Title of Invention	VIRAL DRUG SUSCEPTIBILITY TESTING
Abstract	ABSTRACT "VIRAL DRUG SUSCEPTIBILITY TESTING" A commercial package for testing phenotypic anti-viral drug resistance by testing on a reverse transcriptase (RT) enzyme activity packed into an enveloped retrovirus in a biological sample, wherein the commercial package comprises a RT enzyme assay for determination of the retroviral RT enzyme activity, recombinant reverse transcriptase (RT) controls, at least one anti-retroviral reference drug. optionally an enzyme inactivating agent for inactivating polymerase activity, other than that present in the enveloped virion, optionally a separation gel and and optionally a lysis buffer.

Title of Invention

VIRAL DRUG SUSCEPTIBILITY TESTING

Abstract

ABSTRACT "VIRAL DRUG SUSCEPTIBILITY TESTING" A commercial package for testing phenotypic anti-viral drug resistance by testing on a reverse transcriptase (RT) enzyme activity packed into an enveloped retrovirus in a biological sample, wherein the commercial package comprises a RT enzyme assay for determination of the retroviral RT enzyme activity, recombinant reverse transcriptase (RT) controls, at least one anti-retroviral reference drug. optionally an enzyme inactivating agent for inactivating polymerase activity, other than that present in the enveloped virion, optionally a separation gel and and optionally a lysis buffer.

