Title:  Method and kit for detecting resistance to antiviral drugs

United States Patent:  6,787,126

Issued:  September 7, 2004

Inventors:  Heneine; Walid M. (Atlanta, GA); Lerma; Gerardo Garcia (Atlanta, GA); Yamamoto; Shinji (Kumamoto, JP); Switzer; William M. (Stone Mountain, GA); Folks; Thomas M. (Snellville, GA)

Assignee:  The United States of America as represented by the Department of Health and (Washington, DC)

Appl. No.:  719906

Filed:  July 30, 2001

PCT Filed:  June 18, 1999

PCT NO:  PCT/US99/13957

PCT PUB.NO.:  WO99/66068

PCT PUB. Date:  December 23, 1999

Abstract

An assay and kit for the detection of phenotypic resistance of a retrovirus to a reverse transcriptase inhibitor drug in a biological sample. The assay is based on the direct analysis of the susceptibility of retroviral reverse transcriptase to inhibition by a reverse transcriptase inhibitor drug. The enzymatic activity of the reverse transcriptase is determined by measuring the DNA product produced when an RNA template and a first complementary DNA primer from a suitable region of the encephalomyocarditis virus genome are incubated with a biological sample containing reverse transcriptase in the presence of the drug to which resistance is being determined.

SUMMARY OF THE INVENTION

An assay and kit for the detection of phenotypic resistance to a reverse transcriptase inhibitor drug in a biological sample is provided. Preferably, the biological sample is from a patient infected with a retrovirus. The assay is based on the direct analysis of the susceptibility of retroviral reverse transcriptase to inhibition by a reverse transcriptase inhibitor drug.

The enzymatic activity of the reverse transcriptase enzyme is determined by measuring the DNA product produced when an RNA template and a first complementary DNA primer from a suitable region of the encephalomyocarditis virus genome are incubated with a biological sample containing reverse transcriptase in the presence of the drug to which resistance is being determined. As a control, the enzymatic activity of the reverse transcriptase enzyme is also determined in the absence of the drug. The incubation mixture is reacted under conditions whereby the RNA template and the DNA primer will anneal and a DNA strand will be synthesized as an extension from the DNA primer if the reverse transcriptase in the sample is resistant to and not inhibited by the drug. The DNA product is amplified using a second complementary DNA primer from the encephalomyocarditis virus genome and suitable PCR reagents and conditions, and the amplified product detected in accordance with methods known to those skilled in the art. Detection of the amplified DNA indicates resistance to the drug employed in the assay. The difference in reverse transcriptase activity in the assays with and without drug verifies a finding of resistance and provides an indication as to the degree of resistance to the drug.

Preferably, the biological sample under investigation is a biological fluid, most preferably 0.5 .mu.l to 1.0 ml of blood plasma or serum Preferably, the RNA template consists of the ribonucleotide of SEQ ID NO:4; the first DNA primer consists of the oligonucleotide of SEQ ID NO:2; the second DNA primer consists of the oligonucleotide of SEQ ID NO:1; and PCR amplification is achieved by utilizing 30-40 cycles of heating the synthesized DNA and primer pair to 93 to 97^oC. for 30 to 90 seconds, at 53 to 57^oC. for 30 to 90 seconds, and at 70 to 74^oC. for 30 to 90 seconds. The amplified synthesized DNA is preferably detected by hybridization to an internal specific oligoprobe using an enzyme linked immunosorbent assay (ELISA), Southern blot hybridization methods, or similar methods.

Additionally provided is a kit for determining reverse transcriptase inhibitor drug resistance in a biological sample. The kit contains a suitable region of the encephalomyocarditis virus genome as an RNA template, a first complementary DNA primer for reverse transcriptase, and a second complementary DNA primer for amplification via the polymerase chain reaction, and the RT inhibitor or inhibitors under investigation, whereby each component is provided in separate containers or any combination of the components is provided in a single container. The kit may optionally contain the apparatus and one or more containers for obtaining and storing the sample prior to and during analysis and suitable buffers and other reagents to facilitate nucleic acid hybridization, synthesis, amplification and detection.

Therefore, it is an object of the present invention to provide sensitive methods for detecting drug resistance to reverse transcriptase inhibitor in a retrovirus-infected sample.

It is a further object of the present invention to provide a method for detecting drug resistance that is rapid, reliable, sensitive, and not labor intensive.

It is a further object of the present invention to provide a drug resistance assay that is phenotypic, not genotypic.

It is a further object of the present invention to provide an assay for drug resistance in which only a small amount of sample is needed for highly sensitive analysis.

It is a further object of the invention to provide an assay for the direct testing of biological body fluid samples such as serum, plasma, cerebrospinal fluid, saliva, semen and the like without extensive concentration, culturing or other processing techniques that would be required to increase the levels of reverse transcriptase in the sample under analysis.

