Viral drug susceptibility testing.

The present invention relates to viral drug susceptibility testing, in particular to a method of testing phenotypic drug susceptibility in a drug-treated enveloped virus-infected, such as retrovirus-infected, e.g. human immunodeficiency virus (HIV-1)-infected, mammalian individual by testing on an enzyme packed into an enveloped virus recovered from a biological sample from said individual.

HIV-drug susceptibility is associated with virologic response to new treatments. Standardized drug resistance tests are now available, and data from clinical trials suggest that the use of drug resistance testing may be associated with improved virologic outcome. However, drug resistance is complex in terms of performance, interpretation and clinical application.

Phenotypic tests measure the ability of an HIV isolate to grow in the presence of drug and is performed using assays in which the degree of virus replication inhibition at different drug concentrations is assessed. Results are used to calculate the 50% or 90 % inhibitory concentration of a drug for an isolate. A potential problem with the classic phenotype assay is the effect of genetic drift in the virus population during virus isolation. In worst case the virus clone isolated is the one most fit to grow during in vitro conditions, not the one most abundant in the patient.

Measurement of phenotypic drug susceptibility can now be done via automated assays based on recombinant DNA technology. These approaches involve amplification of plasma RNA coding for HIV protease and reverse transcriptase (RT) and generation of a recombinant virus with the other genes from a laboratory construct (cassette virus). Genotype assays measure the occurrence of certain mutations in the genes targeted by antiretroviral drugs. Whereas phenotypic resistance measures virus-drug susceptibility, genotyping detects the mutations that confer phenotypic resistance. Polymerase chain reaction (PCR) amplification of HIV-1 sequences from plasma containing 500 to 1000 RNA copies per ml is the initial step in both these assays and in the recombinant virus phenotypic assays. Depending on mutations assessed and laboratory performing, the test, genotype assays may differentiate a mutant at a level of 10 to 50 % in a mixture of viruses. The complexity of the data generated from sequencing has led to difficulties in interpreting the results. There may be varying interpretations regarding the level of phenotypic resistance conferred by a specific mutational pattern. As new data are generated, there is a risk of providing inadequate or even incorrect interpretations. The reaction mechanism for all antiretroviral drugs used hitherto is in interference with the enzymatic reaction of either the viral protease or the RT. Depending on the capacity of the enzyme assays and the virus isolation techniques used, the drug sensitivity testing can theoretically be done either on supernatants from virus culture propagation, the primary virus isolation or on virus preparations recovered directly from the patients.

Drug susceptibility testing on RT from the primary virus isolation offers two advantages compared to traditional virus replication inhibition tests. The time for virus propagation is reduced and more important, less selection on the virus population will occur. The ideal situation is finally to characterize enzyme which has been extracted directly from the virus circulating in the blood of the patient. The benefit of such an approach is that the sample will mirror the virus population present in the patient at the time the blood sample was taken. This has so far not been feasible in practice, but the concept has been explored by resistance testing with the PCR based Amp RT assay.

The HIV-1 RT as well as other reverse transcriptases perform three different enzymatic reactions: RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, and degradation of RNA in the DNA-RNA hybrid (RNase H). The HIV RT, encoded by the pol gene, is a heterodimer consisting of a p66 and a p51 subunit. Both RNA-dependent DNA polymerization and DNA-dependent DNA polymerization are performed by the same active site localized in the p66 subunit. The p51 is produced by removal of the C-terminal fragment of the p66 corresponding to the RNase H domain. All RT inhibitors currently approved for clinical use inhibit the polymerase activity of the enzyme. The reaction mechanism of these drugs has mainly been defined according to their action on the RNA-dependent DNA polymerization reaction. The effect on the DNA-dependent DNA polymerization reaction is comparatively less studied.

Conventional RT activity assay is performed by utilising an artificial template-primer construction and labelled deoxynucleotide triphosphate as nucleotide substrate. The template / primer pair poly(rA) /oligo(dT) is the most efficient and most used combination for determination of HTV as well as for other retroviral RTs. A drawback of this type of assay when drug sensitivity testing is concerned, is that only non-nucleoside analogues or analogues that can base pair with rA can be tested. Analogues to the other nucleotide bases will require an assay based on a variable polymer template.

All anti-retroviral drugs approved hitherto interfere with the enzymatic reaction of either the viral protease or the RT. There are in addition candidate drugs in the pipeline which affect the function of the retroviral integrase.

