WO2002022076A2 - Moyens et methodes de suivi d'une therapie antiretrovirale a base d'inhibiteurs de proteases et decisions d'orientation therapeutique dans le traitement du vih et du sida - Google Patents

Moyens et methodes de suivi d'une therapie antiretrovirale a base d'inhibiteurs de proteases et decisions d'orientation therapeutique dans le traitement du vih et du sida Download PDF

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WO2002022076A2
WO2002022076A2 PCT/US2001/028754 US0128754W WO0222076A2 WO 2002022076 A2 WO2002022076 A2 WO 2002022076A2 US 0128754 W US0128754 W US 0128754W WO 0222076 A2 WO0222076 A2 WO 0222076A2
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hiv
mutation
codon
patient
protease
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WO2002022076A3 (fr
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Neil T. Parkin
Rainer A. Ziermann
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Virologic, Inc.
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Priority to EP01970982A priority patent/EP1322779A4/fr
Priority to CA002422815A priority patent/CA2422815A1/fr
Publication of WO2002022076A2 publication Critical patent/WO2002022076A2/fr
Publication of WO2002022076A3 publication Critical patent/WO2002022076A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • This invention relates to antiretroviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) .
  • the invention further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy using phenotypic or genotypic susceptibility assays.
  • the invention also relates to novel vectors, host cells and compositions for carrying out phenotypic susceptibility tests.
  • the invention further relates to the use of various genotypic methodologies to identify patients who do not respond to a particular antiretroviral drug regimen.
  • This invention also relates to the screening of candidate antiretroviral drugs for their capacity to inhibit viral replication, selected viral sequences and/or viral proteins. More particularly, this invention relates to the determination of protease inhibitor (PRI) susceptibility using phenotypic or genotypic susceptibility tests. This invention also relates to a means and method for accurately and reproducibly measuring viral replication fitness .
  • PRI protease inhibitor
  • HIV infection is characterized by high rates of viral turnover throughout the disease process, eventually leading to CD4 depletion and disease progression.
  • the aim of antiretroviral therapy is to achieve substantial and prolonged suppression of viral replication. Achieving sustained viral control is likely to involve the use of sequential therapies, generally each therapy comprising combinations of three or more antiretroviral drugs. Choice of initial and subsequent therapy should, therefore, be made on a rational basis, with knowledge of resistance and cross-resistance patterns being vital to guiding those decisions.
  • the primary rationale of combination therapy relates to synergistic or additive activity to achieve greater inhibition of viral replication.
  • the tolerability of drug regimens will remain critical, however, as therapy will need to be maintained over many years .
  • the target enzyme For antiretroviral drug resistance to occur, the target enzyme must be modified while preserving its function in the presence of the inhibitor. Point mutations leading to an amino acid substitution may result in changes in shape, size or charge of the active site, substrate binding site or in positions surrounding the active site of the enzyme. Mutants resistant to antiretroviral agents have been detected at low levels before the initiation of therapy. (Mohri H, Singh MK, Ching WTW, et al . (1993) Proc Natl
  • protease enzyme of HIV was crystallized and its three-dimensional structure was determined, (Navia MA, Fitzgerald PMD, McKeever BM, Leu CT, Heimbach JC, Herber WK, Sigal IS, Darke PL, Springer JP (1989) Nature 337:615-620 and Winters MA, Schapiro JM, Lawrence J, Merigan TC (1997) In Abstracts of the International Workshop on HIV Drug Resistance, Treatment Strategies and Eradication, St. Russia, Fla.) allowing for the rapid development of protease inhibitors. Initially, it was hypothesized that HIV protease, unlike reverse transcriptase, would be unable to accommodate mutations leading to drug resistance.
  • HIV protease was classified as an aspartic proteinase on the basis of putative active-site homology (Toh H, Ono M, Saigo K, Miyata T (1985) Nature 315:691), its inhibition by peptastin (Richards AD, Roberts R, Dunn BM, Graves MC, Kay J (1989) FEBS Lett 247:113), and its crystal structure (Navia MA, Fitzgerald PMD, McKeever BM, Lau CT, Heimbach JC, Herber WK, Sigal IS, Darke PL, Springer JP (1989) Nature 337:615-620).
  • the enzyme functions as a homodimer composed of two identical 99-amino acid chains (Debouck C, Navia MA, Fitzgerald PMD, McKeever BM, Leu CT, Heimbach JC, Herber WK, Sigal IS, Darke PL, Springer JP (1988) Proc. Natl. Acad. Sci. USA 84:8903-8906), with each chain containing the characteristic Asp-Thr-Gly active-site sequence at positions 25 to 27 (Toh H, Ono M, Saigo K, Miyata T (1985) Nature 315:691).
  • HIV protease processes gag (p55) and gag-pol (pl60) polyprotein products into functional core proteins and viral enzymes
  • gag (p55) and gag-pol (pl60) polyprotein products into functional core proteins and viral enzymes
  • gag NE Diehl RE, Rands E, Davis LJ, Hanobik MG, Wolanski B, Dixon RA (1991) J. Virol. 65:3007-3014 and Kramer RA, Schaber MD, Skalka AM, Ganguly K, Wong-Staal F, Reddy EP (1986) Science 231:1580-1584).
  • the polyproteins are cleaved by the enzyme at nine different cleavage sites to yield the structural proteins (pl7, p24, p7 , and p6) as well as the viral enzymes reverse transcriptase, integrase, and protease (Pettit SC, Michael SF, Swanstrom R (1993) Drug Discov. Des. 1:69-83).
  • the viral protease is a symmetric dimer, it binds its natural substrates or inhibitors asymmetrically (Dreyer, GB, Boehm JC, Chenera B, DesJarlais RL, Hassell AM, Meek TD, Tomaszek TAJ, Lewis M (1993) Biochemistry 32:937-947, Miller MJ, Schneider J, Sathyanarayana BK, Toth MV, Marshall GR, Clawson L, Selk L, Kent SB, Wlodawer A (1989) Science 246:1149-1152).
  • Secondary mutations are usually considered as being compensatory for defects in enzyme activity imposed by primary mutations, or as having enhancing effects on the magnitude of resistance imparted by the primary mutations (e.g. L10I/F/R/V, K20I/M/R/T, L24I, V32I, L33F/V, M36I/L/V, M46I/L/V, I47V, I54L/V, L63X, A71T/V, G73A/S/T, V77I, N88D) .
  • Lists of mutations and corresponding inhibitors are maintained by several organizations, for example: Schinazi et al . , Mutations in retroviral genes associated with drug resistance, Intl . Antiviral News
  • Saquinavir developed by Hoffmann-La Roche, was the first protease inhibitor to undergo clinical evaluation, demonstrating that HIV-1 protease was a valid target for the treatment of HIV infection (Jacobsen H, Brun-Vezinet F, Duncan I, Hanggi M, Ott M, Vella S, Weber J, Mous J
  • Saquinavir is a highly active peptidomimetic protease inhibitor with a 90% inhibitory concentration (IC90) of 6 nM ( id) .
  • IC90 inhibitory concentration
  • saquinavir can select for variants with one or both of two amino acid substitutions in the HIV-1 protease gene, a valine-for-glycine substitution at position 48 (G48V) , a methionine-for-leucine substitution at residue 90 (L90M) , and the double substitution G48V-L90M (Eberle J, Bechowsky B, Rose D, Hauser U, vonder Helm K, Guertler L, Nitschko H
  • Ritonavir developed by Abbott Laboratories, was the second HIV protease inhibitor to be licensed in the United States.
  • Ritonavir is a potent and selective inhibitor of HIV protease that is derived from a C2-symmetric, peptidomimetic inhibitor (Ho DD, Toyoshima T, Mo H, Kempf DJ, Norbeck D, Chen CM, Wideburg NE, Burt SK, Erickson JW, Singh MK (1994) J. Virol. 68:2016-2020).
  • V82F mutation confers an even greater level of resistance, up to 20-fold.
  • substitutions M46I, L63P, and A71V, when introduced into the protease coding region of wild-type NL4-3, did not result in significant changes in drug susceptibility. Based on replication kinetics experiments, these changes are likely to be compensatory for active-site mutations, restoring the impaired replicative capacity of the combined V82F and I84V mutations .
  • Indinavir developed by Merck & Co., is the third HIV protease inhibitor licensed in the United States. Indinavir is a potent and selective inhibitor of HIV-1 and HIV-2 proteases with Ki values of 0.34 and 3.3 nM, respectively (Vacca Jp, Dorsey BD, Schleif WA, Levin RB, McDaniel SL, Darke PL, Weray J, Quintero JC, Blahy OM, Roth E, Sardana VV, Schlabach AJ, Graham PI, Condra JH, Gotlib L, Holloway MK, Lin J, Chen L-w, Vastag K, Ostobich D, Anderson PS, Emini EA, Huff JR (1994) Proc. Natl. Acad.
  • the drug acts as peptidomimetic transition state analogue and belongs to the class of protease inhibitors known as HAPA (hydroxyaminopentane amide) compounds (ibid) .
  • HAPA hydroxyaminopentane amide
  • Indinavir provides enhanced aqueous solubility and oral bioavailability and in cell culture exhibits an IC95 of 50 to 100 nM (Emini EA, Schleif WA, Deutsch P, Condra JH (1996) Antiviral Chemother. 4:327-331.
  • Nelfinavir developed by Agouron Pharmaceuticals, is a selective, nonpeptidic HIV-1 protease inhibitor that was designed by protein structure-based techniques using iterative protein crystallographic analysis (Appelt KR, Bacquet J, Bartlett C, Booth CLJ, Freer ST, Fuhry MM, Gehring MR, Herrmann SM, Howland EF, Janson CA, Jones TR, Kan CC, Kathardekar V, Lewis KK, Marzoni GP, Mathews DA, Mohr C, Moomaw EW, Morse CA, Oatley SJ, Ogden RC, Reddy MR, Reich SH, Schoettlin WS, Smith WW, Varney MD, Villafranca JE, Ward RW, Webber S, Webber SE, Welsh KM, White J (1991) J.
  • nelfinavir was found to be a potent inhibitor of HIV-1 protease with a Ki of 2.0 nM (Kaldor SW, Kalish VJ, Davies JF, Shetty BV, Fritz JE, Appelt K, Burgess JA, Campanale KM, Chirgadze NY, Clawson DK, Dressman BA, Hatch SD, Khalil DA, Kosa MB, Lubbehusen PP, Muesing MA, Patrick AK, Reich SH, Su KS, Tatlock JH (1997) J. Med. Chem. 40:3979-3985).
  • Nelfinavir exhibits additive-to-synergistic effects when combined with other antiretroviral drugs (Partaledis JA, Yamaguchi AK, Tisdale M, Blair EE, Falcione C, Maschera B, Myers RE, Pazhanisamy S, Futer O, Bullinan AB, Stuver CM, Byrn RA, Livingston DJ (1995) J. Virol. 69:5228-5235).
  • Amprenavir is a novel protease inhibitor developed by Vertex Laboratories and designed from knowledge of the HIV-1 protease crystal structure (Kim EE, Baker CT, Dyer MD, Murcko MA, Rao BG, Tung RD, Navia MA (1995) J. Am. Chem. Soc. 117:1181-1182).
  • the drug belongs to the class of sulfonamide protease inhibitors and has been shown to be a potent inhibitor of HIV-1 and HIV-2, with IC50s of 80 and 340 nM, respectively.
  • the mean IC50 for amprenavir against clinical viral isolates was 12 nM (St.
  • HIV-1 variants 100-fold resistant to amprenavir have been selected by in vitro passage experiments ( id) .
  • DNA sequence analysis of the protease of these variants revealed a sequential accumulation of point mutations resulting in amino acid substitutions L10F, M46I, I47V, and I50V.
  • the key resistance mutation in the HIV-1 protease substrate binding site is I50V. As a single mutation it confers a two- to threefold decrease in susceptibility (ibid) .
  • HXB2 HIV-1 infectious clone
  • a triple protease mutant clone containing the mutations M46I, I47V, and I50V was 20-fold less susceptible to amprenavir than wild-type virus.
  • the I50V mutation has not been frequently reported in resistance studies with other HIV protease inhibitors.
  • the catalytic efficiency (k cat /K m ) of the I50V mutant was decreased up to 25-fold, while the triple mutant M46I/I47V/I50V had a 2-fold-higher processing efficiency than the I50V single mutant, confirming the compensatory role of the M46I-and-I47V mutation.
  • the reduced catalytic efficiency (k cat /K for these mutants in processing peptides appeared to be due to both increased K m and decreased k cat values.
  • the relative ability of a given virus or virus mutant to replicate is termed viral fitness. Fitness is dependent on both viral and host factors, including the genetic composition of the virus, the host immune response, and selective pressures such as the presence of anti-viral compounds. Many drug-resistant variants of HIV-1 are less fit than the wild-type, i.e. they grow more slowly in the absence of drug selection. However, since the replication of the wild-type virus is inhibited in the presence of drug, the resistant mutant can outgrow it. The reduction in fitness may be a result of several factors including: decreased ability of the mutated enzyme (i.e. PR or RT) to recognize, its natural substrates, decreased stability of the mutant protein, or decreased kinetics of enzymatic catalysis. See Back et al .
  • Drug resistant viruses that are less fit than wild type may be less virulent i.e. they may cause damage to the host immune system more slowly than a wild type virus .
  • Immunological decline may be delayed after the emergence of drug resistant mutants, compared to the rate of immunological decline in an untreated patient.
  • the defect causing reductions in fitness may be partially or completely compensated for by the selection of viruses with additional amino acid substitutions in the same protein that bears the drug resistance mutations (for example, see Martinez-Picado et al . , J. Virol. 73:3744-3752, 1999), or in other proteins which interact with the mutated enzyme.
  • amino acids surrounding the protease cleavage site in the gag protein may be altered so that the site is better recognized by a drug-resistant protease enzyme (Doyon et al . , J. Virol. 70: 3763-3769, 1996; Zhang et al . , J. Virol. 71: 6662-6670, 1997; Mammano et al., J, Virol. 72: 7632-7637, 1998).