It is a further object of the invention to provide an assay for drug resistance of a retrovirus that does not involve detection or amplification of the nucleic acid molecules of the retrovirus.

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS

An assay and kit for the detection and monitoring of antiviral drug resistance of a retrovirus in a biological sample are provided. The assay is a non-culture, PCR-based phenotypic assay for the detection of drug resistant reverse transcriptase enzyme in the sample. The assay is useful for monitoring a patient's response to treatment with reverse transcriptase inhibitors so that, if resistance to a particular antiviral drug is detected, the treatment can be modified, even before actual symptoms of resistance are observed, thereby keeping viral replication and the onset of opportunistic infection and disease at a minimum. The assay is also useful for isolating and identifying new antiviral drug-resistant retroviral strains and for detecting the transmission of antiviral drug-resistant retroviral strains from patient to patient.

The phenotypic assay is based on the direct analysis of the susceptibility of reverse transcriptase in the sample to inhibition by a reverse transcriptase inhibitor drug such as, but not limited to, zidovudine (ZDV, also known as azidothymidine or AZT), didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), nevirapine (NVP), abacavir (ABC), delavirdine (DLV), loviride (LVD), efavirenz (EFV) or adefovir (bis-POM PMEA).

The reverse transcriptase phenotype is based on the level of inhibition of reverse transcriptase by a fixed concentration of drug, and is determined after calculation of the ratio of units of reverse transcriptase activity/ml from a reverse transcriptase reaction made in the presence of drug to reference reactions in the absence of drug (x100). Drug concentrations resulting in 50% or 90% inhibition (IC₅₀ and IC₉₀) may also be determined by testing the reverse transcriptase with increasing concentrations of drug. The level of inhibition of reverse transcriptase by drug is used to define the susceptibility of the retrovirus to the drug.

The terms "a", "an" and "the" as used herein are defined to mean "one or more" and include the plural unless the context is inappropriate.

The biological sample to be tested may be taken from an individual, such as a wound, blood, secretion, tissue, bone, muscle, cartilage, or skin sample or may be a laboratory research sample such as a cell culture supernatant, viral isolate or viral concentrate. The sample may be may be obtained from any biological source and is preferably taken from a human or animal capable of being infected with or harboring a retrovirus. For example, the sample may be a biological fluid, such as whole blood, blood serum, blood plasma, vaginal lavage, semen, urine, saliva, sputum, cerebrospinal fluid, lacrimal fluid, fermentation fluid, lymph fluid, tissue culture fluid, ascites fluid, synovial fluid, pleural fluid, and the like. The preferred biological sample is a biological fluid from which cells can be removed. The most preferred samples are blood plasma or serum. The sample is collected or obtained using methods well known to those skilled in the art.

The sample may be diluted, purified, concentrated, filtered, dissolved, suspended or otherwise manipulated prior to use in the assay. Preferably, a sample containing particulate matter is diluted, filtered, or both diluted and filtered prior to use. One feature of the present assay is that it is useful for the direct analysis of drug resistance from a biological body fluid sample such as blood serum or plasma, saliva, cerebrospinal fluid and similar body fluids. Therefore, such a sample need not be processed prior to being combined with the assay reagents, thereby facilitating sample analysis and minimizing the amount of labor, materials, time and expenses involved in performing the assay. The sample size for the biological fluid sample is preferably between approximately 0.5 .mu.l and 1 ml.

The retrovirus present in the sample, or infecting the human or animal from which the sample is taken, is a virus characterized by the presence of reverse transcriptase, which transcribes the viral genomic RNA into a double-stranded DNA copy. Exemplary retroviruses for which a determination of drug resistance is sought include lentiviruses such as HIV-1 and HIV-2 and oncoviruses such as human T lymphocytic virus types I and II (HTLV-1 and HTLV-II). It will be understood by those skilled in the art that assays for retroviral drug resistance in species other than humans, such as nonhuman primates, cats, pigs, horses, and mice are included within the scope of the assay described herein.

The enzymatic activity of the reverse transcriptase enzyme of a retrovirus in the sample is determined by measuring the DNA product produced when an RNA template and a first complementary DNA primer from a suitable region of the encephalomyocarditis virus genome are incubated with a biological sample containing reverse transcriptase in the presence one or more drugs to which resistance is being determined, or drug homologs. As a comparative control, the enzymatic activity of the reverse transcriptase enzyme is also determined in the absence of the reverse transcriptase inhibitor. A comparison of these results provides confirmation of drug resistance and an indication as to the extent of resistance.

