.

Instead of pHXBΔCSPR, pHXBΔCSPRTA or pHXBΔCSPR
When TC (> 18 FR) was co-transfected with the PCR product obtained from the M184V RT mutant, the RT phenotype of the virus produced in RVA was introduced, as demonstrated by the gain of lamivudine resistance. The characteristics of the PCR product and the deletion region in the plasmid were also consistent.

[0031] The inclusion or exclusion of the CS mutation, either during growth in RVA or in the sensitivity assay itself, which is a 5-day MT4 cell killing test (see above), indicates that the phenotypic resistance is It was unclear whether it would have any effect on detection. Therefore, CS
Cloned RT-PC from a plasma virus with PI resistance containing mutation
The R product was used to examine the effect of inclusion or exclusion of CS in RVA on drug sensitivity determination (Table 2). These experiments show that, although with the significant difference that the template used to generate the PCR products used in RVA is a clone, it was similar to the plasma virus RVA, so that The possibility of producing a more compatible virus over the mutant and giving false data was ruled out. One of the cloned CS variants had a patient designated as patient A (who received indinavir (IDV) therapy, but viral load data showed that the therapy did not work (previous therapy the were those obtained from zidovudine, lamivudine and stavudine was also included). sequencing of plasma viral obtained from the patient a, the P ₂ position of the p7 / p1 CS (AP 2 V ), a To V. Mutations were evident. Mutations were previously observed in patients who had previously been treated with indinavir (Zhang et al., 1997). Sequencing of the plasma virus from patient A revealed that p7 / pl CS mutations from a to V at P ₂ position _(AP 2 V) is revealed. this mutation, in patients receiving IDV therapy, has been observed previously (Zhang e al., 1997). clone A1 _is, AP 2 V p7
/ P1 CS mutation, and the consensus subtype B PR sequence is I15V,
I54V, R57K, I62V, L63P, H69Y, A71T, I72E, V
82A and I85V were also different. Second patient (patient B) (amprenavir (
Clones were isolated from APV; VX-478; 141W94) therapy (treatment also included zidovudine and lamivudine). Plasma virus from patient B are _{P 1} 'position (LP _1' of p1 / p6 CS F), F from L
Had to gain the mutation. This mutation is associated with the protease inhibitor BILA 1906
In vitro resistance selection using BS or BILA 2185 BS (Doy
on et al. , 1996), in vitro resistance selection during IDV or saquinavir therapy (Zhang et al., 1997; Mammano et a.
l. , 1998), and also after in vitro resistance selection using ABT378 in vitro (Carrillo et al., 1998). Clone B1 has the consensus subtype B PR sequence in addition to the LP1'F p1 / p6 CS mutation and amino acids I15V, E34G, M36I, S37E,
I50V and L63P were different.

The constructs pHXBΔPR and pHXBΔCSPR were cloned from PCR derived from clone A1.
Viruses with similar IDV susceptibility (8.1 and 8.6 FR, respectively) as the product were produced (Table 2). When APV resistance was evaluated using the two constructs, similar results were seen with the virus from clone B1 (2.5 and 2. respectively).
6FR). Thus, the presence or absence of the CS mutation indicates that RV generated from a homogeneous template
It did not significantly affect the drug-sensitive phenotype of the A product. This is not a direct effect of the CS mutation on drug susceptibility, but Doyon et al. (1996). Interestingly, virus B1 appeared to have increased sensitivity to IDV (sensitivity increased by more than 2.4-fold in both recombinants; actual values were outside the scope of this experiment). . A slight increase in the cross-sensitivity of APV-resistant viruses with other PIs has been described in previous literature (Tisdale, 1996).

[0033]

[Table 2]

Experiment 3 Competitive RVA of PI-Resistant Virus with WT When CS is not included in the PCR product used for RVA, the presence of CS mutation in the population of the source plasma virus impairs the growth of the resistant virus. Viruses with fewer mutations and, therefore, lower resistance would be expected to favor growth. The selection of a virus that is less resistant in RVA as a result of the favorable growth can make the determination of resistance in subsequent analysis inaccurate and result in cross-resistance to PI, possibly It can strongly influence decisions made about treatment regimens. A possible selection process in RVA during the growth of a heterogeneous population of plasma-derived recombinant viruses is the transfection PCR obtained from WT virus.
The products were simulated by mixing known ratios of transduced PCR products obtained from PI resistant viruses carrying the CS mutation (FIG. 3). In order to be able to precisely control the ratio of mutant: WT,
Molecular clones were used.