  • Integrase is an attractive target for antivirals because it is essential for HIV replication and, unlike protease and reverse transcriptase, there are no known counterparts in the host cell. Furthermore, integrase uses a single active site to accommodate two different configurations of DNA substrates, which may constrain the ability of HIV to develop drug resistance to integrase inhibitors.
  • HIV-1 integrase is a 32-kDa enzyme that carries out DNA integration in a two-step reaction (Brown, P.O., ibid. ) .
  • 3' processing two nucleotides are removed from each 3' end of the viral DNA made by reverse transcription.
  • DNA strand transfer a pair of transesterification reactions integrates the ends of the viral DNA into the host genome.
  • Integrase is comprised of three structurally and functionally distinct domains, and all three domains are required for each step of the integration reaction (Engel an, A. Bushman, F.D. & Craigie, R., 1993, EMBO J. 12, 3269-3275) .
  • Hazuda et al . (Science 287: 646-650, 2000) have described compounds (termed L-731, 988 and L-708,906) which specifically inhibit the strand-transfer activity of HIV-1 integrase and HIV-1 replication in vitro.
  • Viruses grown in the presence of these inhibitors display reduced inhibitor susceptibility and bear mutations in the integrase coding region at amino acid positions 66 (T66I), 153 (S153Y) , and 154 (M154I) .
  • Site-directed mutants of a laboratory strain of HIV-1 (HXB2) with these amino acid changes confirmed their direct role in conferring reduced integrase inhibitor susceptibility. In addition some of these mutants displayed delayed growth kinetics, suggesting that viral fitness was impaired.
  • Still another object of this invention is to provide a test and methods for evaluating the biological effectiveness of candidate drug compounds which act on specific viruses, viral genes and/or viral proteins particularly with respect to alterations in viral drug susceptibility associated with protease inhibitors. It is also an object of this invention to provide the means and compositions for evaluating HIV antiretroviral drug resistance and susceptibility.
  • viruses including, but not limited to human immunodeficiency virus (HIV) , hepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus).
  • the present invention relates to methods of monitoring, via phenotypic and genotypic methods the clinical progression of human immunodeficiency virus infection and its response to antiviral therapy.
  • the invention is also based, in part, on the discovery that genetic changes in HIV protease (PR) which confer changes in susceptibility to antiretroviral therapy may be rapidly determined directly from patient plasma HIV RNA using phenotypic or genotypic methods.
  • PR HIV protease
  • the methods utilize nucleic acid amplification based assays, such as polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • This invention is based in part on the discovery of mutations at codons 10, 20, 36, 46, 63, 77 and 88 of HIV protease in PRI treated patients in which the presence of certain combinations of these mutations correlate with changes in certain PRI susceptibilities.
  • This invention is also based on the discovery that susceptibility to HIV protease antivirals may not be altered even if primary mutations are present. Additional mutations at secondary positions in HIV protease are required for a reduction in virus susceptibility.
  • This invention established for the first time that a mutation at position 82 of protease (V82A, F, S, or T) in the absence of another primary mutation was not correlated with a reduction in drug susceptibility.
  • Decreased susceptibility to protease inhibitors such as indinavir and saquinavir, in viruses containing V82A, F, S or T was observed in viruses with additional mutations at secondary positions, such as, 24, 71, 54, 46, 10 and/or 63 as described herein.
  • Decreased susceptibility to protease inhibitors such as indinavir and saquinavir, in viruses containing V82A, F, S or T was also observed in viruses with at least 3 or more additional mutations at secondary positions.
  • This inventions also established for the first time that a mutation at position 90 of protease (L90M) in the absence of another primary mutation was not correlated with a reduction in drug susceptibility.
  • Decreased susceptibility to protease inhibitors, such as indinavir and saquinavir, in viruses containing L90M was observed in viruses with additional mutations at secondary positions, such as, 73, 71, 77, and/or 10 as described herein.
  • Decreased susceptibility to protease inhibitors, such as indinavir and saquinavir, in viruses containing L90M was also observed in viruses with at least 3 or more additional mutations at secondary positions.
  • the mutations were found in plasma HIV nucleic acid after a period of time following the initiation of therapy. The development of these mutations, or combinations of these mutations, in HIV PR was found to be an indicator of the development of alterations in phenotypic susceptibility/resistance, which can be associated with virologic failure and subsequent immunological response.
  • a method of assessing the effectiveness of protease antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a plasma sample from the HIV-infected patient; (b) evaluating whether the plasma sample contains nucleic acid encoding HIV protease having a mutation at primary and secondary positions; and (c) determining changes in susceptibility to a protease inhibitor.
  • PCR based assays may be used to detect a substitution at codon 88 from asparagine to a serine residue either alone or in combination with one or more mutations at other codons selected from the group consisting of 10, 20, 36, 46, 63 and/or 77 or a combination thereof of HIV PR.
  • PCR based assays including phenotypic and genotypic assays, may be used to detect mutations at codons 10, 20, 36, 46, 63, 77, and 88 of HIV PR which correlate with changes in susceptibility to antiretroviral therapy and immunologic response. Once mutations at these loci have been detected in a patient undergoing PRI antiretroviral therapy, an alteration in the therapeutic regimen should be considered. The timing at which a modification of the therapeutic regimen should be made, following the assessment of antiretroviral therapy using PCR based assays, may depend on several factors including the patient's viral load, CD4 count, and prior treatment history.
  • PCR based assays may be used to detect a substitution at codon 82 from valine to an alanine (V82A) , phenylalanine (V82F) , serine (V82S) , or threonine (V82T) residue either alone or in combination with one or more mutations at other codons, referred to herein as secondary mutations, selected from the group consisting of 20, 24, 36, 71, 54, 46, 63 and/or 10 or a combination thereof of HIV PR.
  • V82A alanine
  • V82F phenylalanine
  • V82S serine
  • V82T threonine
  • a mutation at codon 82 from a valine residue to a alanine, phenylalanine, serine or threonine in combination with secondary mutations at codons 24 and/or 71 or 20 and/or 36 correlates with a reduction in susceptibility to indinavir and saquinavir, respectively.
  • a mutation at codon 82 from a valine residue to a alanine, phenylalanine, serine or threonine in combination with at least 3 secondary mutations correlates with a reduction in susceptibility to indinavir and saquinavir.
  • PCR based assays may be used to detect a substitution at codon 90 from leucine to a methionine (L90M) residue either alone or in combination with one or more mutations at other codons, referred to herein as secondary mutations, selected from the group consisting of 73, 71, 46 and/or 10 or a combination thereof of HIV PR.
  • L90M methionine
  • secondary mutations selected from the group consisting of 73, 71, 46 and/or 10 or a combination thereof of HIV PR.
  • a mutation at codon 90 from a leucine residue to a methionine alone correlates with susceptibility to certain protease inhibitors including indinavir and saquinavir.
  • a mutation at codon 90 from a leucine residue to a methionine in combination with secondary mutations at codons 73 and/or 71 or 73, 71 and/or 77 correlates with a reduction in susceptibility to indinavir and saquinavir, respectively.
  • a mutation at codon 90 from a leucine residue to a methionine in combination with at least 3 secondary mutations correlates with a reduction in susceptibility to indinavir and saquinavir.
  • a method for assessing the effectiveness of a protease inhibitor antiretroviral drug comprising: (a) introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; (b) culturing the host cell from step (a) ; (c) measuring expression of the indicator gene in a target host cell wherein expression of the indicator gene is dependent upon the patient derived segment; and (d) comparing the expression of the indicator gene from step (c) with the expression of the indicator gene measured when steps (a) - (c) are carried out in the absence of the PRI anti-HIV drug, wherein a test concentration of the PRI, anti-HIV drug is presented at steps (a) - (c) ; at steps (b) - (c); or at step (c) .
  • This invention also provides a method for assessing the effectiveness of protease inhibitor antiretroviral therapy in a patient comprising: (a) developing a standard curve of drug susceptibility for an PRI anti-HIV drug; (b) determining PRI anti-HIV drug susceptibility in the patient using the susceptibility test described above; and (c) comparing the PRI anti-HIV drug susceptibility in step (b) with the standard curve determined in step (a) , wherein a decrease in PRI anti-HIV susceptibility indicates development of anti-HIV drug resistance in the patient's virus and an increase in PRI anti-HIV susceptibility indicates drug hypersensitivity in the patient ' s virus .
  • This invention also provides a method for evaluating the biological effectiveness of a candidate PRI HIV antiretroviral drug compound comprising: (a) introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; (b) culturing the host cell from step (a) ; (c) measuring expression of the indicator gene in a target host cell wherein expression of the indicator gene is dependent upon the patient derived segment; and (d) comparing the expression of the indicator gene from step (c) with the expression of the indicator gene measured when steps (a) - (c) are carried out in the absence of the candidate PRI anti-viral drug compound, wherein a test concentration of the candidate PRI anti-viral drug compound is present at steps (a) - (c) ; at steps (b) - (c) ; or at step (c) .
  • the expression of the indicator gene in the resistance test vector in the target cell is ultimately dependent upon the action of the HIV enzymes (PR and RT) encoded by the patient-derived segment DNA sequences.
  • the indicator gene may be functional or non-functional.
  • this invention is directed to antiretroviral drug susceptibility and resistance tests ' for HIV/AIDS .
  • Particular resistance test vectors of the invention for use in the HIV/AIDS antiretroviral drug susceptibility and resistance test are identified.
  • Yet another aspect of this invention provides for the identification and assessment of the biological effectiveness of potential therapeutic antiretroviral compounds for the treatment of HIV and/or AIDS.
  • the invention is directed to a novel resistance test vector comprising a patient-derived segment further comprising one or more mutations on the PR gene and an indicator gene.
  • Still another aspect of this invention provides for the identification and assessment of the fitness of a virus infecting a patient.
  • the invention is directed to a novel resistance test vector comprising a patient-derived segment further comprising one or more mutations on the PR gene and an indicator gene, enabling the measurement of viral fitness.
  • Resistance Test Vector A diagrammatic representation of the resistance test vector comprising a patient derived segment and an indicator gene.
  • a resistance test vector is generated by cloning the patient-derived segment into an indicator gene viral vector. The resistance test vector is then co-transfected with an expression vector that produces amphotropic murine leukemia virus (MLV) envelope protein or other viral or cellular proteins which enable infection.
  • MMV amphotropic murine leukemia virus
  • Pseudotyped viral particles are produced containing the protease (PR) and the reverse transcriptase (RT) gene products encoded by the patient-derived DNA sequences. The particles are then harvested and used to infect fresh cells. Using defective PR and RT sequences it was shown that luciferase activity is dependent on functional PR and RT . PR inhibitors are added to the cells following transfection and are thus present during particle maturation.
  • RT inhibitors are added to the cells at the time of or prior to viral particle infection.
  • the assay is performed in the absence of drug and in the presence of drug over a wide range of concentrations. Luciferase activity is determined and the percentage (%) inhibition is calculated at the different drug concentrations tested. Fig. 3
  • phenotypic drug susceptibility profiles Data are analyzed by plotting the percent inhibition of luciferase activity vs. loglO concentration. This plot is used to calculate the drug concentration that is required to inhibit virus replication by 50% (IC50) or by 95% (IC95) . Shifts in the inhibition curves towards higher drug concentrations are interpreted as evidence of drug resistance. Three typical curves for a nucleoside reverse transcriptase inhibitor (AZT) , a non-nucleoside reverse transcriptase inhibitor (efavirenz) , and a protease inhibitor (indinavir) are shown.
  • AZA nucleoside reverse transcriptase inhibitor
  • efavirenz non-nucleoside reverse transcriptase inhibitor
  • indinavir protease inhibitor
  • a reduction in drug susceptibility is reflected in a shift in the drug susceptibility curve toward higher drug concentrations (to the right) as compared to a baseline (pre-treatment) sample or a drug susceptible virus reference control, such as pNL4-3 or HXB-2, when a baseline sample is not available.
  • Phenotypic PRI susceptibility profile patient 0732.
  • a PCR-based phenotypic susceptibility assay was carried out giving the phenotypic drug susceptibility profile showing decreased susceptibility to nelfinavir and indinavir, and increased susceptibility to amprenavir.
  • Phenotypic PRI susceptibility profile of a protease mutant generated by site-specific oligonucleotide-directed mutagenesis was carried out giving the phenotypic drug susceptibility profile of a virus having substitutions at codons 63, 77 and 88 (L63P, V77I and N88S) .
  • the profile demonstrates resistance to both nelfinavir and indinavir, and increased susceptibility to amprenavir.
  • a fitness test vector is generated by cloning the patient-derived segment into an indicator gene viral vector. The fitness test vector is then co- transfected with an expression vector that produces amphotropic murine leukemia virus (MLV) envelope protein or other viral or cellular proteins which enable infection.
  • MMV amphotropic murine leukemia virus
  • Pseudotyped viral particles are produced containing the protease (PR) and the reverse transcriptase (RT) gene products encoded by the patient-derived DNA sequences . The particles are then harvested and used to infect fresh cells. Using defective PR and RT sequences it was shown that luciferase activity is dependent on functional PR and RT.
  • the fitness assay is typically performed in the absence of drug.
  • the assay can also be performed at defined drug concentrations. Luciferase activity produced by patient derived viruses is compared to the luciferase activity produced by well- characterized reference viruses. Replication fitness is expressed as a percent of the reference. Figure B .
  • Virus stocks produced from fitness test vectors derived from patient samples were used to infect cells. Luciferase activity was measured at various times after infection. Patient derived viruses may produce more, approximately the same, or less luciferase activity than the reference virus (Ref) and are said to have greater, equivalent, or reduced replication fitness, respectively.
  • the drug susceptibility profiles of three representative patient derived viruses are shown (Pi, P2, P3) .
  • Figure C Identifying alterations in protease or reverse transcriptase function associated with differences in replication fitness of patient viruses .
  • Replication fitness is expressed as a percent of the reference virus (top) .
  • Fitness measurements are compared to protease processing of the p55 gag polyprotein (middle) and reverse transcriptase activity (bottom) .