The concentration of drug added to the assay depends on the drug employed and the concentration of drug normally administered to a patient. For example, the concentration of 3TC added to an assay for a determination of 3TC resistance is preferably between approximately 1 and 10 .mu.M, most preferably approximately 5 .mu.M. The preferred concentration of nevirapine used in the assay is between approximately I and 100 .mu.M, most preferably approximately 50 .mu.M. It will be understood that suitable concentrations for other reverse transcriptase inhibitors can be calculated or experimentally determined using methods known to those skilled in the art.

The term "suitable region" is defined herein as a region of the RNA sequence having no significant secondary structure, less than 50% G-C content and to which complementary DNA primers can be generated which have Tm values within the range of reaction temperatures appropriate for the synthesis of a DNA strand, as described below. The RNA template can be of a length sufficient to produce a DNA product ranging in size from 100 to 500 base pairs in length, most preferably approximately 300 base pairs in length. The RNA template is most preferably the ribonucleotide of SEQ ID NO:4, which has the following sequence:

5' CAUUAGCCAU UUCAACCCAU GCGUUUGAGG AGAAGCGCUU 40 UCUGAUAACC GGUGGUCUCC CAUCAGGUUG UGCAGCGACC 80 UCAAUGCUAA ACACUAUAAU GAAUAAUAUA AUAAUUAGGG 120 CGGGUUUGUA UCUCACGUAU AAAAAUUUUG AAUUUGAUGA 160 UGUGAAGGUG UUGUCGUACG GAGAUGAUCU CCUUGUGGCC 200 ACAAAUUACC AAUUGGAUUU UGAUAAGGUG AGAGCAAGCC 240 UCGCAAAGAC AGGAUAUAAG AUAACUCCCG CUAACACAAC 280 UUCUACCUU CCUCUUAAUU CGACGCUUGA AGACGUUGUC 320 UUCUUAAAAA GAAAGUUUAA GAAAGAGGGC CCUCUGUAUC 360 GGCCUGUCAU GAAC 3'

The incubation mixture is reacted or incubated under conditions whereby the RNA template and the DNA primer will anneal and a DNA strand will be synthesized as an extension from the DNA primer if the reverse transcriptase in the sample is resistant to and therefore not inhibited by the drug. The DNA product is amplified using a second complementary DNA primer from the encephalomyocarditis virus genome and suitable DNA amplification reagents and conditions, and the amplified product detected in accordance with methods known to those skilled in the art. Detection of the amplified DNA indicates resistance to the drug employed in the assay.

As used herein, the term "complementary DNA primer" means an oligonucleotide which anneals to the RNA template in a particular orientation to allow for the synthesis of a nascent DNA strand in the presence of reverse transcriptase in the biological sample under the conditions described herein. Also as used herein, the "condition" under which a DNA strand is synthesized include the presence of nucleotides, cations and appropriate buffering agents in amounts and at temperatures such that the RNA template and the DNA primer will anneal and oligonucleotides will be incorporated into a synthesized DNA strand if reverse transcriptase is not inhibited by the reverse transcriptase inhibitor drug. Exemplary conditions are set forth in the examples below. The described conditions have been optimized from other known RT/cDNA synthesis protocols. It is generally known that other conditions can be established for optimization of a particular reverse transcriptase reaction on the basis of protocols well known to one of ordinary skill in the art. The DNA primer can be the reverse primer of a primer pair to be used in a subsequent amplification, such as, for example, the oligonucleotide of SEQ ID NO:2 (EMCR2), which has the following sequence:

5' GTTCATGACA GGCCGATACA GAGG 3'

Preferably, the second DNA primer consists of the oligonucleotide of SEQ ID NO: 1, which has the following sequence: 5' CATTAGCCAT TTCAACCCAT 3'

The synthesized strand can be amplified by any of the amplification protocols known in the art now or in the future, including but not limited to the polymerase chain reaction (PCR), the ligation amplification reaction (LAR), the ligase-based amplification system (LAS), the self-sustained sequence replication (3SR) system, the transcription-based amplification system (TAS), and the Q.beta. replicase amplification method. The preferred amplification method is PCR.

For amplification by PCR, the conditions for amplification can include 30-40 cycles (preferably 35 cycles) of heating the synthesized DNA and primer pair to 93 to 97^oC. (preferably 95^oC.) for 30 to 90 seconds (preferably one minute), at 53 to 57^oC. (preferably 55^oC.) for 30 to 90 seconds (preferably one minute), and at 70 to 74^oC. (preferably 72^oC.) for 30 to 90 seconds (preferably one minute). The amplified synthesized DNA is preferably detected by an enzyme linked immunosorbent assay (ELISA). Alternatively, the amplified DNA is detected by Southern blot hybridization methods.