[0035] Molecular clones of the CS and Pro genes were generated from the plasma virus of two patients infected with a virus containing the CS mutation. Pro gene, or CS and Pro
The gene was amplified from the clone by PCR and the ratio of mutant to WT was 4: 1 or 9
: 1 was mixed with the WT product. Pro-only product is pHXBΔPro
And electroporated into MT4 cells, and the CS + Pro product was pHXBΔCSPPro
And electroporated. The relative ratio of mutant and WT in the resulting recombinant virus assembly was determined by assessing their susceptibility to indinavir or amprenavir, and the cloned P from the infected cell pellet.
It was determined by sequencing the CR product.

A PCR product covering the CS and Pro genes was prepared from clone A1 using primers CS2 and CP2. A product covering only the Pro gene was created using primer-CS (outside) and CP2. The corresponding WT product was the plasmid pHIVΔBstEII (Kellam and La) as template DNA.
rder, 1994). The concentration of the purified PCR product was 260
The product from clone A1 was mixed with the WT product in a 4: 1 or 9: 1 ratio (variant: WT), as determined by absorbance in nm or by visualization in an agarose gel. Approximately 5 μg of the mixture was electroporated into MT4 cells with 5 μg of linearized pHXBΔCSPPro (for products containing CS) or pHXBΔPro (for products without CS). RVA construct and A1 PCR D
Simultaneous electroporation of NA alone was also performed to create A1-derived viruses with or without CS mutation. After extensive virus CPE occurred over a period of 11-19 days, the susceptibility of the virus in tissue culture supernatant to indinavir was determined. Furthermore, DNA was extracted from the cell pellet, used as a template, and primer RVA was used.
A viral PCR product was created using 5 'and Comb3. The PCR products were cloned and the sequence of 11 or 12 clones obtained from each RVA was determined. The sensitivity and sequence data obtained are shown in Table 3.

As mentioned earlier, the virus reconstructed from clone A1 without the CS mutation (A1-CS) when made from a homogeneous PCR product is different from the reconstructed virus containing the CS mutation (A1 + CS). It had similar indinavir sensitivity (Table 3, Experiment A). A1-CS PCR product is combined with WT-CS product at 4: 1 or 9: 1
, The resulting RVA virus mixture had only slightly stronger resistance to IDV compared to the WT virus. Furthermore, 11 derived from cellular DNA
Or, since all 12 cloned PCR products were WT, the WT virus out-competed A1-CS, giving an RVA product that was not representative of the introduced PCR product. When the A1 + CS PCR product was mixed with the WT + CS product such that the ratio of mutant to WT was 4: 1, the resulting virus population had drug sensitivity equivalent to A1 + CS. In addition, the DNA of the infected cells
Of the 11 cloned PCR products from were A1 + CS. Clearly, A1 + CS is at least as efficient as the WT virus,
As grown in mixed RVA, we conclude that the CS mutation fully compensated for the loss of compatibility with A1-CS and completely restored detection of resistance.

The reconstituted viruses B1 + CS and B1-CS exhibited 4.2 and 2.4 fold resistance to amprenavir, respectively (Table 4). Similar to the experiment with clone A1, competitive RVA of B1-CS DNA with WT DNA resulted in loss of detection of resistance and the superiority of the WT product being amplified from the DNA of infected cells. By including the CS mutation in RVA, the growth of the mutant in a 4: 1 mixture with WT was almost completely restored, and partially restored at a 9: 1 transfer ratio.

Experiment 4 Comparison of Virus Growth The growth rate of the virus clones was compared to the WT virus reconstructed from pHXBΔCSPro. Two 1 × 10 ⁶ MT4 cell cultures were aliquoted into aliquots of virus stock containing 10 ng p24 for each PI resistant virus, or pHXBΔC
Infected with a homogeneous WT virus from SPR. The infected cells were incubated in 10 mL of growth medium, and 2, 4, 6, and 8 days after infection, 0.5 mL aliquots of the supernatant were removed. Thereafter, the p24 concentration in each aliquot was determined to assess the relative virus growth rate. The concentration of HIV-1 p24 in tissue culture supernatant or virus stock was determined by Murex HIV Antigen Ma.
b. Determined using kit (Abbott Diagnostics). The data is shown in FIG.