  • Protease processing is measured by Western blot analysis using an antibody that reacts with the mature capsid protein (p24) .
  • the detection of unprocessed p55 or incompletely processed p41 polyproteins are indicators of reduced cleavage.
  • Reverse transcriptase activity is measured using a quantitative RT-PCR assay and is expressed as a percent of the reference virus.
  • Reduced replication fitness is associated with high numbers of protease mutations, and the L90M mutation.
  • Patient viruses were sorted based on the number of protease mutations. Viruses with large numbers of protease mutations or the L90M protease mutation generally exhibit reduced replication fitness.
  • Low replication capacity is associated with specific protease mutations.
  • Patient viruses were sorted based on replication capacity. Specific protease mutations either alone (D30N) or in combination (L90M plus others) were observed with high frequency in viruses with reduced replication fitness.
  • Figure K Relationship between nelfinavir susceptibility, protease processing and replication fitness. Patient viruses were sorted based on nelfinavir susceptibility ( ⁇ 10 or >10 of reference) . Protease processing and replication fitness were plotted for all patient viruses. Viruses with reduced nelfinavir susceptibility generally exhibited reduced protease processing and reduced replication fitness .
  • Figure L Protease mutations associated with reduced protease processing. Patient viruses were sorted based on protease processing. Specific protease mutations were observed at high frequency in viruses with reduced protease processing.
  • Virus samples were collected weekly during a period of treatment interruption and evaluated for phenotypic drug susceptibility. Values shown represent fold change in susceptibility compared to the reference virus.
  • Virus samples were collected weekly during a period of treatment interruption and evaluated for phenotypic drug susceptibility. Fitness values shown represent percent of the reference virus. The increase in fitness between week 9 and week 10 corresponds to improved protease processing (bottom) and reversion of the drug resistant phenotype to a drug sensitive phenotype ( Figure M) .
  • Figure 0. Increased replication fitness during treatment interruption. Replication fitness was measured at the time of treatment interruption and various times during the period of treatment interruption. Generally, replication fitness was significantly higher in samples that corresponded to timepoints after the virus had reverted from a drug resistant phenotype to a drug sensitive phenotype.
  • the present invention relates to methods of monitoring the clinical progression of HIV infection in patients receiving antiretroviral therapy, particularly protease inhibitor antiretroviral therapy.
  • the present invention provides for a method of evaluating the effectiveness of antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at one or more positions in the PR.
  • the mutation (s) correlate positively with alterations in phenotypic susceptibility.
  • the invention provides for a method of evaluating the effectiveness of PRI antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at codon 88 from an asparagine residue to a serine residue (N88S) .
  • This invention established, using a phenotypic susceptibility assay, that a mutation at codon 88 to a serine residue of HIV protease is correlated with an increase in amprenavir susceptibility.
  • the invention provides for a method of evaluating the effectiveness of PRI antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at codon 88 from an asparagine residue to a serine residue (N88S) either alone or in combination with mutations at codons 63 and/or 77 or a combination thereof.
  • This invention established, using a phenotypic susceptibility assay, that a mutation at codon 88 to a serine residue of HIV protease is correlated with an increase in amprenavir susceptibility and a mutation at codon 88 to a serine residue in combination with mutations at codons 63 and/or 77 or a combination thereof of HIV protease are correlated with an increase in amprenavir susceptibility and a decrease in nelfinavir and indinavir susceptibility.
  • the invention provides for a method of evaluating the effectiveness of PRI antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at codon 88 from an asparagine residue to a serine residue (N88S) either alone or in combination with mutations at codons 46, 63 and/or 77 or a combination thereof.
  • This invention established, using a phenotypic susceptibility assay, that a mutation at codon 88 to a serine residue of HIV protease is correlated with an increase in amprenavir susceptibility and a mutation at codon 88 to a serine residue in combination with mutations at codons 46, 63 and/or 77 or a combination thereof of HIV protease are correlated with an increase in amprenavir susceptibility and a decrease in nelfinavir and indinavir susceptibility.
  • the invention provides for a method of evaluating the effectiveness of PRI antiretroviral therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; and (ii) determining whether the biological sample comprises nucleic acid encoding HIV PR having a mutation at codon 88 from an asparagine residue to a serine residue (N88S) either alone or in combination with mutations at codons 10, 20, 36, 46, 63 and/or 77 or a combination thereof.
  • This invention established, using a phenotypic susceptibility assay, that a mutation at codon 88 to a serine residue of HIV protease is correlated with an increase in amprenavir susceptibility and a mutation at codon 88 to a serine residue in combination with mutations at codons 10, 20, 36, 46, 63 and/or 77 or a combination thereof of HIV protease are correlated with an increase in amprenavir susceptibility and a decrease in nelfinavir and indinavir susceptibility.
  • the phenotypic susceptibility profile and genotypic profile of the HIV virus infecting the patient has been altered reflecting a change in response to the antiretroviral agent.
  • the HIV virus infecting the patient may be resistant to one or more PRIs but hypersensitive to another of the PRIs as described herein. It therefore may be desirable after detecting the mutation (s), to either increase the dosage of the antiretroviral agent, change to another antiretroviral agent, or add one or more additional antiretroviral agents to the patient's therapeutic regimen.
  • the patient's therapeutic regimen may desirably be altered by either (i) changing to a different PRI antiretroviral agent, such as saquinavir, ritonavir or amprenavir and stopping nelfinavir treatment; or (ii) increasing the dosage of nelfinavir; or (iii) adding another antiretroviral agent to the patient's therapeutic regimen.
  • a different PRI antiretroviral agent such as saquinavir, ritonavir or amprenavir and stopping nelfinavir treatment
  • increasing the dosage of nelfinavir or adding another antiretroviral agent to the patient's therapeutic regimen.
  • the effectiveness of the modification in therapy may be further evaluated by monitoring viral burden such as by HIV RNA copy number. A decrease in HIV RNA copy number correlates positively with the effectiveness of a treatment regimen.
  • codon number refers to the position of the amino acid that the codon codes for. Therefore a codon referencing a particular number is equivalent to a "postion" referencing a particular number, such as for example, codon 88" or "position 88".
  • a method of evaluating the effectiveness of PRI therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; (ii) purifying and converting the viral RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the PR gene; (iii) performing PCR using primers that result in PCR products comprising wild type or serine at codon 88; and (iv) determining, via the products of PCR, the presence or absence of a serine residue at codon 88.
  • a method of evaluating the effectiveness of PRI therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; (ii) purifying and converting the viral RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the PR gene; (iii) performing PCR using primers that result in PCR products comprising wild type or serine at codon 88 and mutations at codons 63 and/or 77; and (iv) determining, via the products of PCR, the presence or absence of a serine residue at codon 88 and the presence or absence of mutations at codons 63 and/or 77.
  • a method of evaluating the effectiveness of PRI therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; (ii) purifying and converting the viral RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the PR gene; (iii) performing PCR using primers that result in PCR products comprising wild type or serine at codon 88 and mutations at codons 63, 77 and/or 46 or a combination thereof; and (iv) determining, via the products of PCR, the presence or absence of a serine residue at codon 88 and the presence or absence of mutations at codons 63, 77 and/or 46 or a combination thereof.
  • a method of evaluating the effectiveness of PRI therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; (ii) purifying and converting the viral RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the PR gene; (iii) performing PCR using primers that result in PCR products comprising wild type or serine at codon 88 and mutations at codons 63, 77, 46, 10, 20, and/or 36 or a combination thereof; and (iv) determining, via the products of PCR, the presence or absence of a serine residue at codon 88 and the presence or absence of mutations at codons 63, 77, 46, 10, 20, and/or 36 or a combination thereof.
  • the presence of the mutation at codon 88 to a serine of alone or in combination with mutations at condons 63, 77, 46, 10, 20, and/or 36 or a combination thereof of HIV PR indicates that the effectiveness of the current or prospective PRI therapy may require alteration, since as shown by this invention a mutation at codon 88 to a serine residue alone increases the susceptibility to amprenavir and a mutation at codon 88 to a serine residue in combination with mutations at condons 63, 77, 46, 10, 20, and/or 36 or a combination increases the susceptibility to amprenavir but also reduces the susceptibility to nelfinavir and indinavir. Using the methods of this invention, changes in the PRI therapy would be indicated.
  • Another preferred, non-limiting, specific embodiment of the invention is as follows: a method of evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises nucleic acid encoding HIV protease having a mutation at codon 88 to serine. Using the phenotypic susceptibility assay, it was observed that the presence of the mutation at codon 88 to serine of HIV PR causes a an increase in amprenavir susceptibility.
  • Another preferred, non-limiting, specific embodiment of the invention is as follows: a method of evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises nucleic acid encoding HIV protease having a mutation at codon 88 to serine and additional mutation (s) at codons 63 and/or 77 or a combination thereof.
  • Another preferred, non-limiting, specific embodiment of the invention is as follows: a method of evaluating the effectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises nucleic acid encoding HIV protease having a mutation at codon 88 to serine and additional mutation (s) at codons 63, 77 and/or 46 or a combination thereof.
  • Another preferred, non-limiting, specific embodiment of the invention is as follows: a method of evaluating the e fectiveness of antiretroviral therapy of an HIV-infected patient comprising: (a) collecting a biological sample from an HIV-infected patient; and (b) determining whether the biological sample comprises nucleic acid encoding HIV protease having a mutation at codon 88 to serine and additional mutation (s) at codons 63, 77, 46, 10, 20 and/or 36 or a combination thereof.
  • This invention also provides the means and methods to use the resistance test vector comprising an HIV gene and further comprising a PR mutation for drug screening. More particularly, the invention describes the resistance test vector comprising the HIV protease having a mutation at codon 88 to a serine alone or in combination with mutations at codons 10, 20, 36, 46, 63 and/or 77 or a combination thereof for drug screening.
  • the invention further relates to novel vectors, host cells and compositions for isolation and identification of the HIV-1 protease inhibitor resistant mutant and using such vectors, host cells and compositions to carry out anti-viral drug screening. This invention also relates to the screening of candidate drugs for their capacity to inhibit said mutant.
  • This invention provides a method for identifying a compound which is capable of affecting the function of the protease of HIV-1 comprising contacting the compound with the polypeptide (s) comprising all or part of the HIV-1 protease, wherein codon 88 is changed to a serine residue, wherein a positive binding indicates that the compound is capable of affecting the function of said protease.
  • This invention also provides a method for assessing the viral fitness of patient's virus comprising: (a) determining the luciferase activity in the absence of drug for the reference control using the susceptibility test described above; (b) determining the luciferase activity in the absence of drug for the patient virus sample using the susceptibility test described above; and (c) comparing the luciferase activity determined in step (b) with the luciferase activity determined in step (a) , wherein a decrease in luciferase activity indicates a reduction in viral fitness of the patient's virus.
  • a resistance test vector is constructed using a patient derived segment from a patient virus which is unfit, and the fitness defect is due to genetic alterations in the patient derived segment, then the virus produced from cells transfected with the resistance test vector will produce luciferase more slowly. This defect will be manifested as reduced luciferase activity (in the absence of drug) compared to the drug sensitive reference control, and may be expressed as a percentage of the control.
  • PCR based assays including phenotypic and genotypic assays, may be used to detect mutations at positions 20 and 88 of HIV PR, which correlate with a reduction in viral fitness and immunological response.
  • It is a further embodiment of this invention to provide a means and method for measuring replication fitness for viruses including, but not limited to human immunodeficiency virus (HIV) , hepadnaviruses (human hepatitis B virus) , flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus) .
  • viruses including, but not limited to human immunodeficiency virus (HIV) , hepadnaviruses (human hepatitis B virus) , flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus) .
  • This invention further relates to a means and method for measuring the replication fitness of HIV-1 that exhibits reduced drug susceptibility to reverse transcriptase inhibitors and protease inhibitors.
  • a means and methods are provided for measuring replication fitness for other classes of inhibitors of HIV-1 replication, including, but not limited to integration, virus assembly, and virus attachment and entry.
  • This invention relates to a means and method for identifying mutations in protease or reverse transcriptase that alter replication fitness.
  • a means and methods are provided for identifying mutations that alter replication fitness for other components of HIV-1 replication, including, but not limited to integration, virus assembly, and virus attachment and entry.
  • This invention also relates to a means and method for quantifying the affect that specific mutations in protease or reverse transcriptase have on replication fitness.
  • a means and method are provided for quantifying the affect that specific protease and reverse transcriptase mutations have on replication fitness in other viral genes involved in HIV-1 replication, including, but not limited to the gag, pol, and envelope genes.
  • This invention also relates to the high incidence of patient samples with reduced replication fitness.
  • This invention relates to the correlation between reduced drug susceptibility and reduced replication fitness .
  • This invention further relates to the occurrence of viruses with reduced fitness in patients receiving protease inhibitor and/or reverse transcriptase inhibitor treatment .
  • This invention further relates to the incidence of patient samples with reduced replication fitness in which the reduction in fitness is due to altered protease processing of the gag polyprotein (p55) .
  • This invention further relates to the incidence of protease mutations in patient samples that exhibit low, moderate or normal (wildtype) replication fitness. This invention further relates to protease mutations that are frequently observed, either alone or in combination, in viruses that exhibit reduced replication capacity.
  • This invention also relates to the incidence of patient samples with reduced replication fitness in which the reduction in fitness is due to altered reverse transcriptase activity.
  • This invention relates to the occurrence of viruses with reduced replication fitness in patients failing antiretroviral drug treatment.
  • This invention further relates to a means and method for using replication fitness measurements to guide the treatment of HIV-1.
  • This invention further relates to a means and method for using replication fitness measurements to guide the treatment of patients failing antiretroviral drug treatment.
  • This invention further relates to the means and methods for using replication fitness measurements to guide the treatment of patients newly infected with HIV-1.
  • This invention provides the means and methods for using replication fitness measurements to guide the treatment of viral diseases, including, but not limited to HIV-1, hepadnaviruses (human hepatitis B virus) , flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus) .
  • viral diseases including, but not limited to HIV-1, hepadnaviruses (human hepatitis B virus) , flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus) .