As used herein, the term "primer pair" refers to two primers, one having a forward designation and the other having a reverse designation relative to their respective orientations on a double-stranded DNA molecule which consists of a sense and antisense sequence, such that under the amplification conditions described herein, the forward primer anneals to and primes amplification of the sense sequence and the reverse primer anneals to and primes amplification of the antisense sequence. Primers can be selected for use in the amplification reaction on the basis of having less than 50% G-C content, having minimal complementarity with other primers in the reaction (to minimize the formation of primer dimers) and having Tm values with the range of reaction temperatures appropriate for the amplification method, preferably PCR. In addition, primers can be selected to anneal with specific regions of the RNA template such that the resulting DNA amplification product ranges in size from 100 to 500 base pairs in length and most preferably around 300 base pairs in length. For example, in the conditions described above, the primer pair can consist of the oligonucleotide of SEQ ID NO:1 (EMCF1) as the forward primer and the oligonucleotide of SEQ ID NO:2 (EMCR2) as the reverse primer.

As used herein, the terms "detecting" or "detection" of the amplified DNA refers to qualitatively or quantitatively determining the presence of the amplified DNA strand, which is only synthesized if reverse transcriptase is resistant to the reverse transcriptase inhibitor drug added to the assay mixture. The amplification of the synthesized DNA can be detected by any method for the detection of DNA known in the art. For example, detection of the amplified DNA can be by Southern blot hybridization assay, by visualization of DNA amplification products of specific molecular weight on ethidium bromide stained agarose gels, by measurement of the incorporation of radiolabeled nucleotides into the synthesized DNA strand by autoradiography or scintillation measurement, by ELISA modified for the capture of a detectable moiety bound to the amplified DNA, or any other detection method known to one of ordinary skill in the art. The preferred detection method is by hybridization of the amplified DNA to an internal specific oligoprobe using techniques such as ELISA. Southern blot hybridization or similar method, most preferably using the specific hybridization probe of SEQ ID NO:3, which has the following sequence:

5' TGCTCTCACC TRATCAAAAT CCAAT 3'

Additionally provided is a kit for determining reverse transcriptase inhibitor drug resistance in a biological sample. The kit contains a suitable region of the encephalomyocarditis virus genome as an RNA template, a first complementary DNA primer for reverse transcriptase, and a second complementary DNA primer for amplification via the polymerase chain reaction, whereby each component is provided in separate containers or any combination of the components is provided in a single container. The kit may optionally contain a sample of the drug to which resistance is being determined, a hybridization probe for detection of the amplified DNA product, an apparatus for conducting the assay, an apparatus for assay detection, one or more containers for obtaining and storing the sample prior to and during analysis, and suitable buffers and other reagents to facilitate nucleic acid hybridization, synthesis, amplification and detection.

A preferred embodiment of the assay method described herein for the detection of 3TC-, ddC-, ddI-, AZT-, and NVP-resistant HIV-1 reverse transcriptase activity in plasma is shown as a flow chart in FIG. 1A. The protocol for the assay is set forth in FIG. 1B. The preferred assay, also referred to as the Amp-RT assay, is also described in copending application, Ser. No. 08/763,762, which is incorporated by reference herein.

In contrast to culture-based phenotypic assays, the reverse transcriptase-based phenotypic assay described herein is a highly sensitive, rapid and simple method for the direct analysis of phenotypic resistance to reverse transcriptase inhibitor drugs, and therefore provides a feasible tool for clinical monitoring and management of drug resistance. The assay is useful for the determination of phenotypic resistance to both known and unknown genotypic mutations. This assay approach is expandable to analysis of resistance to a wide variety of reverse transcriptase inhibitor drugs, and may also be useful for surveillance of transmission of drug-resistant viruses.

Claim 1 of 8 Claims

What is claimed is:

1. A method for the detection of drug resistance of a retrovirus comprising:

a) incubating a first sample comprising the retrovims with a first added quantity of a reverse transriptase inhibitor antiviral drug, an RNA template, and a first complementary DNA primer, wherein the RNA template and first complementary DNA primer are oligonucleotides from a region of the encephalomyocarditis virus genome having no significant secondary stucture and less than 50% G-C content, wherein the RNA template directs the synthesis of a DNA product, and

b) incubating a second sample comprising the retrovirus with a second added quantity of a reverse transcriptase inhibitor antiviral drug, an RNA template, and a first complementary DNA primer, wherein the RNA template and first complementary DNA primer are oligonucleotides from a region of the encephalomyocarditis virus genome having no significant secondary structure and less than 50% G-C content, wherein the second added quantity of the reverse transriptase inhibitor drug is measurably less than the first quantity of drug used in a) and wherein the RNA template directs the synthesis of a DNA product; and

c) detecting the DNA product produced in a) and b), wherein the quantity of DNA produced in a) relative to the quantity of DNA produced in b) is not reduced, thereby detecting drug resistance or the retrovirus.

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