The results were consistent with the effects of CS variants observed by others, and with competitive RVA experiments (Doyon et al., 1996; Zhang et al.)
al. , 1997; Carrillo et al. , 1998; Mammman
o et al. , 1998). Compared to the WT virus, the growth of A1-CS was impaired, but when the CS mutation was included (A1 + CS), the deletion was almost completely recovered. B1-CS was the virus whose growth was most significantly affected (p24 levels were 86-fold lower than WT levels on day 4). However, since B1 + CS remained significantly defective compared to WT, inclusion of the mutant CS region only partially improved B1 proliferation (p24 levels were less than 4 days). Eye was 7.4 times lower than the WT). Thus, the reduction in virus compatibility induced by the resistance mutations was due to PR and CS
However, in the case of virus B1, the compensation at these sites was insufficient to fully restore compatibility.

[0041]

[Table 3]

[0042]

[Table 4]

Discussion We have identified three known sites of resistance to current anti-HIV-1 drugs (RT, Pr
o, and a plasmid containing the cloned HXB2 provirus with all deleted p7 / p1 and p1 / p6 Pro Cs) was constructed. With RVA using PCR products from the virus in the plasma of untreated patients, we have a rapid method to simultaneously detect resistance and cross-resistance to all current drugs . The novel constructs should play an important role in patient management by facilitating phenotypic analysis of drug-resistant variants arising from the complex multiple drug combinations used in HIV-1 therapy.

The WT virus sequenced 11 or 12 clones when CS was not included in the competitive RVA of the Pro mutant carrying the CS mutation and the heterogeneous PCR assembly obtained from the WT. Occasionally, they grew out of the mutant without producing detectable mutants, producing a drug-sensitive virus population. The introduced mutant PCR product is W
Even when 9: 1 more than T, WT virus propagation occurred. Loss of virus compatibility is restored when CS from indinavir escape mutant A1 is introduced, allowing detection of resistance and introducing CS from amprenavir-resistant virus clone B1. The loss of virus compatibility was partially restored. Loss of virus compatibility with inadequate polypeptide cleavage by mutant Pro has a significant effect on RVA and, in the clones examined, p7 / p1 or p1 / p6 We conclude that the inclusion of the mutation is important to minimize the growth of more compatible viruses.

The PI resistance mutation results in multiple Gag and Gag-Pol cleavage defects (Do
yon et al. Maschera et al., 1996; , 1996
a, b; Mammano et al. , 1998; Zennow et al.
. , 1998). For this reason, sites outside of the introduced PCR product can be selected for the growth of resistant viruses, and when examining resistance to Pro inhibitors with RVA, mutations can occur in CS other than p7 / p1 and p1 / p6. Sex should be considered. Nevertheless, the p7 / p1 and p1 / p6 sites appear to be sites that are preferentially mutated in response to reduced viral compatibility. HI for 58 passages
When V-1 strain IIIB was exposed to the inhibitor BILA 2185 BS, no mutations were observed in any of the CSs in gag and pol, and 1 in the Pro gene.
Eight mutations were created that confer a 500-fold resistance (Doyon et al., 19).
96). Similar results were seen with BILA 1906 BS. In another study,
From all of the late passages of the three different viruses, when all CS in gag and pol from three clones obtained using PI ABT-378 were sequenced, the mutations were at the p7 / p1 and p1 / p6 junctions. (Carrill
o et al. , 1998). In a virus obtained from a patient receiving ritonavir or saquinavir, a substitution was observed in MA / CACS, a substitution of CA / p2 was observed in patients receiving ritonavir, and the CS mutation was p7 / p1
And what can happen outside of p1 / p6 (Mammano et a
l. , 1998). It has not previously been shown that replacement of MA / CA with CA / p2 plays a role in improving virus compatibility. The observation that p7 / p1 and p1 / p6 CS are the rate-limiting sites for cleavage in PI-resistant viruses, and thus preferentially mutate, suggests that the inefficiencies of cleavage at these sites in WT virus are (Darke et al., 1)
988; Tozser et al. , 1991; Wonrak et al.
, 1993). However, it is important to note that different PR variants may behave differently, and that cleavage may be affected by varying degrees at different CSs. Because multiple Gag and Gag-Pol cleavage deficiencies occur in PI-resistant viruses, it may not be possible to completely eliminate selection for PI-resistant viruses in culture-based sensitivity assays. However, our novel RVA constructs and assays overcome the most significant deleterious effects identified to date, and our competitive RVA experiments demonstrate that they significantly improve the detection of resistance with RVA. ing.