  • the invention provides a method for determining replication capacity for a patient's virus comprising:
  • step (c) harvesting viral particles from step (b) and infecting target host cells
  • step (f) normalizing the expression of the indicator gene by measuring an amount of virus in step (c) .
  • patient-derived segment encompasses segments derived from human and various animal species . Such species include, but are not limited to chimpanzees, horses, catties, cats and dogs.
  • Patient-derived segments can also be incorporated into resistance test vectors using any of several alternative cloning techniques as set forth in detail in US Patent Number 5,837,464 (International Publication Number WO 97/27319) which is hereby incorporated by reference. For example, cloning via the introduction of class II restriction sites into both the plasmid backbone and the patient-derived segments or by uracil DNA glycosylase primer cloning.
  • the patient-derived segment may be obtained by any method of molecular cloning or ⁇ gene amplification, or modifications thereof, by introducing patient sequence acceptor sites, as described below, at the ends of the patient-derived segment to be introduced into the resistance test vector.
  • restriction sites corresponding to the patient-sequence acceptor sites can be incorporated at the ends of the primers used in the PCR reaction.
  • restriction sites can be incorporated at the ends of the primers used for first or second- strand cDNA synthesis, or in a method such as primer-repair of DNA, whether cloned or uncloned DNA, said restriction sites can be incorporated into the primers used for the repair reaction.
  • the patient sequence acceptor sites and primers are designed to improve the representation of patient-derived segments. Sets of resistance test vectors having designed patient sequence acceptor sites provide representation of patient-derived segments that may be underrepresented in one resistance test vector alone.
  • Resistance test vector means one or more vectors which taken together contain DNA comprising a patient-derived segment and an indicator gene. Resistance test vectors are prepared as described in US Patent Number 5,837,464
  • a resistance test vector (also referred to as a resistance test vector system) is prepared by introducing patient sequence acceptor sites into a packaging vector, amplifying or cloning patient-derived segments and inserting the amplified or cloned sequences precisely into the packaging vector at the patient sequence acceptor sites and co-transfecting this packaging vector with an indicator gene viral vector.
  • Preferred examples of an indicator gene is the E.
  • the indicator or indicator gene may be functional or non-functional as described in US Patent Number 5,837,464 (International Publication Number WO 97/27319) .
  • the phenotypic drug susceptibility and resistance tests of this invention may be carried out in one or more host cells as described in US Patent Number 5,837,464 ( International Publication Number WO 97/27319) which is incorporated herein by reference.
  • Viral drug susceptibility is determined as the concentration of the anti-viral agent at which a given percentage of indicator gene expression is inhibited (e.g. the IC50 for an anti-viral agent is the concentration at which 50% of indicator gene expression is inhibited) .
  • a standard curve for drug susceptibility of a given anti-viral drug can be developed for a viral segment that is either a standard laboratory viral segment or from a drug-naive patient
  • viral drug resistance is a decrease in viral drug susceptibility for a given patient compared to such a given standard or when making one or more sequential measurements in the same patient over time, as determined by decreased susceptibility in virus from later time points compared to that from earlier time points .
  • the antiviral drugs being added to the test system are added at selected times depending upon the target of the antiviral drug.
  • HIV protease inhibitors including saquinavir, ritonavir, indinavir, nelfinavir and amprenavir
  • they are added to packaging host cells at the time of or shortly after their transfection with a resistance test vector, at an appropriate range of concentrations.
  • HIV reverse transcriptase inhibitors including AZT, ddl, ddC, d4T, 3TC, abacavir, nevirapine, delavirdine and efavirenz are added to target host cells at the time of or prior to infection by the resistance test vector viral particles, at an appropriate range of concentration.
  • the antiviral drugs may be present throughout the assay.
  • the test concentration is selected from a range of concentrations which is typically between about 8 X 10" 6 ⁇ M and about 2mM and more specifically for each of the following drugs: saquinavir, indinavir, nelfinavir and amprenavir, from about 2.3 X 10 "5 ⁇ M to about 1.5 ⁇ M and ritonavir, from about 4.5 X 10 "5 ⁇ M to about 3 ⁇ M.
  • a candidate PRI antiretroviral compound is tested in the phenotypic drug susceptibility and resistance test using the resistance test vector comprising PR having a mutation at codon 88 to a serine.
  • the candidate antiviral compound is added to the test system at an appropriate range of concentrations and at the transfection step.
  • more than one candidate antiviral compound may be tested or a candidate antiviral compound may be tested in combination with an approved antiviral drug such as AZT, ddl , ddC, d4T, 3TC, abacavir, delavirdine, nevirapine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, or a compound which is undergoing clinical trials such as adefovir and ABT-378.
  • the effectiveness of the candidate antiviral will be evaluated by measuring the expression or inhibition of the indicator gene.
  • the drug susceptibility and resistance test may be used to screen for viral mutants.
  • mutants resistant to either known antiretrovirals or candidate antiretrovirals Following the identification of mutants resistant to either known antiretrovirals or candidate antiretrovirals the resistant mutants are isolated and the DNA is analyzed. A library of viral resistant mutants can thus be assembled enabling the screening of candidate PRI antiretrovirals, alone or in combination. This will enable one of ordinary skill to identify effective PRI antiretrovirals and design effective therapeutic regimens.
  • a method of assessing the effectiveness of protease antiretroviral therapy of an HIV-infected patient comprising:
  • step (c) determines changes in susceptibility to saquinavir.
  • the method is provided , wherein the mutation at codon 82 codes for alanine (A), phenylalanine (F) , serine (S), or threonine (T) .
  • the method is provided , wherein the mutation at codon 82 is a substitution of alanine (A) , phenylalanine (F) , serine (S), or threonine (T) for valine (V) .
  • the method is provided, wherein the mutation at codon 90 codes for methionine (M) .
  • the method is provided , wherein the mutation at codon 90 is a substitution of methionine (M) for leucine (L) .
  • a method for evaluating the biological effectiveness of a candidate HIV protease antiretroviral drug compound comprising:
  • step (b) culturing the host cell from step (a) ;
  • step (d) comparing the measurement of the indicator gene from step (c) with the measurement of the indicator gene measured when steps (a) - (c) are carried out in the absence of the candidate antiretroviral drug compound; wherein a test concentration of the candidate antiretroviral drug compound is present at steps (a) (c) ; at steps (b) - (c) ; or at step (c) .
  • a resistance test vector comprising an HIV patient-derived segment further comprising protease having a mutation at codon 82 or codon 90 and an indicator gene, wherein the expression of the indicator gene is dependent upon the patient-derived segment .
  • the resistance test vector is provided , wherein the patient-derived segment having a mutation at codon 82 codes for alanine (A) , phenylalanine (F) , serine (S) , or threonine (T) .
  • the resistance test vector of is provided , wherein the patient-derived segment having a mutation at codon 82 is a substitution of alanine (A) , phenylalanine (F) , serine (S) , or threonine (T) for valine (V) .
  • the resistance test vector is provided , wherein the patient-derived segment having a mutation at codon 90 codes for methionine (M) .
  • the resistance test vector is provided, wherein the patient- derived segment having a mutation at codon 90 is a substitution of methionine (M) for leucine (L) .
  • a method for determining replication capacity for a patient's virus comprising:
  • step (c) harvesting viral particles from step (b) and infecting target host cells; (d) measuring expression of the indicator gene in the target host cell, wherein the expression of the indicator gene is dependent upon the patient-derived segment; and (e) comparing the expression of the indicator gene from (d) with the expression of the indicator gene measured when steps (a) through (d) are carried out in a control resistance test vector.
  • the method further comprises the step of:
  • step (f) normalizing the expression of the indicator gene by measuring an amount of virus in step (c) .
  • the method is provided wherein the patient-derived segment comprises nucleic acid encoding HIV integrase having a mutation at codon 66.
  • the method is provided wherein the patient-derived segment comprises nucleic acid encoding HIV integrase having a mutation at codon 154.
  • the method is provided wherein the patient-derived segment comprises nucleic acid encoding HIV integrase having mutations at codon 66 and codon 153. In another embodiment of this invention, the method is provided wherein the patient-derived segment comprises nucleic acid encoding HIV integrase having mutations at codon 66 and codon 154.
  • Retrovirally infected cells shed a membrane virus containing a diploid RNA genome.
  • the virus studded with an envelope glycoprotein (which serves to determine the host range of infectivity) , attaches to a cellular receptor in the plasma membrane of the cell to be infected. After receptor binding, the virus is internalized and uncoated as it passes through the cytoplasm of the host cell.
  • the reverse transcriptase molecules resident in the viral core drive the synthesis of the double-stranded DNA provirus, a synthesis that is primed by the binding of a tRNA molecule to the genomic viral RNA.
  • the double-stranded DNA provirus is subsequently integrated in the genome of the host cell, where it can serve as a transcriptional template for both mRNAs encoding viral proteins and virion genomic RNA, which will be packaged into viral core particles .
  • core particles move through the cytoplasm, attach to the inside of the plasma membrane of the newly infected cell, and bud, taking with them tracts of membrane containing the virally encoded envelope glycoprotein gene product. This cycle of infection - reverse transcription, transcription, translation, virion assembly, and budding - repeats itself over and over again as infection spreads.
  • the viral RNA and, as a result, the proviral DNA encode several cis-acting elements that are vital to the successful completion of the viral lifecycle.
  • the virion RNA carries the viral promoter at its 3' end. Replicative acrobatics place the viral promoter at the 5 ' end of the proviral genome as the genome is reverse transcribed,. Just 3 ' to the 5 ' retroviral LTR lies the viral packaging site.
  • the retroviral lifecycle requires the presence of virally encoded transacting factors.
  • the viral-RNA-dependent DNA polymerase (po-I) -reverse transcriptase is also contained within the viral core and is vital to the viral life cycle in that it is responsible for the conversion of the genomic RNA to the integrative intermediate proviral DNA.
  • the viral envelope glycoprotein, env is required for viral attachment to the uninfected cell and for viral spread.
  • transactivators transcriptional trans-activating factors, so called transactivators, that can serve to modulate the level of transcription of the integrated parental provirus.
  • replication-competent (non-defective) viruses are self-contained in that they encode all of these trans-acting factors. Their defective counterparts are not self-contained.
  • RNA virus such as a hepadnavirus
  • rc-DNA asymmetric relaxed circle DNA
  • cccDNA covalently closed circle DNA
  • This process which occurs within the nucleus of infected liver cells, involves completion of the DNA positive-strand synthesis and ligation of the DNA ends.
  • the cccDNA is transcribed by the host RNA polymerase to generate a 3.5 kB RNA template (the pregenome) .
  • This pregenome is complexed with protein in the viral core.
  • the third step involves the synthesis of the first negative-sense DNA strand by copying the pregenomic RNA using the virally encoded P protein reverse transcriptase.
  • the P protein also serves as the minus strand DNA primer.
  • the synthesis of the second positive-sense DNA strand occurs by copying the first DNA strand, using the P protein DNA polymerase activity and an oligomer of viral RNA as primer.
  • the pregenome also transcribes mRNA for the major structural core proteins.
  • Vectors Indicator gene c a s s e 11 e + V i r a 1 vector (functional/nonfunctional (genomic or subgenomic) indicator gene)
  • Resistance Test Vector Host Cell - a packaging host cell transfected with a resistance test vector
  • Target Host Cell - a host cell to be infected by a resistance test vector viral particle produced by the resistance test vector host cell
  • Resistance test vector means one or more vectors which taken together contain DNA or RNA comprising a patient-derived segment and an indicator gene.
  • the resistance test vector comprises more than one vector the patient-derived segment may be contained in one vector and the indicator gene in a different vector.
  • Such a resistance test vector comprising more than one vector is referred to herein as a resistance test vector system for purposes of clarity but is nevertheless understood to be a resistance test vector.
  • the DNA or RNA of a resistance test vector may thus be contained in one or more DNA or RNA molecules.
  • the resistance test vector is made by insertion of a patient-derived segment into an indicator gene viral vector.
  • the resistance test vector is made by insertion of a patient-derived segment into a packaging vector while the indicator gene is contained in a second vector, for example an indicator gene viral vector.
  • patient-derived segment refers to one or more viral segments obtained directly from a patient using various means, for example, molecular cloning or polymerase chain reaction (PCR) amplification of a population of patient-derived segments using viral DNA or complementary DNA (cDNA) prepared from viral RNA, present in the cells (e.g. peripheral blood mononuclear cells, PBMC) , serum or other bodily fluids of infected patients.
  • PCR polymerase chain reaction
  • viral segment refers to any functional viral sequence or viral gene encoding a gene product (e.g., a protein) that is the target of an anti-viral drug.
  • functional viral sequence refers to any nucleic acid sequence (DNA or RNA) with functional activity such as enhancers, promoters, polyadenylation sites, sites of action of trans-acting factors, such as tar and RRE, packaging sequences, integration sequences, or splicing sequences.
  • patient-derived segments corresponding to each said viral gene would be inserted in the resistance test vector.
  • patient-derived segments corresponding to each functional viral sequence or viral gene product would be inserted in the resistance test vector.
  • the patient-derived segments are inserted into unique restriction sites or specified locations, called patient sequence acceptor sites, in the indicator gene viral vector or for example, a packaging vector depending on the particular construction being used as described herein.
  • patient-derived segment encompasses segments derived f om human and various animal species . Such species include, but are not limited to chimpanzees, horses, catties, cats and dogs. Patient-derived segments can also be incorporated into resistance test vectors using any of several alternative cloning techniques. For example, cloning via the introduction of class II restriction sites into both the plasmid backbone and the patient-derived segments or by uracil DNA glycosylase primer cloning (refs) .
  • refs uracil DNA glycosylase primer cloning
  • the patient-derived segment may be obtained by any method of molecular cloning or gene amplification, or modifications thereof, by introducing patient sequence acceptor sites, as described below, at the ends of the patient-derived segment to be introduced into the resistance test vector.
  • patient sequence acceptor sites as described below
  • restriction sites corresponding to the patient-sequence acceptor sites can be incorporated at the ends of the primers used in the PCR reaction.