Full Text	FORM 2 THE PATENTS ACT 1970 [39 OF 1970] COMPLETE SPECIFICATION [See Section 10 ; rule 13]] VIRAL DRUG SUSCEPTIBILITY TESTING CAVIDI TECH AB, a Swedish corporation of Uppsala Science Park, SE-751 83 Uppsala, Sweden, The following specification particularly describes the nature of the invention and the manner in which it is to be performed:- The present invention relates to viral drug susceptibility testing, in particular to a method of 5 testing phenotypic drug susceptibility in a drug-treated enveloped virus-infected, such as retro virus-infected, e.g. human immunodeficiency virus (HIV-l)-infected, mamrnalian individual by testing on an enzyme packed into an enveloped virus recovered from a biological sample from said individual. Background 10 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. 15 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 20 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 25 recombinant virus with the other genes from a laboratory construct (cassette virus) [ 1 ]. 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 30 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 WO 02/103040 PCT/SE02/01156 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 5 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 10 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 15 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 [5], 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 [6]. 20 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 25 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. Description of the invention 30 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. WO 02/103040 PCT/SE02/01156 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 5 drug susceptibility in an enveloped virus-infected mammalian individual by testing on an enzyme packed into an enveloped virus recovered from a biological sample from said individual, comprising the steps of a) adding an enzyme inactivating agent to the sample for inactivating polymerase activity other than that present in the enveloped virion, 10 b) removing the enzyme inactivating agent, enzyme activity blocking antibodies, endogenous enzyme activity inhibitors and antiviral drugs, c) lysing the virus particle to release the enzyme, d) recovering the concentrated purified viral enzyme resulting from c) and determining the drug sensitivity profile of the individual from the recovered 15 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 20 a human immunodeficiency virus (HIV). The enzyme is in the last mentioned case preferably a HTV 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 25 drug sensitivity profile at several points of time to monitor the development of the infection and the viral drug treatment in said individual. The invention is also directed to a commercial package comprising written or data carrier instructions for testing phenotypic drug susceptibility in an enveloped virus-infected mammalian individual according to the invention, and an enzyme inactivating agent 30 for inactivating polymerase activity, a sensitive enzyme assay, and at least one reference drug. The invention will now be illustrated by the following unlimiting description of embodiments and drawings of the invention. The teachings of the cited literature is incorporated herein by reference. WO 02/103040 PCT/SE02/01156 Short description of the drawings. 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 5 drug susceptibility assays and the IC50 values found. Symbols: ( O ) Efavirenz wild type RT, (♦ )Efavirenz L1001 RT, ( O) AZT-TP wild type RT, (• ) AZT-TP T215Y RT. Description of embodiments The procedure used consists of four different steps. I) Inactivation of host 10 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. EI) Recovering the concentrated purified viral enzyme. IV) Determining the drug sensitivity profile of the recovered enzyme. 15 (Steps I-III) 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. 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 20 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 25 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 pre- treatment tube. 3) Incubate the samples with the gel slurry for 90 minutes at room temperature with the tubes 30 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 WO 02/103040 PCT/SE02/01156 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 5 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- 10 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. 15 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 20 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 [7]. 25 μl lysate obtained according to the current protocol is 25 sufficient for determination of the RT activity in the sample. The Remaining 575 ul 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 30 (containing e.g. the mutation Y181C) on the other hand require addition of a sulfhydryl reducing agent i.e. cystein or cysteamine to the lysis buffer. WO 02/103040 PCT/SE02/011S6 (Step IV) Protocol for determination of drug susceptibility of the RT activity in the lysates. 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 5 of a 96 well microtiter plate serves as template for the incorporation of 5-bromo- deoxyuridine 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 10 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. 15 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 20 lysate dilution. The final nucleotide substrate (BrdUTP) concentration was 16 uM 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 μ1 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 ul lysate dilution. The final nucleotide 25 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. 