[0046]

[References]

[0047]

[0048]

[Brief description of the drawings]

FIG. 1-1 illustrates the construction of three CS deletion plasmids for RVA. (A) Pro-deleted HIV containing an ApaI site at the vector cloning site.
1 shows a pIBI20 vector containing the -1 sequence pHXBΔPro. (B) shows pHXBΔProA created by removing the ApaI site from pHXBΔPro. ;

FIG. 1-2 illustrates the construction of three CS deletion plasmids for RVA. (C) shows pHXBΔProA created by removing the ApaI site from pHXBΔPro. (D) a plasmid pHXBΔ produced by removing a 200 bp fragment encompassing p7 / p1 and p1 / p6 CS from pHXBΔProA;
CSPro is shown. (E) shows the plasmid pHXBΔCSPRTA generated by extending the deletion in pHXBΔCSPro to codon 232 to include RT. (F) shows the plasmid pHXBΔCSPRTC in which the deletion of pHXBΔCSPro has been extended to codon 483 of RT.

FIG. 2 (A) illustrates the regions of the HIV-1 Gag and Gag-Pol polyproteins, and the position of Pro CS is indicated by Δ (TF: -transframe protein; NC:- Nucleocapsid protein). (B) shows the positions of the primers used for sequencing and preparation of the PCR product for the assay (the direction from 5 'to 3' is indicated by an arrow). (C) by means of the bidirectional arrows in parts a) and c) to e), FIGS.
1E illustrates the region of the HIV-1 genome deleted in the RVA plasmid shown in FIGS.

FIG. 3 illustrates an overview of the competitive RVA experiment of Example 3.

FIG. 4 shows the relative growth rates of recombinant drug-resistant viruses made from RVA vectors containing CS regions from plasma isolates or experimental strains.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA , ZW (72) Inventor Blair, Edward Duncan United Kingdom, S.G. 1-2 NW, Hartfordshire, Stephen Age, Gunnells Wood Road, GlaxoSmithskline (no address) (72) Invention Robinson, Lawrence Henry United Kingdom, ESG 1.2 NW, Hartfordshire, Stephen Age, Gunnels Wood Road, Glaxo Sos Smith Klein (None) (72) Inventor Snowden, Barbara Wendy, England, S.G.1-2 N.W., Hartfordshire, Stephen Age, Gunnells Wood Road, in GlaxoSmithskline (no address) (72) Invention Tisdale, Sylvia Margaret United Kingdom, S.G. 1.2 N.W., Hartfordshire, Stephen Age, Gunnells Wood Road, GlaxoSmithskline (no address) F-term (reference) 4B024 AA14 BA32 CA04 CA06 DA02 EA02 EA04 GA11 HA12 4B063 QA01 QQ42 QR32 QR55 QS34

Claims (27)