  • restriction sites can be incorporated at the ends of the primers used for first or second strand cDNA synthesis, or in a method such as primer-repair of DNA, whether cloned or uncloned DNA, said restriction sites can be incorporated into the primers used for the repair reaction.
  • the patient sequence acceptor sites and primers are designed to improve the representation of patient-derived segments. Sets of resistance test vectors having designed patient sequence acceptor sites provide representation of patient-derived segments that would be underrepresented in one resistance test vector alone.
  • Resistance test vectors are prepared by modifying an indicator gene viral vector (described below) by introducing patient sequence acceptor sites, amplifying or cloning patient-derived segments and inserting the amplified or cloned sequences precisely into indicator gene viral vectors at the patient sequence acceptor sites.
  • the resistance test vectors are constructed from indicator gene viral vectors which are in turn derived from genomic viral vectors or subgenomic viral vectors and an indicator gene cassette, each of which is described below. Resistance test vectors are then introduced into a host cell.
  • a resistance test vector also referred to as a resistance test vector system
  • a resistance test vector system is prepared by introducing patient sequence acceptor sites into a packaging vector, amplifying or cloning patient-derived segments and inserting the amplified or cloned sequences precisely into the packaging vector at the patient sequence acceptor sites and co-transfecting this packaging vector with an indicator gene viral vector.
  • the resistance test vector may be introduced into packaging host cells together with packaging expression vectors, as defined below, to produce resistance test vector viral particles that are used in drug resistance and susceptibility tests that are referred to herein as a "particle-based test.”
  • the resistance test vector may be introduced into a host cell in the absence of packaging expression vectors to carry out a drug resistance and susceptibility test that is referred to herein as a "non-particle-based test.”
  • a "packaging expression vector” provides the factors, such as packaging proteins (e.g. structural proteins such as core and envelope polypeptides), transacting factors, or genes required by replication-defective retrovirus or hepadnavirus .
  • a replication-competent viral genome is enfeebled in a manner such that it cannot replicate on its own.
  • the packaging expression vector can produce the trans-acting or missing genes required to rescue a defective viral genome present in a cell containing the enfeebled genome, the enfeebled genome cannot rescue itself.
  • “Indicator or indicator gene” refers to a nucleic acid encoding a protein, DNA or RNA structure that either directly or through a reaction gives rise to a measurable or noticeable aspect, e.g. a color or light of a measurable wavelength or in the case of DNA or RNA used as an indicator a change or generation of a specific DNA or RNA structure.
  • Preferred examples of an indicator gene is the E. coli lacZ gene which encodes beta-galactosidase, the luc gene which encodes luciferase either from, for example, Photonis pyralis (the firefly) or Renilla reniformis (the sea pansy) , the E.
  • indicator gene which encodes alkaline phosphatase, green fluorescent protein and the bacterial CAT gene which encodes chloramphenicol acetyltransferase .
  • Additional preferred examples of an indicator gene are secreted proteins or cell surface proteins that are readily measured by assay, such as radioimmunoassay (RIA) , or fluorescent activated cell sorting (FACS) , including, for example, growth factors, cytokines and cell surface antigens (e.g. growth hormone, 11-2 or CD4, respectively).
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • “Indicator gene” is understood to also include a selection gene, also referred to as a selectable marker.
  • Suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR) , thymidine kinase, hygromycin, neomycin, zeocin or E. coli gpt .
  • DHFR dihydrofolate reductase
  • the indicator gene and the patient-derived segment are discrete, i.e. distinct and separate genes.
  • a patient-derived segment may also be used as an indicator gene.
  • one of said viral genes may also serve as the indicator gene.
  • a viral protease gene may serve as an indicator gene by virtue of its ability to cleave a chromogenic substrate or its ability to activate an inactive zymogen which in turn cleaves a chromogenic substrate, giving rise in each case to a color reaction.
  • the indicator gene may be either "functional” or “non-functional” but in each case the expression of the indicator gene in the target cell is ultimately dependent upon the action of the patient-derived segment.
  • the indicator gene may be capable of being expressed in a "packaging host cell/resistance test vector host cell" as defined below, independent of the patient-derived segment, however the functional indicator gene could not be expressed in the target host cell, as defined below, without the production of functional resistance test vector particles and their effective infection of the target host cell.
  • the indicator gene cassette comprising control elements and a gene encoding an indicator protein, is inserted into the indicator gene viral vector with the same or opposite transcriptional orientation as the native or foreign enhancer/promoter of the viral vector.
  • One example of a functional indicator gene in the case of HIV or HBV places the indicator gene and its promoter (a CMV IE enhancer/promoter) in the same or opposite transcriptional orientation as the HIV-LTR or HBV enhancer-promoter, respectively, or the CMV IE enhancer/promoter associated with the viral vector.
  • a CMV IE enhancer/promoter a CMV IE enhancer/promoter
  • the indicator gene may be "non-functional" in that the indicator gene is not efficiently expressed in a packaging host cell transfected with the resistance test vector, which is then referred to a resistance test vector host cell, until it is converted into a functional indicator gene through the action of one or more of the patient-derived segment products.
  • An indicator gene is rendered non-functional through genetic manipulation according to this invention.
  • an indicator gene is rendered non-functional due to the location of the promoter, in that, although the promoter is in the same transcriptional orientation as the indicator gene, it follows rather than precedes the indicator gene coding sequence.
  • This misplaced promoter is referred to as a "permuted promoter.”
  • the orientation of the non-functional indicator gene is opposite to that of the native or foreign promoter/enhancer of the viral vector.
  • the coding sequence of the non-functional indicator gene can neither be transcribed by the permuted promoter nor by the viral promoters .
  • the non-functional indicator gene and its permuted promoter is rendered functional by the action of one or more of the viral proteins.
  • T7 promoter T7 phage RNA polymerase promoter
  • the indicator gene cannot be transcribed by the T7 promoter as the indicator gene cassette is positioned upstream of the T7 promoter.
  • the non-functional indicator gene in the resistance test vector is converted into a functional indicator gene by reverse transcriptase upon infection of the target cells, resulting from the repositioning of the T7 promoter, by copying from the 5' LTR to the 3' LTR, relative to the indicator gene coding region.
  • a nuclear T7 RNA polymerase expressed by the target cell transcribes the indicator gene.
  • a non-functional indicator gene with a permuted promoter places an enhancer-promoter region downstream or 3' of the indicator gene both having the same transcriptional orientation.
  • the indicator gene cannot be transcribed by the enhancer-promoter as the indicator gene cassette is positioned upstream.
  • the non-functional indicator gene in the resistance test vector is converted into a functional indicator gene by reverse transcription and circularization of the HBV indicator gene viral vector by the repositioning of the enhancer-promoter upstream relative to the indicator gene coding region.
  • a permuted promoter may be any eukaryotic or prokaryotic promoter which can be transcribed in the target host cell.
  • the promoter will be small in size to enable insertion in the viral genome without disturbing viral replication.
  • a promoter that is small in size and is capable of transcription by a single subunit RNA polymerase introduced into the target host cell such as a bacteriophage promoter, will be used. Examples of such bacteriophage promoters and their cognate RNA polymerases include those of phages T7, T3 and Sp6.
  • a nuclear localization sequence may be attached to the RNA polymerase to localize expression of the RNA polymerase to the nucleus where they may be needed to transcribed the repaired indicator gene.
  • Such an NLS may be obtained from any nuclear-transported protein such as the SV40 T antigen.
  • an internal ribosome entry site such as the EMC virus 5' untranslated region (UTR) may be added in front of the indicator gene, for translation of the transcripts which are generally uncapped.
  • the permuted promoter itself can be introduced at any position within the 5' LTR that is copied to the 3' LTR during reverse transcription so long as LTR function is not disrupted, preferably within the U5 and R portions of the LTR, and most preferably outside of functionally important and highly conserved regions of U5 and R.
  • the permuted promoter can be placed at any position that does not disrupt the cis acting elements that are necessary for HBV DNA replication. Blocking sequences may be added at the ends of the resistance test vector should there be inappropriate expression of the non-functional indicator gene due to transfection artifacts (DNA concatenation) .
  • such a blocking sequence may consist of a T7 transcriptional terminator, positioned to block readthrough transcription resulting from DNA concatenation, but not transcription resulting from repositioning of the permuted T7 promoter from the 5' LTR to the 3' LTR during reverse transcription.
  • an indicator gene is rendered non-functional due to the relative location of the 5 ' and 3 ' coding regions of the indicator gene, in that, the 3' coding region precedes rather than follows the 5' coding region.
  • This misplaced coding region is referred to as a "permuted coding region.”
  • the orientation of the non-functional indicator gene may be the same or opposite to that of the native or foreign promoter/enhancer of the viral vector, as mRNA coding for a functional indicator gene will be produced in the event of either orientation.
  • the non-functional indicator gene and its permuted coding region is rendered functional by the action of one or more of the patient-derived segment products.
  • a second example of a non-functional indicator gene with a permuted coding region in the case of HIV places a 5' indicator gene coding region with an associated promoter in the 3' LTR U3 region and a 3 ' indicator gene coding region in an upstream location of the HIV genome, with each coding region having the same transcriptional orientation as the viral LTRs .
  • the 5' and 3' coding regions may also have associated splice donor and acceptor sequences, respectively, which may be heterologous or artificial splicing signals.
  • the indicator gene cannot be functionally transcribed either by the associated promoter or viral promoters, as the permuted coding region prevents the formation of functionally spliced transcripts.
  • the non-functional indicator gene in the resistance test vector is converted into a functional indicator gene by reverse transcriptase upon infection of the target cells, resulting from the repositioning of the 5' and 3' indicator gene coding regions relative to one another, by copying of the 3' LTR to the 5' LTR.
  • RNA splicing can join the 5' and 3' coding regions to produce a functional indicator gene product.
  • a non-functional indicator gene with a permuted coding region in the case of HBV, places a 3' indicator gene coding region upstream or 5' of the enhancer-promoter and the 5' coding region of the indicator gene.
  • the transcriptional orientation of the indicator gene 5' and 3' coding regions are identical to one another, and the same as that of the indicator gene viral vector. However, as the indicator gene 5' and 3' coding regions are permuted in the resistance test vectors (i.e., the 5' coding region is downstream of the 3' coding region) , no mRNA is transcribed which can be spliced to generate a functional indicator gene coding region. Following reverse transcription and circularization of the indicator gene viral vector, the indicator gene 3' coding region is positioned downstream or 3' to the enhancer-promoter and 5' coding regions thus permitting the transcription of mRNA which can be spliced to generate a functional indicator gene coding region.
  • the indicator gene is rendered non-functional through use of an "inverted intron," i.e. an intron inserted into the coding sequence of the indicator gene with a transcriptional orientation opposite to that of the indicator gene.
  • the overall transcriptional orientation of the indicator gene cassette including its own, linked promoter is opposite to that of the viral control elements, while the orientation of the artificial intron is the same as the viral control elements. Transcription of the indicator gene by its own linked promoter does not lead to the production of functional transcripts as the inverted intron cannot be spliced in this orientation.
  • the indicator gene Transcription of the indicator gene by the viral control elements does, however, lead to the removal of the inverted intron by RNA splicing, although the indicator gene is still not functionally expressed as the resulting transcript has an antisense orientation.
  • the indicator gene can be functionally transcribed using its own linked promoter as the inverted intron has been previously removed. In this case, the indicator gene itself may contain its own functional promoter with the entire transcriptional unit oriented opposite to the viral control elements.
  • the non-functional indicator gene is in the wrong orientation to be transcribed by the viral control elements and it cannot be functionally transcribed by its own promoter, as the inverted intron cannot be properly excised by splicing.
  • transcription by the viral promoters results in the removal of the inverted intron by splicing.
  • the indicator gene can now be functionally transcribed by its own promoter.
  • the inverted intron consisting of a splice donor and acceptor site to remove the intron, is preferably located in the coding region of the indicator gene in order to disrupt translation of the indicator gene.
  • the splice donor and acceptor may be any splice donor and acceptor.
  • a preferred splice donor-receptor is the CMV IE splice donor and the splice acceptor of the second exon of the human alpha globin gene ("intron A”) .
  • indicator gene viral vector refers to a vector (s) comprising an indicator gene and its control elements and one or more viral genes .
  • the indicator gene viral vector is assembled from an indicator gene cassette and a "viral vector, " defined below.
  • the indicator gene viral vector may additionally include an enhancer, splicing signals, polyadenylation sequences, transcriptional terminators, or other regulatory sequences. Additionally the indicator gene viral vector may be functional or nonfunctional. In the event that the viral segments which are the target of the anti-viral drug are not included in the indicator gene viral vector they are provided in a second vector.
  • An "indicator gene cassette” comprises an indicator gene and control elements.
  • “Viral vector” refers to a vector comprising some or all of the following: viral genes encoding a gene product, control sequences, viral packaging sequences, and in the case of a retrovirus, integration sequences.
  • the viral vector may additionally include one or more viral segments one or more of which may be the target of an anti-viral drug.
  • Two examples of a viral vector which contain viral genes are referred to herein as an "genomic viral vector” and a “subgenomic viral vector.”
  • a “genomic viral vector” is a vector which may comprise a deletion of a one or more viral genes to render the virus replication incompetent, but which otherwise preserves the mRNA expression and processing characteristics of the complete virus.
  • the genomic viral vector comprises the HIV gag-pol , vif, vpr, ta t, rev, vpu, and nef genes (some, most or all of env may be deleted) .
  • a "subgenomic viral vector” refers to a vector comprising the coding region of one or more viral genes which may encode the proteins that are the target (s) of the anti-viral drug.
  • a preferred embodiment is a subgenomic viral vector comprising the HIV gag-pol gene.
  • HBV a preferred embodiment is a subgenomic viral vector comprising the HBV P gene.
  • HXB2 Fisher et al . , (1986) Nature, 320, 367-371) and NL4-3, (Adachi et al . , (1986) J. Virol . , 59, 284-291) .
  • HBV a large number of full length genomic sequences have been characterized and could be used for construction of HBV viral vectors: GenBank Nos. M54923, M38636, J02203 and X59795.