30 WO 02/103040 PCT/SE02/01156 Materials 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. 5 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 10 with 0.87 M Tris(hydroxymethyl)aminomethane (pH 8.3). Mild sulfhydryl reducing agent, e.g. 33 mM cysteamine in water. Antiviral drugs: 3'-azido-2', 3'-deoxythymidine triphosphate (AZT-TP) and (2',3-didehydro-3'- deoxythymidine triphosphate (d4T-TP) was bought from Moravek Biochemicals, California. 15 Nevirapine (1 l-cyclopropyl-5,1 l-dihydro-4-methyl-6H-dipyrido[3,2-b:2,,3'-f][l,4]dia2epin-6- one) (NVP), Delaviridine, l-(5-methanesulphonamido-lH-indol-2-yl-carbonyl)-4-[3-(l- methylethyl-amino)pyridinyl]piperazine monomethane sulphonate (DLV) and Efavirenz, (-)6- chloro-4-cyclopropylethynyl-4-trifluoromethyl-l,4-dihydro-2H-3,l-benzoxazin-2-one)(EFV) were bought from Apoteksbolaget, Uppsala, Sweden. 20 Plasma samples from HIV infected individuals. 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 25 standard HIV 1 RNA PCR (Cobas, Roche Diagnostica) and the viral genotype present was analyzed with TRUGENE™ HIV-1 Genotyping kit. (Visible Genetics). Recombinant RT enzymes: NNRTI resistant mutant forms of RT were produced (L100I, K103N, L100I/IC103N, Y181C). As template for the mutations was used the pETRT expression 30 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 of RT were isolated as previously described [8]. WO 02/103040 PCT/SE02/01156 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, 5 QuikChange (Stratagene). The mutated cloned expression vectors were transformed into E. coli strain XL 1-Blue and the genotypes were verified by DNA sequence analysis. Buffers used: 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- 10 Hydroxyethyipiperazine-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 % 15 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 uM BrdUTP, 80 ng/ml odT22, 4mM 20 MgCl2,0.5 g/1 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/1, spermine 2mM, Triton-X 100 0.5 %(v/v), EGTA 0.2 mM and BSA 0.5 mg/ml. 25 Examples Example 1. Correlation between HIV-1 RT recovered and viral RNA measured with RNA PCR. 1 ml samples of EDTA plasma from HIV infected individuals were processed 30 according to "Protocol for isolation of viral RTfrom material which contains RT blocking antibodies, based on destruction of soluble cellular enzymes followed by isolation of viral RT from mini columns." and the amount of RT activity recovered from each sample was WO 02/103040 PCT/SE02/01156 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 HTV 1 RNA PCR (Roche Amplicor). The plot is restricted to samples with PCR values > 500 copies / ml. 5 A strong correlation was found between amount of plasma RT recovered and amount of HTV 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 HTV 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 10 tests, which can be used for determination of the drug sensitivity profile of the isolated RT. Example 2. Influence of the amount of RT used in the drug susceptibility tests. 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 15 activity in the lysates " using indicated RT concentrations. Fig 2 exemplifies the relation between the amount of RT 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 20 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 25 amounts are added to the test makes drug sensitivity determination feasible without previous standardization of the amount of RT used in each assay. Example 3. Comparison of the effects of non-nucleoside inhibitors on recombinant HIV 1 RT with defined mutations. 30 The effects of three non-nucleoside inhibitors on indicated recombinant HTV 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 WO 02/103040 PCT/SE02/01156 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 1). 5 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). Example 4. Comparison of the effect of dT anlogues on recombinant HIV 1 RT with defined 10 mutations. The effects of AZT-TP and d4T on indicated recombinant HIV I 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 15 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 20 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 25 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). Example 5. 30 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 WO 02/103040 PCT7SE02/0115 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 of Nevirapine, Efavirenz, and Delavirdine according to Protocol for determination of drug susceptibility of 5 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 10 sensitive to all three NNRTIs. Six of which had no relevant mutation in their RT genes while the remaining two had either VI791 or V179T/A/I mutations, which not is known to affect the sensitivity to NNRTIs. Example 6. Determination of the susceptibility to AZT-TP and d4T-TP using plasma derived RTs. 15 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 RTfrom 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. 20 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 25 drugs increased in parallel with accumulation of mutations known to be involved in resistance to NRTIs (Table 4). The mutations D69N or Ml 84V 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 30 or Ml84V 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 [8] and the other contained the classic K70R WO 02/103040 PCT/SE02/01156 References 1. Petropoulos CJ, Parkin NT, Limoli KL, Lie YS, Wrin T, Huang W, Tian H, Smith D, Winslow GA, Capon DJ, Whitcomb JM. A novel phenotypic drug susceptibility assay for 5 human immunodeficiency virus type 1. Antimicrob Agents Chemother. 2000 Apr; 44(4):920-8. 2. The Euroguidelines Group for HIV resistance. Clinical and laboratory guidelines for the use of HIV-1 drug resistance testing as part of treatment management: recommendations 10 for the European setting. AIDS. 2001 Feb 16;15(3):309-20. 