The RT inhibitors are either nucleoside analogues or non-nucleoside analogues. The non- nucleoside inhibitors bind to a hydrophobic pocket in the RT enzyme close to, but not contiguous with the active site. HIV-1 replication is inhibited allosterically by displacing the catalytic aspartate residues relative to the polymerase binding site. Resistance usually emerges rapidly when non-nucleosides are administered as monotherapy or in the presence of incomplete virus suppression. Currently only three non-nucleoside inhibitors: Nevirapine, Efavirenz and Delavirdine are approved for clinical use by FDA.

The nucleoside inhibitors used today terminate the DNA chain elongation as they lack a 3'-hydroxyl group. Prolonged therapy with nucleoside inhibitors commonly leads to the development of resistant virus. This process is associated with the gradual appearance of mutations in the virus pol gene, each leading to defined amino acid substitutions. The effects of these substitutions at the enzymatic levels are complicated and includes enhancement of a primitive DNA editing function. This reaction is nucleotide dependent and produces dinucleoside polyphosphate and an extendible DNA 3'end.

HIV therapy today is based on multidrug therapy. The regimens are based on combinations of all three types of drugs available: nucleoside analogues, non-nucleoside analogues and protease inhibitors. The strategy is to minimise the probability for a mutant virus to survive. Facing virologic failure current therapy guidelines recommend switch to an entirely new batch of drugs. This is frustrating since many HIV positive people do not have three or more untried drugs from which to choose. Further it may also be a wasteful decision to remove a drug that in fact still is effective. With improved drug resistance testing it might be possible to weed out the ineffective drug or drugs in a given combination.

The present invention provides a procedure to perform phenotypic drug resistance testing on enzyme that is recovered directly from patient plasma samples. It is based on a combination of techniques for recovering viral enzymes essentially free from their cellular counterparts followed by their measurement using sensitive enzyme assays. The enzyme isolation technique described can be used for any enzyme packed into an enveloped virus, but its use is in the present specification only explored by utilization of plasma derived RT for drug resistance testing.

Thus, one aspect of the invention is directed to a method of testing phenotypic drug susceptibility in an enveloped virus-infected mammalian individual by testing on a polymerase packed into an enveloped virus recovered from a biological sample from said individual, comprising the steps of

• a) adding an enzyme inactivating agent selected from cysteine modifyin agents, such as 5,5'-dithiobis-(2-nitrobenzoic acid), to the sample for inactivating polymerase activity other than that present in the enveloped virion,
• b) purifying and concentrating the enveloped virus by removing enzyme activity blocking antibodies, endogenous enzyme activity inhibitors, antiviral drugs, and the enzyme inactivating agent,,
• c) lysing the virus particle to release the enzyme,
• d) recovering the concentrated purified viral enzyme resulting from b) and c) and determining the drug sensitivity profile of the individual from the recovered enzyme by using sensitive enzyme assays.

In an embodiment of the invention the mammalian individual is a human being.

In a presently preferred embodiment the biological sample is a blood sample, such as a plasma sample.

In another preferred embodiment the enveloped virus is a retrovirus, such as a human immunodeficiency virus (HIV). The enzyme is in the last mentioned case preferably a HIV reverse transcriptase (RT).

The drug sensitivity profile of an individual obtained with the method of the invention may be used for selecting drug treatment therapy for the individual. In practice, the enveloped virus-infected mammalian individual will be subjected to testing of the drug sensitivity profile at several points of time to monitor the development o the infection and the viral drug treatment in said individual.

The invention is also directed to a commercial package for testing phenotypic drug susceptibility in an enveloped virus-infected mammalian individual according to the invention, comprising an enzyme inactivating agent for inactivating polymerase activity, a sensitive enzyme assay, and at least one reference drug. It may optionally further contain written or data carrier instructions.

The invention will now be illustrated by the following unlimiting description of embodiments and drawings of the invention.

• Figure 1. shows the correlation between HIV-1 RT recovered and viral RNA measured with RNA PCR.
• Figure 2 exemplifies the relationship between the amount of RT used in the drug susceptibility assays and the IC₅₀ values found. Symbols: (◇) Efavirenz wild type RT, (◆)Efavirenz L100I RT, (○) AZT-TP wild type RT, (•) AZT-TP T215Y RT.