[Claims] 1. To produce a viable recombinant virus having the resistance profile of a sample of a virus by recombination of a DNA vector with a nucleotide sequence derived from the sample of the virus which is thought to contain a drug-resistant virus. An assay for detecting a virus having resistance to an antiviral drug, wherein the DNA vector comprises a deletion of at least a part of a sequence encoding a target of the antiviral drug, and a compensatory mutation site. An assay comprising a wild-type laboratory virus strain genome carrying an additional deletion of the sequence, wherein the nucleotide sequence from a sample of the virus comprises a sequence substantially corresponding to a sequence deleted from the DNA vector. 2. The method of claim 1, wherein the target of the drug is a viral protease, polymerase,
Or the assay according to claim 1, which is a reverse transcriptase. 3. The assay according to claim 1, wherein the virus is HIV-1. 4. The assay according to any one of the preceding claims, wherein the DNA vector comprises a deletion of at least a fragment of the viral protease-encoding sequence and one or more protease cleavage sites. Assays carrying additional deletions of the coding sequence. 5. The assay according to claim 3, wherein the DNA vector is derived from a wild-type HIV-1 provirus and has a deletion of a sequence encoding an HIV-1 p7 / p1 protease cleavage site. Assay to carry. 6. The assay according to claim 3, 4 or 5, wherein said D
The NA vector is derived from the wild-type HIV-1 provirus and has HIV-1 p1 / p
6 Assay carrying deletion of sequence encoding protease cleavage site. 7. The assay according to any one of the preceding claims, wherein the DNA vector carries a deletion of at least a fragment of a sequence encoding a viral polymerase. 8. The assay of claim 7, wherein said vector carries a deletion of at least a fragment of one or more sequences encoding a polymerase auxiliary protein. 9. The assay according to claim 7 or 8, wherein the vector carries a deletion of at least a fragment of one or more viral polymerase binding sites or activation sites. 10. The assay according to any one of the preceding claims, wherein:
Assay wherein said DNA vector carries a deletion of at least a fragment of a sequence encoding a viral reverse transcriptase. 11. The assay according to claim 10, wherein said vector comprises:
Assays carrying deletions of at least a fragment of one or more sequences encoding a reverse transcriptase auxiliary protein. 12. The assay according to claim 10 or 11, wherein said vector carries a deletion of at least one fragment of one or more reverse transcriptase binding sequences or activation sequences. 13. The assay according to any one of the preceding claims, wherein:
The DNA vector may have the deletion of one or both of the p7 / p1 and p1 / p6 protease cleavage sites of HIV-1, deletion of the entire sequence encoding the viral protease, and deletion of the entire sequence encoding the viral reverse transcriptase. Assays carrying deletions. 14. An assay for detecting, in a sample of a virus, a virus having resistance to an antiviral drug, comprising: (1) recombination of a DNA vector with a nucleotide sequence derived from the sample; Creating a recombinant virus having a drug resistance profile of the sample, wherein the DNA vector comprises a deletion of at least a portion of the sequence encoding the target of the antiviral agent and a candidate site for a compensatory mutation Including the genome of a wild-type experimental virus strain carrying a further deletion of the sequence; (2) infecting the cells with the recombinant virus thus produced; and Incubating with the drug, (4) measuring the sensitivity of the recombinant virus to the drug, After Yubeshon, the assay comprising the step of detecting the viability of virus or infected cells. 15. Recombination of a vector with a nucleotide sequence from a sample of a virus suspected of containing a drug-resistant virus to produce a viable recombinant virus having the resistance profile of the sample of said virus. Claims: 1. A DNA vector for use in an assay for detecting a virus resistant to a viral drug, said vector comprising a deletion of at least a fragment of said sequence encoding a target of an antiviral drug, A DNA vector comprising the genome of a wild-type laboratory virus strain carrying a further deletion of the sequence containing the site of mutation. 16. The DNA vector according to claim 15, wherein the target of the drug is a viral protease, polymerase, or reverse transcriptase. 17. The DNA vector according to claim 15, wherein the virus is HIV-1, and the sequence of the virus is derived from a wild-type HIV-1 provirus. 18. The DNA vector according to any one of claims 15 to 17, which carries a deletion of at least a fragment of a viral protease and a further deletion of one or more protease cleavage sites. 19. The p-1 of HIV-1 derived from a wild-type HIV-1 provirus.
19. The DNA vector according to claims 17 and 18, which carries a deletion of one or both of the 7 / p1 and p1 / p6 protease cleavage sites. 20. The DNA vector according to any one of claims 15 to 19, which carries deletion of at least a fragment of a sequence encoding a viral reverse transcriptase. 21. Deletion of one or both of the p7 / p1 and p1 / p6 protease cleavage sites of HIV-1, deletion of the entire sequence encoding the viral protease, and entire sequence encoding the reverse transcriptase of the virus Claim 15 carrying the deletion of
21. The DNA vector according to any one of to 20. 22. The method according to claim 15, which is pHXBΔCSPro.
The DNA vector according to Item. 23. The DNA according to claim 19, which is pHXBΔCSPRTA.
vector. 24. The DNA according to claim 19, which is pHXBΔCSPRTC.
vector. 25. A kit for performing the assay according to claim 1 or 14, wherein the kit comprises the DNA vector according to any one of claims 15 to 24. 26. Use of the DNA vector according to any one of claims 15 to 24 in an assay for detecting a virus having resistance to an antiviral drug. 27. The DNA vector according to any one of claims 15 to 24 for use in the assay according to claim 1 or 14.