3. Vazquez-Rosales G, Garcia Lerma JG, Yamamoto S, Switzer WM, Havlir D, Folks TM, Richman DD, Heneine W. Rapid screening of phenotypic resistance to nevirapine by direct analysis of HIV type 1 reverse transcriptase activity in plasma. AIDS Res Hum 15 Retroviruses. 1999 Sep 1;15(13):1191-200. 4. Goff, S.P. (1990) Retrovirus reverse transcriptase: Synthesis, Structure, and Function. Review. J Acquir Imm Defic Syndr 3: 817-831, 20 5. Vandamme AM, Van Vaerenbergh K, De Clercq E. Anti-human immunodeficiency virus drug combination strategies. Antivir Chem Chemother. 1998 May; 9(3): 187-203. 6. Meyer PR, Matsuura SE, Mian AM, So AG, Scott WA. Related Articles. A mechanism of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant 25 HIV-1 reverse transcriptase. Mol Cell. 1999 Jul;4(l):35-43 7. Ekstrand DH, Awad RJ, Kallander CF, Gronowitz JS. A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human- 30 immunodeficiency-virus isolation. Biotechnol Appl Biochem. 1996 Apr;23 (Pt 2):95- 105. WO 02/103040 PCT/SE02/01156 8. Lindberg J, Sigurethsson S, Lowgren S, O Andersson H, Sahlberg C, Noreen R, Fridborg K, Zhang H, Unge T. Structural basis for the inhibitory efficacy of efavirenz (DMP-266), MSC194 and PNU142721 towards the HTV-1 RT K103N mutant. Eur J Biochem. 2002 5 Mar;269(6):1670-1677 9. Shirasaka, T., Kavlick, M. F., Ueno, T., Gao, W.-Y., Kojima, E., Alcaide, M. L., Chokekijchai, S., Roy, B. M., Arnold, E., Yarchoan, R., and Mitsuya, H. Emergence of human immunodeficiency virus type 1 variants with resistance to multiple 10 dideoxynucleosides in patients receiving therapy with dideoxynucleosides. Proc Natl Acad Sci USA 1995, 92: 2398-2402. 10. Larder BA. 3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase 15 inhibitors. Antimicrob Agents Chemother. 1992 Dec;36(12):2664-9. 11. Larder BA. Interactions between drug resistance mutations in human immunodeficiency virus type 1 reverse transcriptase. J Gen Virol. 1994 May;75 (Pt 5):951-7. 20 12. Boucher CA, Cammack N, Schipper P, Schuurman R, Rouse P, Wainberg MA, Cameron JM. High-level resistance to (-) enantiomeric 2'-deoxy-3'-thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother. 1993 Oct;37(10):2231-4 Table 1. Comparison of the effect of non-nucleoside inhibitors on recombinant HIV 1 RT with defined mutations. Phenotype data: S = sensitive, L= Low level resistance (2-10 times increase in inhibitory dose), I = Intermediate resistance (10-100 times increase), H = High level resistance (>100 times increase), "nd not done. Phenotype data: RS= the mutations gives re-sensitation of resistant virus. S = sensitive, L= Low level resistance, I = Intermediate resistance, H = High level resistance. and not done.. WO 02/103040 PCT/SE02/01156 Table 3. Effect of NNRTIs on HIV RT recovered from patient plasma ^ *Hetrogenicity in the sequence V179T/A/I. WE CLAIM: 1. A commercial package for testing phenotypic anti-viral drug resistance by testing on a reverse transcriptase (RT) enzyme activity packed into an enveloped retrovirus in a biological sample, wherein the commercial package comprises a RT enzyme assay for determination of the retroviral RT enzyme activity, recombinant reverse transcriptase (RT) controls, at least one anti-retroviral reference drug. optionally an enzyme inactivating agent for inactivating polymerase activity, other than that present in the enveloped virion, optionally a separation gel and and optionally a lysis buffer. 2. A commercial package as claimed in claim 1, wherein the anti-retroviral reference drug is a human immunodeficiency virus (HIV) drug. 3. A commercial package as claimed in claim 2, wherein the HIV-drug is a nucleoside RT-inhibitor and/or a non-nucleoside RT-inhibitior. 4. A commercial package as claimed in claim 3, wherein the nucleoside RT-inhibitor and/or a non-nucleoside RT-inhibitior is/are selected from the group consisting of 3'-azido-2', 3'-deoxythymidine triphosphate (AZT-TP). (2', 3'-didehydro-3'deoxythymidine triphosphate (d4T-TP), Nevirapine (11-cyclopropyl- 5. ll-dihydro-4-methyl-6H-dipyrido [3,2-b:2, 3'-f] [1, 4] diazepin-6one)(NVP), Delaviridine,l- (5-methanesulphonamido-lH-indol-2-yl-carbonyl)-4- [3- (1-methylethyl-amino) pyridinyl] piperazin monomethane sulphonate (DLV) and Efavirenz, (-) chloro-4-cyclopropylethynyl-4-trifluoromethyl-l, 4-dihydro-2H-3,l-benzoxazin-2-one) (EFV). 5. A commercial package as claimed in claims 1 to 4, wherein the recombinant reverse transcriptase (RT) controls are selected from the group consisting of wild type human immunodeficiency virus (HIV) reverse transcriptase and HIV reverse transcriptase comprising site-directed mutations. 6. A method of testing phenotypic drug susceptibility in an enveloped retrovirus-infected mammalian individual by testing on an enzyme packed into an enveloped retrovirus recovered from a biological sample from said individual, comprising the steps of: a) adding an enzyme inactivating agent to the sample for inactivating polymerase activity other than that present in the enveloped virion, b) removing the enzyme inactivating agent, enzyme activity blocking antibodies, endogenous enzyme activity inhibitors and antiviral drugs, c) lysing the virus particle to release the enzyme, d) recovering the concentrated purified viral enzyme resulting from c) and determining the drug sensitivity profile of the individual from the recovered enzyme by using sensitive enzyme assays. 7. The method as claimed in claim 6, wherein the mammalian individual is a human being. 8. The method as claimed in claim 6 or 7, wherein the biological sample is a blood sample. 9. The method as claimed in claim 8, wherein the blood sample is a plasma sample.