The procedure used consists of four different steps. I) Inactivation of host polymerase activity that is present in the sample without affecting the viral enzymes present in the enveloped virion. II) Removal of the enzyme inactivating agent, enzyme activity blocking antibodies, endogenous enzyme activity inhibitors and antiviral drugs. III) Recovering the concentrated purified viral enzyme. IV) Determining the drug sensitivity profile of the recovered enzyme.

• 1) Label the 4.5 ml plastic tubes to be used. Place them in a Nalgene box. Add 1 ml of sample (e.g. EDTA plasma from HIV infected individuals) to each labelled tube. Add 100 µl of a 66 mM solution of 5,5'-dithiobis-(2-nitrobenzoic acid) in buffered water, vortex and incubate the samples for one hour at room temperature. The activity of the free plasma enzymes is destroyed during this procedure while the enzymes contained within the virions remain intact. The virions can then be purified from 5,5'-dithiobis-(2-nitrobenzoic acid), enzyme activity blocking antibodies and other substances that may interfere with quantification of viral RT by several separation procedures. The protocol below is based on the use of Fractogel® EMD TMAE Hicap gel.
• 2) Suspend the separation gel carefully and transfer 1500 µl gel slurry to each sample pretreatment tube.
• 3) Incubate the samples with the gel slurry for 90 minutes at room temperature with the tubes lying down horizontally on an orbital shaker.
• 4) Label the desired amount of 10 ml plastic mini columns to identify the samples being analyzed. Mount the columns in a column washing device i.e. a Supelco Visiprep solid phase extraction vacuum manifold. Transfer the contents in the binding tubes to their corresponding columns. Before transfer vortex the tube briefly to evenly distribute the gel.
• 5) When all the columns are filled, apply the vacuum and suck the gels dry. Turn off the vacuum and start the washing by filling each column with 9 ml buffer A. When all columns have been filled, apply the vacuum and suck the gels dry.
• 6) Repeat step 5 three more times, giving a total of four washes. Suck the gels dry after each wash. After sucking the gels dry after the fourth wash, turn off the vacuum and proceed to step 7. The washing step removes unbound RT blocking antibodies and 5,5'-dithiobis-(2-nitrobenzoic acid) from the system.
• 7) Add to all dry gels 9 ml of conditioning buffer (B). After one minute apply vacuum and suck the gels dry.
• 8) Repeat step 7. Before turning off the vacuum check that all conditioning buffer (B) has been removed from all gels.
• 9) Lift off the upper part of the column wash device. Mount the tube holder with the labelled tubes into a clean container. Refit the upper part of the device. Control that the small tubings from each column go down in their corresponding tubes.
• 10) Add 600 µl lysis buffer (C) to each column. Let the buffer stand in the column for five minutes. Then apply the vacuum slowly and suck the gels dry. This will in each tube give approximately 600 µl of virus lysate from the connected gel.

The RT activity recovered in the lysates from step 10 are essentially free from RT blocking antibodies, drugs and cellular polymerase activity, and can be quantified with a sensitive RT activity assay, i.e. the Cavidi HS-kit Lenti RT, which is based on the method described by Ekstrand et al. 25 µl lysate obtained according to the current protocol is sufficient for determination of the RT activity in the sample. The Remaining 575 µl sample should be frozen at -70°C or below for later use in the drug sensitivity test. Note: RT enzymes that are not sensitive to cystein modifying agents e.g. wild type HIV 1 RT can optionally be assayed in the presence of up to 5 mM 5,5'-dithiobis-(2-nitrobenzoic acid). Sensitive enzymes such as MULV RT and RT from certain therapy resistant HTV 1 strains (containing e.g. the mutation Y 181 C) on the other hand require addition of a sulfhydryl reducing agent i.e. cystein or cysteamine to the lysis buffer.