  • the viral coding genes may be under the control of a native enhancer/promoter or a foreign viral or cellular enhancer/promoter.
  • a preferred embodiment for an HIV drug susceptibility and resistance test is to place the genomic or subgenomic viral coding regions under the control of the native enhancer/promoter of the HIV-LTR U3 region or the CMV immediate-early (IE) enhancer/promoter.
  • a preferred embodiment for an HBV drug susceptibility and resistance test is to place the genomic or subgenomic viral coding regions under the control of the CMV immediate-early (IE) enhancer/promoter.
  • an indicator gene viral vector that contains one or more viral genes which are the targets or encode proteins which are the targets of an anti-viral drug(s) then said vector contains the patient sequence acceptor sites. The patient-derived segments are inserted in the patient sequence acceptor site in the indicator gene viral vector which is then referred to as the resistance test vector, as described above.
  • Patient sequence acceptor sites are sites in a vector for insertion of patient-derived segments and said sites may be: 1) unique restriction sites introduced by site-directed mutagenesis into a vector; 2) naturally occurring unique restriction sites in the vector; or 3) selected sites into which a patient-derived segment may be inserted using alternative cloning methods (e.g. UDG cloning) .
  • the patient sequence acceptor site is introduced into the indicator gene viral vector.
  • the patient sequence acceptor sites are preferably located within or near the coding region of the viral protein which is the target of the anti-viral drug.
  • the viral sequences used for the introduction of patient sequence acceptor sites are preferably chosen so that no change, or a conservative change, is made in the amino acid coding sequence found at that position.
  • the patient sequence acceptor sites are located within a relatively conserved region of the viral genome to facilitate introduction of the patient-derived . ' segments.
  • the patient sequence acceptor sites are located between functionally important genes or regulatory sequences.
  • Patient-sequence acceptor sites may be located at or near regions in the viral genome that are relatively conserved to permit priming by the primer used to introduce the corresponding restriction site into the patient-derived segment.
  • primers may be designed as degenerate pools to accommodate viral sequence heterogeneity, or may incorporate residues such as deoxyinosine (I) which have multiple base-pairing capabilities.
  • Sets of resistance test vectors having patient sequence acceptor sites that define the same or overlapping restriction site intervals may be used together in the drug resistance and susceptibility tests to provide representation of patient-derived segments that contain internal restriction sites identical to a given patient sequence acceptor site, and would thus be underrepresented in either resistance test vector alone.
  • the resistance test vector is introduced into a host cell.
  • Suitable host cells are mammalian cells.
  • Preferred host cells are derived from human tissues and cells which are the principle targets of viral infection.
  • HIV include human cells such as human T cells, monocytes, macrophage, dendritic cells, Langerhans cells, hematopoeitic stem cells or precursor cells, and other cells .
  • suitable host cells include hepatoma cell lines (HepG2, Huh 7), primary human hepatocytes, mammalian cells which can be- infected by pseudotyped HBV, and other cells.
  • Host cells will assure that the anti-viral drug will enter the cell efficiently and be converted by the cellular enzymatic machinery into the metabolically relevant form of the anti-viral inhibitor.
  • Host cells are referred to herein as a "packaging host cells,” “resistance test vector host cells,” or “target host cells.”
  • a "packaging host cell” refers to a host cell that provides the trans-acting factors and viral packaging proteins required by the replication defective viral vectors used herein, such as the resistance test vectors, to produce resistance test vector viral particles .
  • the packaging proteins may be provided for by the expression of viral genes contained within the resistance test vector itself, a packaging expression vector(s), or both.
  • a packaging host cell is a host cell which is transfected with one or more packaging expression vectors and when transfected with a resistance test vector is then referred to herein as a "resistance test vector host cell” and is sometimes referred to as a packaging host cell/resistance test vector host cell.
  • Preferred host cells for use as packaging host cells for HIV include 293 human embryonic kidney cells (293, Graham, F.L. et al., J. Gen Virol. 36: 59, 1977), BOSC23 (Pear et al., Proc . Natl . Acad . Sci . 90, 8392, 1993), tsa54 and tsa201 cell lines (Heinzel et al . , J. Virol .
  • a "target host cell” refers to a cell to be infected by resistance test vector viral particles produced by the resistance test vector host cell in which expression or inhibition of the indicator gene takes place.
  • Preferred host cells for use as target host cells include human T cell leukemia cell lines including Jurkat (ATCC T1B-152), H9 (ATCC HTB-176) , CEM (ATCC CCL-119), HUT78 (ATCC T1B-161), and derivatives thereof.
  • replication capacity is defined herein is a measure of how well the virus replicates. This may also be referred to as viral fitness. In one embodiment, replication capacity can be measured by evaluating the ability of the virus to replicate in a single round of replication.
  • control resistance test vector is defined as a resistance test vector comprising a standard viral sequence (for example, HXB2, PNL4-3) and an indicator gene.
  • normalizing is defined as standardizing the amount of the expression of indicator gene measured relative to the number of viral particles giving rise to the expression of the indicator gene. For example, normalization is measured by dividing the amount of luciferase activity measured by the number of viral particles measured at the time of infection.
  • Plasmids and vectors are designated by a lower case p followed by letters and/or numbers .
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures .
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • Plasmids of the invention employs standard ligation and restriction techniques which are well understood in the art (see Ausubel et al., (1987) Current Protocols in Molecular Biology, Wiley Interscience or Maniatis et al . , (1992) in Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory, N.Y.). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired. The sequences of all DNA constructs incorporating synthetic DNA were confirmed by DNA sequence analysis (Sanger et al . (1977) Proc. Natl. Acad. Sci. 74, 5463-5467).
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences, restriction sites, in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements are known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution. Alternatively, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37°C are workable, although variations can be tolerated.
  • cleaved fragments After each incubation, protein is removed by extraction with phenol/chloroform and the nucleic acid recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A 'general description of size separations is found in Methods of Enzymology 65:499-560 (1980) .
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 minutes at 20°C in 50 mM Tris (pH 7.6) 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 5-10 mM dNTPs.
  • the Klenow fragment fills in at 5' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present.
  • selective repair can be performed by supplying only one of the dNTPs, or with selected dNTPs, within the limitations dictated by the nature of the sticky ends .
  • the mixture is extracted with phenol/chloroform and ethanol precipitated.
  • Treatment under appropriate conditions with SI nuclease or Bal-31 results in hydrolysis of any single-stranded portion.
  • Ligations are performed in 15-50 ⁇ l volumes under the following standard conditions and temperatures: 20 mM Tris-CI pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 mg/ml BSA, 10 M- 50 mM NaCl, and either 40 ⁇ M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end” ligation) or lmM ATP, 0.3 - 0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end” ligation) .
  • Intermolecular "sticky end” ligations are usually performed at 33-100 ⁇ g/ml total DNA concentrations (5-100 mM total end concentration) .
  • Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at l ⁇ M total ends concentration.
  • Transient expression refers to unamplified expression within about one day to two weeks of transfection.
  • the optimal time for transient expression of a particular desired heterologous gene may vary depending on several factors including, for example, any transacting factors which may be employed, translational control mechanisms and the host cell.
  • Transient expression occurs when the particular plasmid that has been transfected functions, i.e., is transcribed and translated. During this time the plasmid DNA which has entered the cell is transferred to the nucleus. The DNA is in a nonintegrated state, free within the nucleus. Transcription of the plasmid taken up by the cell occurs during this period. Following transfection the plasmid DNA may become degraded or diluted by cell division. Random integration within the cell chromatin occurs .
  • promoters and control sequences which are derived from species compatible with the host cell are used with the particular host cell.
  • Promoters suitable for use with prokaryotic hosts illustratively include the beta-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan
  • trp tac promoter system and hybrid promoters such as tac promoter.
  • other functional bacterial promoters are suitable.
  • eukaryotic microbes such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available.
  • Promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, simian virus 40
  • SV40 adenovirus
  • retroviruses hepatitis B virus and preferably cytomegalovirus
  • heterologous mammalian promoters e.g. ⁇ -actin promoter.
  • the early and late promoters of the SV 40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment.
  • promoters from the host cell or related species also are useful herein.
  • the vectors used herein may contain a selection gene, also termed a selectable marker.
  • a selection gene encodes a protein, necessary for the survival or growth of a host cell transformed with the vector.
  • suitable selectable markers for mammalian cells include the dihydrofolate reductase gene (DHFR) , the ornithine decarboxylase gene, the multi-drug resistance gene ( dr) , the adenosine deaminase gene, and the gluta ine synthase gene.
  • the first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media.
  • the second category is referred to as dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin (Southern and Berg (1982)
  • Transfection means introducing DNA into a host cell so that the DNA is expressed, whether functionally expressed or otherwise; the DNA may also replicate either as an extrachromosomal element or by chromosomal integration.
  • the method used herein for transfection of the host cells is the calcium phosphate co-precipitation method of Graham and van der Eb (1973) Virology 52, 456-457.
  • Alternative methods for transfection are electroporation, the DEAE-dextran method, lipofection and biolistics (Kriegler (1990) Gene Transfer and Expression: A Laboratory Manual, Stockton Press).
  • Host cells may be transfected with the expression vectors of the present invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes.
  • Host cells are cultured in F12:DMEM (Gibco) 50:50 with added glutamine.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • patient-derived segment (s) corresponding to the HIV protease and reverse transcriptase coding regions were either patient-derived segments amplified by the reverse transcription-polymerase chain reaction method (RT-PCR) using viral RNA isolated from viral particles present in the serum of HIV-infected individuals or were mutants of wild type HIV-1 made by site directed mutagenesis of a parental clone of resistance test vector DNA.
  • Isolation of viral RNA was performed using standard procedures (e.g. RNAgents Total RNA Isolation System, Promega, Madison WI or RNAzol, Tel-Test, Friendswood, TX) .
  • the RT-PCR protocol was divided into two steps.
  • a retroviral reverse transcriptase [e.g.
  • Moloney MuLV reverse transcriptase (Roche Molecular Systems, Inc., Branchburg, NJ) , or avian myeloblastosis virus (AMV) reverse transcriptase, (Boehringer Mannheim, Indianapolis, IN)] was used to copy viral RNA into cDNA.
  • the cDNA was then amplified using a thermostable DNA polymerase [e.g.
  • thermostable polymerases as described for the performance of "long PCR” (Barnes, W.M., (1994) Proc. Natl. Acad. Sci, USA 91, 2216-2220) [e.g. Expand High Fidelity PCR System (Taq -fc Pwo) , (Boehringer Mannheim. Indianapolis, IN) OR GeneAmp XL PCR kit (Tth + Vent), (Roche Molecular Systems, Inc., Branchburg, NJ) ] .
  • PCR6 (Table 5, #1) is used for reverse transcription of viral RNA into cDNA.
  • the primers, Apal primer (PDSApa, Table 5, #2) and Agel primer (PDSAge, Table 5, #3) used to amplify the "test" patient-derived segments contained sequences resulting in Apal and Agel recognition sites being introduced into both ends of the PCR product, respectively.
  • Resistance test vectors incorporating the "test" patient-derived segments were constructed as described in US Patent Number 5,837,464 (International Publication Number WO 97/27319) (see Fig.
  • the plasmid DNA corresponding to the resultant resistance test vector comprises a representative sample of the HIV viral quasi-species present in the serum of a given patient, many (>100) independent E. coli transformants obtained in the construction of a given resistance test vector were pooled and used for the preparation of plasmid DNA.
  • a packaging expression vector encoding an amphotrophic MuLV 4070A env gene product enables production in a resistance test vector host cell of resistance test vector viral particles which can efficiently infect human target cells.
  • Resistance test vectors encoding all HIV genes with the exception of env were used to transfect a packaging host cell (once transfected the host cell is referred to as a resistance test vector host cell) .
  • the packaging expression vector which encodes the amphotrophic MuLV 4070A env gene product is used with the resistance test vector to enable production in the resistance test vector host cell of infectious pseudotyped resistance test vector viral particles .
  • Resistance tests performed with resistance test vectors were carried out using packaging host and target host cells consisting of the human embryonic kidney cell line 293 (Cell Culture Facility, UC San Francisco, SF, CA) or the Jurkat leukemic T-cell line (Arthur Weiss, UC San Francisco, SF, CA) .
  • Resistance tests were carried out with resistance test vectors ' using two host cell types. Resistance test vector viral particles were produced by a first host cell (the resistance test vector host cell) that was prepared by transfecting a packaging host cell with the resistance test vector and the packaging expression vector. The resistance test vector viral particles were then used to infect a second host cell (the target host cell) in which the expression of the indicator gene is measured (see Fig. 2) .
  • the resistance test vectors containing a functional luciferase gene cassette were constructed and host cells were transfected with the resistance test vector DNA.
  • the resistant test vectors contained patient-derived reverse transcriptase and protease DNA sequences that encode proteins which were either susceptible or resistant to the antiretroviral agents, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors.
  • the resistance test vector viral particles produced by transfecting the resistance test vector DNA into host cells, either in the presence or absence of protease inhibitors, were used to infect target host cells grown either in the absence of NRTI or NNRTI or in the presence of increasing concentrations of the drug.
  • Luciferase activity in infected target host cells in the presence of drug was compared to the luciferase activity in infected target host cells in the absence of drug.
  • Drug resistance was measured as the concentration of drug required to inhibit by 50% the luciferase activity detected in the absence of drug (inhibitory concentration 50%, IC50 ) .
  • the IC50 values were determined by plotting percent drug inhibition vs. log 10 drug concentration.
  • Host cells were seeded in 10-cm-diameter dishes and were transfected one day after plating with resistance test vector plasmid DNA and the envelope expression vector. Transfections were performed using a calcium-phosphate co-precipitation procedure. The cell culture media containing the DNA precipitate was replaced with fresh medium, from one to 24 hours, after transfection. Cell culture media containing resistance test vector viral particles was harvested one to four days after transfection and was passed through a 0.45-mm filter before being stored at " 80°C. HIV capsid protein (p24) levels in the harvested cell culture media were determined by an EIA method as described by the manufacturer (SIAC; Frederick, MD) . Before infection, target cells (293 and 293/T) were plated in cell culture media.