Full Text

FORM 2 THE PATENTS ACT 1970 [39 OF 1970]
COMPLETE SPECIFICATION
[See Section 10 ; rule 13]] VIRAL DRUG SUSCEPTIBILITY TESTING
CAVIDI TECH AB, a Swedish corporation of Uppsala Science Park, SE-751 83 Uppsala, Sweden,
The following specification particularly describes the nature of the invention and the manner in which it is to be performed:-

The present invention relates to viral drug susceptibility testing, in particular to a method of
5 testing phenotypic drug susceptibility in a drug-treated enveloped virus-infected, such as
retro virus-infected, e.g. human immunodeficiency virus (HIV-l)-infected, mamrnalian
individual by testing on an enzyme packed into an enveloped virus recovered from a
biological sample from said individual.
Background
10 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.
15 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
20 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
25 recombinant virus with the other genes from a laboratory construct (cassette virus) [ 1 ].
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
30 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

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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 5 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 10 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
15 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 [5], 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 [6].
20 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 25 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.
Description of the invention
30 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.

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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 5 drug susceptibility in an enveloped virus-infected mammalian individual by testing on an enzyme packed into an enveloped virus recovered from a biological sample from said individual, comprising the steps of
a) adding an enzyme inactivating agent to the sample for inactivating polymerase
activity other than that present in the enveloped virion,
10 b) removing the enzyme inactivating agent, enzyme activity blocking antibodies,
endogenous enzyme activity inhibitors and antiviral drugs,
c) lysing the virus particle to release the enzyme,
d) recovering the concentrated purified viral enzyme resulting from c) and determining the drug sensitivity profile of the individual from the recovered
15 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 20 a human immunodeficiency virus (HIV). The enzyme is in the last mentioned case preferably a HTV 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 25 drug sensitivity profile at several points of time to monitor the development of the infection and the viral drug treatment in said individual.
The invention is also directed to a commercial package comprising written or data carrier instructions for testing phenotypic drug susceptibility in an enveloped virus-infected mammalian individual according to the invention, and an enzyme inactivating agent 30 for inactivating polymerase activity, a sensitive enzyme assay, and at least one reference drug. The invention will now be illustrated by the following unlimiting description of embodiments and drawings of the invention.
The teachings of the cited literature is incorporated herein by reference.

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Short description of the drawings.
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 5 drug susceptibility assays and the IC50 values found. Symbols: ( O ) Efavirenz wild type RT, (♦ )Efavirenz L1001 RT, ( O) AZT-TP wild type RT, (• ) AZT-TP T215Y RT.
Description of embodiments
The procedure used consists of four different steps. I) Inactivation of host 10 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. EI) Recovering the concentrated purified viral enzyme. IV) Determining the drug sensitivity profile of the recovered enzyme. 15 (Steps I-III) 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.
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
20 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 25 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 pre-
treatment tube.
3) Incubate the samples with the gel slurry for 90 minutes at room temperature with the tubes
30 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

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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
5 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-
10 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.
15 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
20 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 [7]. 25 μl lysate obtained according to the current protocol is 25 sufficient for determination of the RT activity in the sample. The Remaining 575 ul 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 30 (containing e.g. the mutation Y181C) on the other hand require addition of a sulfhydryl reducing agent i.e. cystein or cysteamine to the lysis buffer.