A modification of the colorimetric RT assay (Cavidi® HS-kit Lenti RT), available from Cavidi Tech, Uppsala, Sweden was used for the determination of the level of RT activity in the virus preparations studied. In short, poly(rA) covalently bound to the wells of a 96 well microtiter plate serves as template for the incorporation of 5-bromodeoxyuridine 5'-triphosphate (BrdUTP) during the reverse transcription step at 33°C. The amount of bromodeoxyuridine monophosphate (BrdUMP) incorporated into DNA, is detected with an alkaline phosphatase (Ap) conjugated anti-BrdU monoclonal antibody. An Ap substrate, 4-methylumbelliferyl phosphate , is finally used for fluorimetric detection.The amount of isolate RT used in the IC₅₀ determinations was for the nucleoside analogues standardised to an activity corresponding to 5 fg (4.3 x 10^-20 mol) reference HIV-1 RT per well, while 1.7 fg (1.5 x 10^-20 mol) RT was used for the non-nucleoside analogues. The lysates were diluted in "mock lysates", i.e. lysates obtained from mock separation of foetal calf serum.The RT inhibition studies were performed in two different modified versions of the HS Lenti RT assay.

The non-nucleoside analogues were serially diluted in five fold steps in RT reaction mixture and 25 µl aliquots were transferred to each well in the microtiter plate, mixed with 125 µl RT Reaction mixture and the enzyme reaction was initiated by addition of 25 µl lysate dilution. The final nucleotide substrate (BrdUTP) concentration was 16 µM and the primer (odT₂₂) amount 12 ng per well. The dT analogues were serially diluted in five fold steps in Chain-termination reaction mixture and 25 µl aliquots were transferred to each well in the microtiter plate, mixed with 125 µl RT Chain-termination reaction mixture and the enzyme reaction was initiated by addition of 25 µl lysate dilution. The final nucleotide substrate (BrdUTP) concentration was 1.5 µM and the primer (odT₂₂) amount 12 ng per well. For both nucleoside and non-nuclosides RT inhibitors the RT reaction was allowed to proceed over-night (16-24 hours at 33°C). Thereafter the reaction was terminated by a wash of the plate. The IC₅₀ value was defined as the concentration of drug giving 50% inhibition of the RT activity studied.

Separation gel: e.g. Fractogel® EMD TMAE or Fractogel® EMD TMAE Hicap in 314 mM (2-(N-Morpholino)ethanesulfonic acid) (MES) pH 5.1, 413 mM Potassium iodide and Heparin 0.5 mg / ml. Mini columns, e.g. Biorad Poly-Prep® (7311553) Mini column washing device, i.e. Supelco Visiprep solid phase extraction vacuum manifold. Plastic tubes, e.g. Nunc 4.5 ml cryogenic tubes. Microtiter plates with immobilised prA, i.e. Nalge Nunc NucleoLinck® Cysteine modifying agent, e.g. 66 mM 5,5'-dithiobis-(2-nitrobenzoic acid) in water buffered with 0.87 M Tris(hydroxymethyl)aminomethane (pH 8.3). Mild sulfhydryl reducing agent, e.g. 33 mM cysteamine in water.

3'-azido-2', 3'-deoxythymidine triphosphate (AZT-TP) and (2',3'-didehydro-3'-deoxythymidine triphosphate (d4T-TP) was bought from Moravek Biochemicals, California. Nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2',3'-f][1,4]diazepin-6-one) (NVP), Delaviridine, 1-(5-methanesulphonamido-1H-indol-2-yl-carbonyl)-4-[3-(1-methylethyl-amino)pyridinyl]piperazine monomethane sulphonate (DLV) and Efavirenz, (-)6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one) (EFV) were bought from Apoteksbolaget, Uppsala, Sweden.

Plasma samples from naive treatment patients or from patients treated with ordinary combination therapy were selected retrospectively. The coded samples were delivered frozen as two different panels, representing patients with resistance to NNRTIs and to T-analogues respectively. The amount of HIV-1 RNA in each sample was measured by standard HIV 1 RNA PCR (Cobas, Roche Diagnostica) and the viral genotype present was analyzed with TRUGENE™ HIV-1 Genotyping kit. (Visible Genetics).

NNRTI resistant mutant forms ofRT were produced (L100I, K103N, L100I/K103N, Y181C). As template for the mutations was used the pETRT expression vector, which was constructed from the BH10 isolate. Mutations were generated using commercial site-directed mutagenesis kits, QuikChange (Stratagene). The mutations were verified by DNA sequence analysis. The mutated and native forms ofRT were isolated as previously described.

The recombinant RTs with AZT specific mutations were produced by introducing the mutation into the RT-coding region of a wild type HXB2-D EcoRI-Ndel restriction enzyme fragment cloned into the expression vector pKK233-2 (Amersham Biotech). Mutations were generated using commercial site-directed mutagenesis kits, QuikChange (Stratagene). The mutated cloned expression vectors were transformed into E. coli strain XL1-Blue and the genotypes were verified by DNA sequence analysis.