  • Control infections were performed using cell culture media from mock transfections (no DNA) or transfections containing the resistance test vector plasmid DNA without the envelope expression plasmid. One to three or more days after infection the media was removed and cell lysis buffer (Promega) was added to each well. Cell lysates were assayed for luciferase activity. The inhibitory effect of the drug was determined using the following equation:
  • % luciferase inhibition [1 - (RLUluc [drug] RLUluc) ] x 100
  • RLUluc [drug] is the relative light unit of luciferase activity in infected cells in the presence of drug and RLUluc is the Relative Light Unit of luciferase activity in infected cells in the absence of drug.
  • IC50 values were obtained from the sigmoidal curves that were generated from the data by plotting the percent inhibition of luciferase activity vs. the log 10 drug concentration. Examples of drug inhibition curves are shown in (Fig. 3) .
  • Resistance test vectors are constructed as described in example 1. Resistance test vectors, or clones derived from the resistance test vector pools, are tested in a phenotypic assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
  • This panel of anti-retroviral drugs may comprise members of the classes known as nucleoside-analog reverse transcriptase inhibitors
  • NRTIs non-nucleoside reverse transcriptase inhibitors
  • NRTIs neuropeptide kinase inhibitors
  • PRIs protease inhibitors
  • the panel of drugs can be expanded as new drugs or new drug targets become available.
  • An IC50 is determined for each resistance test vector pool for each drug tested. The pattern of susceptibility to all of the drugs tested is examined and compared to known patterns of susceptibility.
  • a patient sample can be further examined for genotypic changes correlated with the pattern of susceptibility observed. Genotypic analysis of patient HIV samples
  • Resistance test vector DNAs are analyzed by any of the genotyping methods described in
  • Example 1 In one embodiment of the invention, patient
  • HIV sample sequences are determined using viral RNA purification, RT/PCR and ABI chain terminator automated sequencing. The sequence that is determined is compared to control sequences present in the database or is compared to a sample from the patient prior to initiation of therapy, if available. The genotype is examined for sequences that are different from the control or pre-treatment sequence and correlated to the observed phenotype. Phenotypic susceptibility analysis of site directed mutants
  • Genotypic changes that are observed to correlate with changes in phenotypic patterns of drug susceptibility are evaluated by construction of resistance test vectors containing the specific mutation on a defined, wild-type
  • Mutations may be incorporated alone and/or in combination with other mutations that are thought to modulate the susceptibility of HIV to a certain drug or class of drugs. Mutations are introduced into the resistance test vector through any of the widely known methods for site-directed mutagenesis. In one embodiment of this invention the mega-primer PCR method for site-directed mutagenesis is used. A resistance test vector containing the specific mutation or group of mutations are then tested using the phenotypic susceptibility assay described above and the susceptibility profile is compared to that of a genetically defined wild-type (drug susceptible) resistance test vector which lacks the specific mutations. Observed changes in the pattern of phenotypic susceptibility to the antiretroviral drugs tested are attributed to the specific mutations introduced into the resistance test vector.
  • Phenotypic analysis of Patient 0732 A resistance test vector was constructed as described in example 1 from a patient sample designated as 0732. This patient had been previously treated with nelfinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-0732. RTV-0732 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
  • This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz) , and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
  • An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
  • a pattern of susceptibility to the PRIs was observed for patient sample RTV-0732 in which there was a decrease in both nelfinavir and indinavir susceptibility (increased resistance) and an increase in amprenavir susceptibility (see Fig. 4 and Table 1) .
  • Patient sample 0732 was examined further for genotypic changes associated with the pattern of susceptibility.
  • RTV-0732 DNA was analyzed by ABI chain terminator automated sequencing.
  • the nucleotide sequence was compared to the consensus sequence of a wild type clade B HIV-1 (HIV Sequence Database Los Alamos, NM) .
  • the nucleotide sequence was examined for sequences that are different from the control sequence.
  • PR mutations were noted at positions K14R, I15V, K20T, E35D, M36I, R41K, I62V, L63Q and N88S.
  • K14R, I15V, E35D, R41K and I62V are naturally occurring polymorphisms in HIV-1 PR and are not associated with reduced susceptibility to any drug.
  • M36I has previously been described to be associated with resistance to ritonavir and nelfinavir (Shihazi, 1998).
  • N88S has previously been described to be associated with resistance to nelfinavir (Patick AAC, 42: 2637 (1998) and an investigational PRI, SC55389A (Smidt, 1997) .
  • a resistance test vector was constructed as described in example 1 from a patient sample designated as 627. This patient had been treated with indinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-627. RTV-627 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
  • This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz) , and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
  • An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
  • a pattern of susceptibility to the PRIs was observed for patient sample RTV-627 in which there was a decrease in indinavir and nelfinavir susceptibility (increased resistance) and an increase in amprenavir and saquinavir susceptibility.
  • Patient sample 627 was examined further for genotypic changes associated with the pattern of susceptibility.
  • RTV-627 DNA was analyzed by ABI chain terminator automated sequencing.
  • the nucleotide sequence was compared to the consensus sequence of a wild type clade B HIV-1 (HIV Sequence Database Los Alamos, NM) .
  • the nucleotide sequence was examined for sequences that are different from the control sequence.
  • PR mutations were noted at positions 13I/V, E35D, M46L, L63P, I64V, I73V and N88S.
  • I13V, E35D and I64V are naturally occurring polymorphisms in HIV-1 PR and are not associated with reduced susceptibility to any drug.
  • M46L has previously been described to be associated with resistance to indinavir and amprenavir.
  • L63P has previously been described to be associated with resistance to indinavir and nelfinavir.
  • N88S has previously been described to be associated with resistance to nelfinavir (Patick, 1998) and an investigational PRI, SC55389A (Smidt, 1997) .
  • a resistance test vector was constructed as described in example 1 from a patient sample designated as 1208. This patient had been previously treated with nelfinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-1208. RTV-1208 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs .
  • This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz) , and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
  • An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
  • a pattern of susceptibility to the PRIs was observed for patient sample RTV-1208 in which there was a decrease in indinavir and nelfinavir susceptibility (increased resistance) and an increase in amprenavir susceptibility.
  • Patient sample 1208 was examined further for genotypic changes associated with the pattern of susceptibility.
  • RTV-1208 DNA was analyzed by ABI chain terminator automated sequencing. The nucleotide sequence was compared to the consensus sequence of a wild type clade B
  • HIV-1 HIV Sequence Database Los Alamos, NM
  • the nucleotide sequence was examined for sequences that are different from the control sequence. PR mutations were noted at positions I62V, L63P, V77I, and N88S.
  • I62V is a naturally occurring polymorphism in HIV-1 PR and is not associated with reduced susceptibility to any drug.
  • L63P has previously been described to be associated with resistance to indinavir and nelfinavir.
  • V77I has previously been described to be associated with resistance to nelfinavir.
  • N88S has previously been described to be associated with resistance to nelfinavir (Patick, 1998) and an investigational PRI, SC55389A (Smidt, 1997) .
  • a resistance test vector was constructed as described in example 1 from a patient sample designated as 360. This patient had been previously treated with indinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-360. RTV-360 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
  • This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz) , and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
  • An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
  • a pattern of susceptibility to the PRIs was observed for patient sample RTV-360 in which there was a decrease in indinavir and nelfinavir susceptibility (increased resistance) and an increase in amprenavir susceptibility.
  • Patient sample 360 was examined further for genotypic changes associated with the pattern of susceptibility. Determination of genotype of patient 360
  • RTV-360 DNA was analyzed by ABI chain terminator automated sequencing.
  • the nucleotide sequence was compared to the consensus sequence of a wild type clade B HIV-1 (HIV Sequence Database Los Alamos, NM) .
  • the nucleotide sequence was examined for sequences that are different from the control sequence.
  • PR mutations were noted at positions I13V, K20M, M36V, N37A, M46I, I62V, L63P, N88S, and I93L.
  • I13V, N37A and I62V are naturally occurring polymorphisms in HIV-1 PR and are not associated with reduced susceptibility to any drug.
  • K20M has previously been described to be associated with resistance to indinavir.
  • M46I has previously been described to be associated with resistance to indinavir, ritonavir, nelfinavir and amprenavir.
  • L63P has previously been described to be associated with resistance to indinavir and nelfinavir.
  • N88S has previously been described to be associated with resistance to nelfinavir (Patick, 1998) and an investigational PRI, SC55389A (Smidt, 1997) .
  • a resistance test vector was constructed as described in example 1 from a patient sample designated as 0910. This patient had been previously treated with nelfinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-0910. RTV-0910 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
  • This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz) , and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
  • An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
  • a pattern of susceptibility to the PRIs was observed for patient sample RTV-0910 in which there was a decrease in indinavir and nelfinavir susceptibility (increased resistance) and an increase in amprenavir susceptibility.
  • Patient sample 0910 was examined further for genotypic changes associated with the pattern of susceptibility.
  • RTV-0910 DNA was analyzed by ABI chain terminator automated sequencing.
  • the nucleotide sequence was compared to the consensus sequence of a wild type clade B HIV-1 (HIV Sequence Database Los Alamos, NM) .
  • the nucleotide sequence was examined for sequences that are different from the control sequence.
  • PR mutations were noted at positions M46I, L63P, V77I, N88S and I93I/L.
  • I13V, K14R, N37D and I193L are naturally occuring polymorphism in HIV-1 PR and is not associated with reduced susceptibility to any drug.
  • V77I has previously been described to be associated with resistance to nelfinavir.
  • M46I has previously been described to be associated with resistance to indinavir, ritonavir, nelfinavir and amprenavir.
  • L63P has previously been described to be associated with resistance to indinavir and nelfinavir.
  • N88S has previously been described to be associated with resistance to nelfinavir (Patick, 1998) and an investigational PRI, SC55389A (Smidt, 1997) .
  • a resistance test vector was constructed as described in example 1 from a patient sample designated as 3542. This patient had been treated with indinavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-3542. RTV-3542 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
  • This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz) , and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
  • An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
  • a pattern of susceptibility to the PRIs was observed for patient sample RTV-3542 in which there was a decrease in indinavir, nelfinavir and ritonavir susceptibility (increased resistance) and an increase in amprenavir susceptibility.
  • Patient sample 3542 was examined further for genotypic changes associated with the pattern of susceptibility.
  • RTV-3542 DNA was analyzed by ABI chain terminator automated sequencing.
  • the nucleotide sequence was compared to the consensus sequence of a wild type clade B HIV-1 (HIV Sequence Database Los Alamos, NM) .
  • the nucleotide sequence was examined for sequences that are different from the control sequence.
  • PR mutations were noted at positions I13V, K14R, N37D, M46I, L63P, N88S and I93L.
  • K14R and N37A/D are naturally occurring polymorphisms in HIV-1 PR and are not associated with reduced susceptibility to any drug.
  • M46I has previously been described to be associated with resistance to indinavir, ritonavir, nelfinavir and amprenavir.
  • L63P has previously been described to be associated with resistance to indinavir and nelfinavir.
  • N88S has previously been described to be associated with resistance to nelfinavir
  • a resistance test vector was constructed as described in example 1 from a patient sample designated as 3654. This patient had been previously treated with ritonavir. Isolation of viral RNA and RT/PCR was used to generate a patient derived segment that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segment was inserted into an indicator gene viral vector to generate a resistance test vector designated RTV-3654. RTV-3654 was tested using a phenotypic susceptibility assay to determine accurately and quantitatively the level of susceptibility to a panel of anti-retroviral drugs.
  • This panel of anti-retroviral drugs comprised members of the classes known as NRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir) , NNRTIs (delavirdine, nevirapine and efavirenz) , and PRIs (indinavir, nelfinavir, ritonavir, saquinavir and amprenavir) .
  • An IC50 was determined for each drug tested. Susceptibility of the patient virus to each drug was examined and compared to known patterns of susceptibility.
  • a pattern of susceptibility to the PRIs was observed for patient sample RTV-3654 in which there was a decrease in indinavir and nelfinavir susceptibility (increased resistance) and an increase in amprenavir susceptibility.
  • Patient sample 3654 was examined further for genotypic changes associated with the pattern of susceptibility.
  • RTV-3654 DNA was analyzed ⁇ by ABI chain terminator automated sequencing.
  • the nucleotide sequence was compared to the consensus sequence of a wild type clade B HIV-1 (HIV Sequence Database Los Alamos, NM) .
  • the nucleotide sequence was examined for sequences that are different from the control sequence.
  • PR mutations were noted at positions I13V, R41K, M46I, L63P, V77I, N88S and I93L.
  • I13V, R41K and I93L are naturally occurring polymorphism in HIV-1 PR and is not associated with reduced susceptibility to any drug.
  • M46I has previously been described to be associated with resistance to indinavir, ritonavir, nelfinavir and amprenavir.
  • L63P has previously been described to be associated with resistance to indinavir and nelfinavir.
  • V77I has previously been described to be associated with resistance to nelfinavir.
  • N88S has previously been described to be associated with resistance to an investigational PRI, SC55389A (Smidt, 1997) .
  • Site directed mutagenesis Resistance test vectors were constructed containing the N88S mutation alone and in combination with other substitutions in PR (L63P, V77I and M46L) known to modulate the HIV susceptibility to PRIs. Mutations were introduced into the resistance test vector using the mega-primer PCR method for site-directed mutagenesis . (Sakar G and Sommar SS (1994) Biotechniques 8(4), 404-407) . First, a resistance test vector was constructed that harbors a unique RsrII restriction site 590 bp downstream of the Apal restriction site.
  • the 590 bp Apal - RsrII fragment thus contains the entire protease region. This site was introduced by site-specific oligonucleotide-directed mutagenesis using primer #4. All subsequent mutants were constructed by f agment-exchange of the wild-type Apal - RsrII fragment in the parent vector with the equivalent fragment carrying the respective mutations.
  • a resistance test vector containing the N88S mutation (N88S-RTV) was tested using the phenotypic susceptibility assay described above and the results were compared to that of a genetically defined resistance test vector that was wild type at position 88.