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(Step IV) Protocol for determination of drug susceptibility of the RT activity in the lysates.
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 5 of a 96 well microtiter plate serves as template for the incorporation of 5-bromo-
deoxyuridine 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
10 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.
15 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
20 lysate dilution. The final nucleotide substrate (BrdUTP) concentration was 16 uM 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 μ1 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 ul lysate dilution. The final nucleotide
25 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.
30

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Materials
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. 5 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 10 with 0.87 M Tris(hydroxymethyl)aminomethane (pH 8.3).
Mild sulfhydryl reducing agent, e.g. 33 mM cysteamine in water.
Antiviral drugs:
3'-azido-2', 3'-deoxythymidine triphosphate (AZT-TP) and (2',3-didehydro-3'-
deoxythymidine triphosphate (d4T-TP) was bought from Moravek Biochemicals, California. 15 Nevirapine (1 l-cyclopropyl-5,1 l-dihydro-4-methyl-6H-dipyrido[3,2-b:2,,3'-f][l,4]dia2epin-6-
one) (NVP), Delaviridine, l-(5-methanesulphonamido-lH-indol-2-yl-carbonyl)-4-[3-(l-
methylethyl-amino)pyridinyl]piperazine monomethane sulphonate (DLV) and Efavirenz, (-)6-
chloro-4-cyclopropylethynyl-4-trifluoromethyl-l,4-dihydro-2H-3,l-benzoxazin-2-one)(EFV)
were bought from Apoteksbolaget, Uppsala, Sweden. 20 Plasma samples from HIV infected individuals.
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 25 standard HIV 1 RNA PCR (Cobas, Roche Diagnostica) and the viral genotype present was
analyzed with TRUGENE™ HIV-1 Genotyping kit. (Visible Genetics).
Recombinant RT enzymes:
NNRTI resistant mutant forms of RT were produced (L100I, K103N,
L100I/IC103N, Y181C). As template for the mutations was used the pETRT expression 30 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 of RT were isolated as
previously described [8].

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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, 5 QuikChange (Stratagene). The mutated cloned expression vectors were transformed into E. coli strain XL 1-Blue and the genotypes were verified by DNA sequence analysis. Buffers used:
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-
10 Hydroxyethyipiperazine-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 %
15 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 uM BrdUTP, 80 ng/ml odT22, 4mM
20 MgCl2,0.5 g/1 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/1, spermine 2mM, Triton-X
100 0.5 %(v/v), EGTA 0.2 mM and BSA 0.5 mg/ml.
25
Examples Example 1.
Correlation between HIV-1 RT recovered and viral RNA measured with RNA PCR.
1 ml samples of EDTA plasma from HIV infected individuals were processed 30 according to "Protocol for isolation of viral RTfrom material which contains RT blocking antibodies, based on destruction of soluble cellular enzymes followed by isolation of viral RT from mini columns." and the amount of RT activity recovered from each sample was

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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 HTV 1 RNA PCR (Roche Amplicor). The plot is restricted to samples with PCR values > 500 copies / ml. 5 A strong correlation was found between amount of plasma RT recovered and amount of HTV 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 HTV 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 10 tests, which can be used for determination of the drug sensitivity profile of the isolated RT.
Example 2.
Influence of the amount of RT used in the drug susceptibility tests.
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 15 activity in the lysates " using indicated RT concentrations. Fig 2 exemplifies the relation
between the amount of RT 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 20 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 25 amounts are added to the test makes drug sensitivity determination feasible without previous
standardization of the amount of RT used in each assay.
Example 3.
Comparison of the effects of non-nucleoside inhibitors on recombinant HIV 1 RT with
defined mutations.
30 The effects of three non-nucleoside inhibitors on indicated recombinant HTV 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