• A) Wash buffer: 20 mM MES pH 5.4, 500 mM Potassium acetate (KAc)
• B) Conditioning buffer. An RT assay compatible buffer e.g. 50 mM (N-(2-Hydroxyethylpiperazine-N'-(2-ethanesulfonic acid) (Hepes) pH 7.6, KAc 25 mM, magnesium chloride (MgCl₂) 20 mM, EthyleneGlycol-bis(β-aminoethyl Ether) N,N,N',N'-Tetraacetic Acid (EGTA) 0.2 mM, spermine 2 mM and heat inactivated bovine serum albumin (BSA) 0.5 mg / ml.
• C) Lysis buffer: An RT assay compatible buffer including a detergent e.g. 1.25 % Polyoxyethylene 4 Lauryl Ether (Brij 30), 13 ng / ml odT₂₂ and the same components as in the conditioning buffer (B). A sulfhydryl reducing agent, i.e. 0.2 mM cysteamine is optionally added when processing viruses with RT that are sensitive to SH oxidation /modification.
• D) RT Reaction mixture e.g. 10 mM Hepes pH 7.6,19 µM BrdUTP, 80 ng/ml odT₂₂, 4mM MgCl₂, 0.5 g/l dextrane sulphate, 2 mM spermine, Triton-X 100 0.5 %(v/v), EGTA 0.2 mM and BSA 0.5 mg/ml.
• E) Chain-termination reaction mixture : Hepes 10 mM pH 7.0, BrdUTP 1.75 µM, odT₂₂ 80 ng/ml, MgCl₂ 10 mM, ATP 7 mM, dextrane sulphate 0.05 g/l, spermine 2mM, Triton-X 100 0.5 %(v/v), EGTA 0.2 mM and BSA 0.5 mg/ml.

1 ml samples of EDTA plasma from HIV infected individuals were processed according to "Protocol for isolation of viral RT from material which contains RT blocking antibodies, based on destruction of soluble cellular enzyrnes followed by isolation of viral RT from mini columns." and the amount of RT activity recovered from each sample was determined in an overnight RT assay using Cavidi® HS-kit Lenti RT. The RT activities obtained were recalculated into fg HIV 1 RT / ml plasma according to an internal standard curve. The amount of HIV 1 RNA in each sample was measured by standard HIV 1 RNA PCR (Roche Amplicor). The plot is restricted to samples with PCR values > 500 copies / ml. A strong correlation was found between amount of plasma RT recovered and amount of HIV RNA measured with PCR (r =0.93, n=33, p<<0.001). See Figure 1.

From the example it can be extracted that processing of 1 ml plasma from an individual with 50 000 HIV RNA copies per ml plasma will result in recovery of approximately 200 fg RT activity. Using 1.7 fg RT per test this amount corresponds to 118 tests, which can be used for determination of the drug sensitivity profile of the isolated RT.

The effects of NNRTIs, AZT-TP and d4T-TP on indicated recombinant HTV 1 RT were determined according to "Protocol for determination of drug susceptibility of the RT activity in the lysates " using indicated RT concentrations. Fig 2 exemplifies the relation between the amount ofRT used in the drug susceptibility assays and the IC₅₀ values found. The IC₅₀ values for NNRTIs like Efavirenz was not at all influenced by variations in the RT amount within the range investigated. Among the NRTIs studied AZT-TP exhibit the largest variation in relation to the amount of RT included in the assay. This variation was maximal for wild type RT and the IC₅₀ increased from 0.13 to 0.22 µM when increasing the RT amount from 2 to 162 fg / well (Fig. 2).

The amount of RT activity available in the plasma samples sets the limits of the current tests. A signal at least five times background is required to obtain reproducible IC₅₀ values for the drugs tested. The low variability of the IC₅₀ values when increasing RT amounts are added to the test makes drug sensitivity determination feasible without previous standardization of the amount ofRT used in each assay.

The effects of three non-nucleoside inhibitors on indicated recombinant HIV 1 RT were determined according to "Protocol for determination of drug susceptibility of the RT activity in the lysates. " The amount of each RT was standardized to correspond to the activity of 1.7 fg / well of our reference RT. The duration of the RT reaction was 19 hours and the activities obtained were recalculated into % of the activity with the same RT incubated in absence of inhibitor.

The phenotype data was extracted from the literature (International antiviral news and httn://stanford.edu/hiv database) (Table 1).

There was in general a strong correlation between the IC₅₀ values from the RT inhibition assay and the phenotype data according to the literature. It was always possible to discern RTs from highly or intermediate resistant virus (Table 1).

The effects of AZT-TP and d4T on indicated recombinant HIV 1 RT were determined according to "Protocol for determination of drug susceptibility of the RT activity in the lysates. " The amount of each RT was standardized to correspond to the activity of 5 fg /well of our reference RT. The duration of the RT reaction was 19 hours and the activities obtained were recalculated into % of the activity with the same RT incubated in absence of inhibitor. The phenotype data was extracted from the literature (International antiviral news and http://stanford.edu/hiv database) (Table 2).

The Chain-termination reaction mixture used for the determination of susceptibility to T-analogue drugs should be able to support an energy dependent phosphorolysis reaction. Regrettably an efficient phosphorolysis reaction is promptly reflected by a decrease in polymerization velocity and as a consequence also a decrease in detection sensitivity. Therfore this assay require more RT activity, compared to the corresponding assay for the NNRTIs. Another consequence is that the span between resistant and sensitive RTs is smaller than in the NNRTI assay. However, there was in general a strong correlation between the IC₅₀ values from the RT inhibition assay and the phenotype data according to the literature. It was always possible to discern RTs from highly or intermediate resistant virus. (Table 2).

Determination of the susceptibility to NNRTIs using plasma derived RTs. One ml samples of plasma from 17 HIV infected individuals from Stockholm, Sweden were processed according to "Protocol for isolation of viral RT from material which contains RT blocking antibodies, based on destruction of soluble cellular enzymes followed by isolation of viral RT from mini columns." 15 of these contained enough RT to allow drug susceptibility testing. The PCR value for these samples was 17 000 copies / ml or larger. Each plasma RT and two control enzymes were titrated towards a set of serial dilutions ofNevirapine, Efavirenz, and Delavirdine according to Protocol for determination of drug sresceptibility of the RT activity in the lysates..

Using an activity corresponding to 1.7 fg/well reference RT HIV-1 RT from each RT extract we found that 7 of the 15 samples contained RTs that were highly resistant to all three NNRTIs (Tabel 3). All had the substitution K103N or in one case (sample 656) the combination of the substitutions A98G and Y181C in their RT genes. Eight samples were sensitive to all three NNRTIs. Six of which had no relevant mutation in their RT genes while the remaining two had either V 179I or V179T/A/I mutations, which not is known to affect the sensitivity to NNRTIs.

One ml samples of plasma from 27 HIV infected individuals from Stockholm, Sweden were processed according to "Protocol for isolation of viral RT from material which contains RT blocking antibodies, based on destruction of soluble cellular enzymes followed by isolation of viral RT from mini columns." Only 13 of these contained enough RT to allow drug susceptibility testing. The PCR value for these samples was 43 000 copies / ml or larger. Each plasma RT and two control enzymes were titrated towards a set of serial dilutions of AZT-TP and d4T-TP according to Protocol for determination of drug susceptibility of the RT activity in the lysates. See Table 4.

Using an activity corresponding to 5 fg/well reference RT HIV-1 RT from each RT extract and the Chain-termination reaction mixture we found that the IC₅₀ values for both drugs increased in parallel with accumulation of mutations known to be involved in resistance to NRTIs (Table 4). The mutations D69N or M184V alone did as expected not result in increased IC₅₀ values. Sample 656 and 1320 contained RTs which in spite of presence of several amino acid substitutions known to affect resistance to NRTIs exhibited intermediate IC₅₀ values for both AZTT-TP and d4T-TP. These isolates were found to also contain Y181C or M184V substitutions which are known to cause resensitation towards T-analogue drugs. Two RTs in the current panel exhibited IC₅₀ values which were elevated at least 20 times compared to the wild type control RT (Table 4). One of these had a set of amino acid (aa) insertions representative for the Q151M complex and the other contained the classic K70R substitution and in addition a L210S substitution. Using the IC₅₀ values 1µM and 0.1 µM as cut of values for AZT-TP and d4T-TP respectively the extracts can be classified as containing 9 sensitive and 4 resistant RTs, which is in concordance with the results from the genotype assay.

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