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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 1).
5 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). Example 4. Comparison of the effect of dT anlogues on recombinant HIV 1 RT with defined
10 mutations.
The effects of AZT-TP and d4T on indicated recombinant HIV I 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
15 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
20 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
25 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). Example 5.
30 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

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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 of Nevirapine, Efavirenz, and Delavirdine according to Protocol for determination of drug susceptibility of 5 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
10 sensitive to all three NNRTIs. Six of which had no relevant mutation in their RT genes while the remaining two had either VI791 or V179T/A/I mutations, which not is known to affect the sensitivity to NNRTIs. Example 6. Determination of the susceptibility to AZT-TP and d4T-TP using plasma derived RTs.
15 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 RTfrom 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.
20 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
25 drugs increased in parallel with accumulation of mutations known to be involved in resistance to NRTIs (Table 4). The mutations D69N or Ml 84V 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
30 or Ml84V 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 [8] and the other contained the classic K70R

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References
1. Petropoulos CJ, Parkin NT, Limoli KL, Lie YS, Wrin T, Huang W, Tian H, Smith D,
Winslow GA, Capon DJ, Whitcomb JM. A novel phenotypic drug susceptibility assay for
5 human immunodeficiency virus type 1. Antimicrob Agents Chemother. 2000 Apr;
44(4):920-8.
2. The Euroguidelines Group for HIV resistance. Clinical and laboratory guidelines for the
use of HIV-1 drug resistance testing as part of treatment management: recommendations
10 for the European setting. AIDS. 2001 Feb 16;15(3):309-20.
3. Vazquez-Rosales G, Garcia Lerma JG, Yamamoto S, Switzer WM, Havlir D, Folks TM,
Richman DD, Heneine W. Rapid screening of phenotypic resistance to nevirapine by
direct analysis of HIV type 1 reverse transcriptase activity in plasma. AIDS Res Hum
15 Retroviruses. 1999 Sep 1;15(13):1191-200.
4. Goff, S.P. (1990) Retrovirus reverse transcriptase: Synthesis, Structure, and Function. Review. J Acquir Imm Defic Syndr 3: 817-831,
20 5. Vandamme AM, Van Vaerenbergh K, De Clercq E. Anti-human immunodeficiency virus drug combination strategies. Antivir Chem Chemother. 1998 May; 9(3): 187-203.
6. Meyer PR, Matsuura SE, Mian AM, So AG, Scott WA. Related Articles. A mechanism
of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant
25 HIV-1 reverse transcriptase. Mol Cell. 1999 Jul;4(l):35-43
7. Ekstrand DH, Awad RJ, Kallander CF, Gronowitz JS. A sensitive assay for the
quantification of reverse transcriptase activity based on the use of carrier-bound template
and non-radioactive-product detection, with special reference to human-
30 immunodeficiency-virus isolation. Biotechnol Appl Biochem. 1996 Apr;23 (Pt 2):95-
105.

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8. Lindberg J, Sigurethsson S, Lowgren S, O Andersson H, Sahlberg C, Noreen R, Fridborg
K, Zhang H, Unge T. Structural basis for the inhibitory efficacy of efavirenz (DMP-266),
MSC194 and PNU142721 towards the HTV-1 RT K103N mutant. Eur J Biochem. 2002
5 Mar;269(6):1670-1677
9. Shirasaka, T., Kavlick, M. F., Ueno, T., Gao, W.-Y., Kojima, E., Alcaide, M. L.,
Chokekijchai, S., Roy, B. M., Arnold, E., Yarchoan, R., and Mitsuya, H. Emergence of
human immunodeficiency virus type 1 variants with resistance to multiple
10 dideoxynucleosides in patients receiving therapy with dideoxynucleosides. Proc Natl
Acad Sci USA 1995, 92: 2398-2402.
10. Larder BA. 3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring
human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase
15 inhibitors. Antimicrob Agents Chemother. 1992 Dec;36(12):2664-9.
11. Larder BA. Interactions between drug resistance mutations in human immunodeficiency
virus type 1 reverse transcriptase. J Gen Virol. 1994 May;75 (Pt 5):951-7.
20 12. Boucher CA, Cammack N, Schipper P, Schuurman R, Rouse P, Wainberg MA, Cameron JM. High-level resistance to (-) enantiomeric 2'-deoxy-3'-thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother. 1993 Oct;37(10):2231-4

Table 1. Comparison of the effect of non-nucleoside inhibitors on recombinant HIV 1 RT with defined mutations.

*Phenotype data: S = sensitive, L= Low level resistance (2-10 times increase in inhibitory dose), I = Intermediate resistance (10-100 times increase), H = High level resistance (>100 times increase), "nd not done.

*Phenotype data: RS= the mutations gives re-sensitation of resistant virus. S = sensitive, L= Low level resistance, I = Intermediate resistance, H = High level resistance. and not done..

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Table 3. Effect of NNRTIs on HIV RT recovered from patient plasma

^
*Hetrogenicity in the sequence V179T/A/I.

WE CLAIM:
1. A commercial package for testing phenotypic anti-viral drug resistance by
testing on a reverse transcriptase (RT) enzyme activity packed into an
enveloped retrovirus in a biological sample, wherein the commercial package
comprises
a RT enzyme assay for determination of the retroviral RT enzyme activity,
recombinant reverse transcriptase (RT) controls,
at least one anti-retroviral reference drug.
optionally an enzyme inactivating agent for inactivating polymerase activity,
other than that present in the enveloped virion,
optionally a separation gel and
and optionally a lysis buffer.
2. A commercial package as claimed in claim 1, wherein the anti-retroviral reference drug is a human immunodeficiency virus (HIV) drug.
3. A commercial package as claimed in claim 2, wherein the HIV-drug is a nucleoside RT-inhibitor and/or a non-nucleoside RT-inhibitior.
4. A commercial package as claimed in claim 3, wherein the nucleoside RT-inhibitor and/or a non-nucleoside RT-inhibitior is/are selected from the group consisting of 3'-azido-2', 3'-deoxythymidine triphosphate (AZT-TP). (2', 3'-didehydro-3'deoxythymidine triphosphate (d4T-TP), Nevirapine (11-cyclopropyl-
5. ll-dihydro-4-methyl-6H-dipyrido [3,2-b:2, 3'-f] [1, 4] diazepin-6one)(NVP), Delaviridine,l- (5-methanesulphonamido-lH-indol-2-yl-carbonyl)-4- [3- (1-methylethyl-amino) pyridinyl] piperazin monomethane sulphonate (DLV) and Efavirenz, (-) chloro-4-cyclopropylethynyl-4-trifluoromethyl-l, 4-dihydro-2H-3,l-benzoxazin-2-one) (EFV).

5. A commercial package as claimed in claims 1 to 4, wherein the recombinant
reverse transcriptase (RT) controls are selected from the group consisting of
wild type human immunodeficiency virus (HIV) reverse transcriptase and HIV
reverse transcriptase comprising site-directed mutations.
6. A method of testing phenotypic drug susceptibility in an enveloped
retrovirus-infected mammalian individual by testing on an enzyme packed into
an enveloped retrovirus recovered from a biological sample from said individual,
comprising the steps of:
a) adding an enzyme inactivating agent to the sample for inactivating polymerase activity other than that present in the enveloped virion,
b) removing the enzyme inactivating agent, enzyme activity blocking antibodies, endogenous enzyme activity inhibitors and antiviral drugs,

c) lysing the virus particle to release the enzyme,
d) recovering the concentrated purified viral enzyme resulting from c) and determining the drug sensitivity profile of the individual from the recovered enzyme by using sensitive enzyme assays.

7. The method as claimed in claim 6, wherein the mammalian individual is a human being.
8. The method as claimed in claim 6 or 7, wherein the biological sample is a blood sample.
9. The method as claimed in claim 8, wherein the blood sample is a plasma sample.

VIRAL DRUG SUSCEPTIBILITY TESTING

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