  • the pattern of phenotypic susceptibility to the PRIs in the N88S-RTV was altered as compared to wild type.
  • the N88S-RTV was more susceptible to both amprenavir and ritonavir and slightly less susceptible to nelfinavir compared to the wild type control RTV (see Table 2) .
  • a resistance test vector containing the N88S mutation along with the L63P mutation was tested using the phenotypic susceptibility assay described above and the results were compared to that of a genetically defined resistance test vector that was wild type at positions 63 and 88.
  • the L63P-N88S-RTV showed decreased susceptibility to both indinavir and nelfinavir and an increase in the susceptibility to amprenavir compared the wild-type control RTV (see Table 2) .
  • L63P-N88S-RTV showed decreased susceptibility to both indinavir and nelfinavir and an increase in the susceptibility to amprenavir compared the wild-type control RTV (see Table 2) .
  • a resistance test vector containing the N88S mutation along with the L63P mutation and the V77I mutation was tested using the phenotypic susceptibility assay described above and the results were compared to that of a genetically defined resistance test vector that was wild type at positions 63 and 77 and 88.
  • the RTV containing mutations at these positions, L63P-V77I-N88S-RTV showed a decrease in susceptibility to both indinavir and nelfinavir and an increase in the susceptibility to amprenavir compared to the wild-type control RTV (see Fig. 5 and Table 2) .
  • a resistance test vector containing the N88S mutation along with the M46L mutation, the L63P mutation, and the V77I mutation was tested using the phenotypic susceptibility assay described above and the results were compared to that of a genetically defined resistance test vector that was wild type at positions 46, 63, 77 and 88.
  • the RTV containing mutations at these four positions, M46L-L63P-V77I-N88S-RTV showed a decrease in susceptibility to nelfinavir and indinavir and an increase in the susceptibility to amprenavir compared to the wild-type control RTV (see Fig. 5 and Table 2).
  • a resistance test vector containing the L63P mutation (L63P-RTV) was tested using the phenotypic susceptibility assay described above and the results were compared to that of a genetically defined resistance test vector that was wild type at position 63.
  • the pattern of phenotypic susceptibility to the PRIs in the L63P-RTV was similar to wild type with no significant changes in susceptibility to the PRIs observed.
  • the L63P mutation was also introduced into an RTV containing an additional mutation at position V77I.
  • the L63P-V77I-RTV showed a slight decrease in susceptibility to nelfinavir compared to the wild-type control RTV (see Fig. 5 and Table 2) .
  • changes in the amino acid at position 88 of the protease protein of HIV-1 is evaluated using the following method • comprising: (i) collecting a biological sample from an HIV-1 infected subject; (ii) evaluating whether the biological sample contains nucleic acid encoding HIV-1 protease having an asparagine to serine mutation at codon 88 (N88S); and (iii) determining susceptibility to protease inhibitors
  • the biological sample comprises whole blood, blood components including peripheral mononuclear cells (PBMC) , serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin) , tissue biopsies, cerebral spinal fluid (CSF) , or other cell, tissue or body fluids.
  • PBMC peripheral mononuclear cells
  • plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
  • tissue biopsies tissue biopsies
  • cerebral spinal fluid (CSF) cerebral spinal fluid
  • the HIV-1 nucleic acid (genomic RNA) or reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample. Evaluating whether the amino acid at position 88 of the HIV-1 protease is mutated to serine, can be performed using various methods, such as direct characterization of the viral nucleic acid encoding protease or direct characterization of the protease protein itself.
  • Defining the amino acid at position 88 of protease can be performed by direct characterization of the protease protein by conventional or novel amino acid sequencing methodologies, epitope recognition by antibodies or other specific binding proteins or compounds.
  • the amino acid at position 88 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
  • Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR) , NASBA, SDA, RCR, or 3SR.
  • the nucleic acid sequence encoding HIV protease at codon 88 can be determined by direct nucleic acid sequencing using various primer extension-chain termination (Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam and Gilbert) methodologies or more recently developed sequencing methods such as matrix assisted laser desorption-ionization time of flight (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems).
  • the nucleic acid sequence encoding amino acid position 88 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
  • evaluation of protease inhibitor susceptibility and of whether amino acid position 88 of HIV-1 protease was wild type or serine was carried out using a phenotypic susceptibility assay or genotypic assay, respectively, using resistance test vector DNA prepared from the biological sample.
  • the plasma sample was collected, viral RNA was purified and an RT-PCR methodology was used to amplify a patient derived segment encoding the HIV-1 protease and reverse transcriptase regions. The amplified patient derived segments were then incorporated, via DNA ligation and bacterial transformation, into an indicator gene viral vector thereby generating a resistance test vector.
  • Resistance test vector DNA was isolated from the bacterial culture and the phenotypic susceptibility assay was carried out as described in Example 1.
  • the results of the phenotypic susceptibility assay with a patient sample having an N88S mutation in PR is shown in Figure 4.
  • the nucleic acid (DNA) sequence of the patient derived HIV-1 protease and reverse transcriptase regions from patient sample 0732 was determined using a fluorescence detection chain termination cycle sequencing methodology (ABI/PE) . The method was used to determine a consensus nucleic acid sequence representing the combination of sequences of the mixture of HIV-1 variants existing in the subject sample (representing the quasispecies) , and to determine the nucleic acid sequences of individual variants.
  • Phenotypic and genotypic correlation of mutations at amino acid 88 of HIV-1 Protease Phenotypic susceptibility profiles of patient samples and site directed mutants showed that amprenavir susceptibility correlated with the presence of the N88S mutation in HIV-1 protease. Phenotypic susceptibility profiles of patient samples and site directed mutants showed that a significant increase in amprenavir susceptibility (decreased resistance) correlated with a mutation in the nucleic acid sequence encoding the amino acid serine (S) at position 88 of HIV-1 protease.
  • Phenotypic susceptibility profiles of patient samples and site directed mutants showed reduction in amprenavir susceptibility (decreased resistance) and a decrease in susceptibility to nelfinavir and indinavir with the amino acid serine at position 88 when the PR mutations at positions 63, 77 or 46 were also present (L63P, V77I, or M46L) .
  • EXAMPLE 6 Using Resistance test vectors and site directed mutants to correlate genotypes associated with alterations in PRI susceptibility with viral itness .
  • Luciferase activity measured in the absence of drug for the seven resistance test vectors constructed from the patient viruses containing the N88S PR mutation ranged from 0.7 to 16% of control (Table 3). Although these viruses also contain multiple mutations in reverse transcriptase, which could also contribute to a reduction in viral fitness, the data suggest that viruses containing the N88S mutation are less fit than wild type. To confirm this observation, the luciferase expression level for the site-directed mutant resistance test vectors was also examined.
  • Viruses containing N88S as the only substitution produced only 1.0% of the luciferase activity in the absence of drug (Table 4). This reduction was substantially alleviated by the addition of the L63P substitution (20.7%) or by addition of the combinations of L63P/V77I (29.3%) or M46L/L63P (28.0%).
  • the L63P or L63P/V77I mutants had equivalent or increased relative luciferase activity compared to wild type (163.9 and 75.6%, respectively) .
  • EXAMPLE 7 Predicting Response to Protease Inhibitors by Characterization of Amino Acid 82 of HIV-1 Protease.
  • changes in the amino acid at position 82 of the protease protein of HIV-1 are evaluated using the following method comprising: (i) collecting a biological sample from an HIV-1 infected subject; (ii) evaluating whether the biological sample contains nucleic acid encoding HIV-1 protease having a valine to alanine (V82A) , phenylalanine (V82F) , serine (V82S) , or threonine (V82T) substitution at codon 82; and (iii) determining susceptibility to protease inhibitors (PRI) .
  • V82A valine to alanine
  • V82F phenylalanine
  • V82S serine
  • V82T threonine
  • the biological sample comprises whole blood, blood components including peripheral mononuclear cells (PBMC) , serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin), tissue biopsies, cerebral spinal fluid (CSF) , or other cell, tissue or body fluids.
  • PBMC peripheral mononuclear cells
  • plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
  • tissue biopsies tissue biopsies
  • cerebral spinal fluid (CSF) cerebral spinal fluid
  • RNA genomic RNA
  • reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample. Evaluating whether the amino acid at position 82 of the HIV-1 protease is mutated to alanine, phenylalanine, serine, or threonine, can be performed using various methods, such as direct characterization of the viral nucleic acid encoding protease or direct characterization of the protease protein itself. Defining the amino acid at position 82 of protease can be performed by direct characterization of the protease protein by conventional or novel amino acid sequencing methodologies, epitope recognition by antibodies or other specific binding proteins or compounds.
  • the amino acid at position 82 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
  • Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR) , NASBA, SDA, RCR, or 3SR.
  • the nucleic acid sequence encoding HIV protease at codon 82 can be determined by direct nucleic acid sequencing using various primer extension-chain termination (Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam and Gilbert) methodologies or more recently developed sequencing methods such as matrix assisted laser desorption-ionization time of flight (MALDI-TOF) or mass spectrometry (Sequeno , Gene Trace Systems) .
  • the nucleic acid sequence encoding amino acid position 82 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affy etrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
  • evaluation of protease inhibitor susceptibility and of whether amino acid position 82 of HIV-1 protease was wild type or alanine, phenylalanine, serine, or threonine was carried out using a phenotypic susceptibility assay or genotypic assay, respectively, using resistance test vector DNA prepared from the biological sample.
  • the plasma sample was collected, viral RNA was purified and an RT-PCR methodology was used to amplify a patient derived segment encoding the HIV-1 protease and reverse transcriptase regions. The amplified patient derived segments were then incorporated, via DNA ligation and bacterial transformation, into an indicator gene viral vector thereby generating a resistance test vector.
  • Resistance test vector DNA was isolated from the bacterial culture and the phenotypic susceptibility assay was carried out and analyzed as described in Example 1.
  • the nucleic acid (DNA) sequence of the patient derived HIV-1 protease and reverse transcriptase regions was determined using a fluorescence detection chain termination cycle sequencing methodology (ABI/PE) . The method was used to determine a consensus nucleic acid sequence representing the combination of sequences of the mixture of HIV-1 variants existing in the subject sample
  • Genotypes are analyzed as lists of amino acid differences between virus in the patient sample and a reference laboratory strain of HIV-1, NL4-3. Genotypes and corresponding phenotypes (fold-change in IC50 values) are entered in a relational database linking these two results with patient information. Large datasets can then be assembled from patient virus samples sharing particular characteristics, such as the presence of any given mutation, or combination of mutations or reduced susceptibility to any drug or combination of drugs .
  • Phenotypic susceptibility profiles of 75 patient virus samples which contained a mutation at position 82 (V82A, F, S, or T) , but no other primary mutations, were analyzed. According to most published guidelines, such viruses are expected to be resistant to ritonavir, nelfinavir, indinavir, and saquinavir. However, 8%, 20%, 23%, and 73% of these samples were phenotypically susceptible to these four protease inhibitors, respectively (see Table 6) . Thus, particularly for indinavir and saquinavir, there was poor correlation between the presence of mutations at position 82 and drug susceptibility.
  • Indinavir resistance in viruses containing mutations at position 82 was evaluated with respect to the presence of other specific mutations. Decreased indinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing V82A, F, S, or T but no other primary mutations was correlated with the presence of mutations at secondary positions. Reduced indinavir susceptibility was observed in 20 samples containing mutations at both positions 24 and 82 (100%) and in 27 samples with both 71 and 82 (100%) (See Table 7) . The combination of mutations at position 82 with mutations at other positions (e.g. 54, 46, 10, and 63) also significantly increased the proportion of samples that had reduced indinavir susceptibility (Table 7) .
  • Saquinavir resistance in viruses containing mutations at position 82 was evaluated with respect to the presence of other specific mutations .
  • Decreased saquinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing V82A, F, S, or T but no other primary mutations was correlated with the presence of mutations at secondary positions.
  • Reduced saquinavir susceptibility was observed in 4 of 5 samples containing mutations at both positions 20 and 82 (80%) and in 8 of 11 samples with both 36 and 82 (73%) (See Table 8) .
  • the combination of mutations at position 82 with mutations at other positions e.g. 24, 71, 54, and 10) also significantly increased the proportion of samples that had reduced saquinavir susceptibility (Table 8).
  • Indinavir resistance in viruses containing mutations at position 82 was evaluated with respect to the presence of a defined number of other mutations. Decreased indinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing V82A, F, S, or T but no other primary mutations was correlated with the number of mutations at secondary positions. Reduced indinavir susceptibility was observed in 100% of samples with V82A, F, S, or T and at least 6 other secondary mutations (See Table 9) . The proportion of samples that had reduced indinavir susceptibility increased significantly in samples with V82A, F, S, or T combined with 3 to 5 other secondary mutations (Table 9) .
  • changes in the amino acid at position 90 of the protease protein of HIV-1 are evaluated using the following method comprising: (i) collecting a biological sample from an HIV-1 infected subject; (ii) evaluating whether the biological sample contains nucleic acid encoding HIV-1 protease having a leucine to methionine (L90M) substitution at codon 90; and (iii) determining susceptibility to protease inhibitors (PRI) .
  • the biological sample comprises whole blood, blood components including peripheral mononuclear cells (PBMC) , serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin) , tissue biopsies, cerebral spinal fluid (CSF) , or other cell, tissue or body fluids.
  • PBMC peripheral mononuclear cells
  • plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
  • tissue biopsies tissue biopsies
  • cerebral spinal fluid (CSF) cerebral spinal fluid
  • the HIV-1 nucleic acid (genomic RNA) or reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample. Evaluating whether the amino acid at position 90 of the HIV-1 protease is mutated to methionine, can be performed using various methods, such as direct characterization of the viral nucleic acid encoding protease or direct characterization of the protease protein itself.
  • Defining the amino acid at position 90 of protease can be performed by direct characterization of the protease protein by conventional or novel amino acid sequencing methodologies, epitope recognition by antibodies or other specific binding proteins or compounds.
  • the amino acid at position 90 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
  • Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR), NASBA, SDA, RCR, or 3SR.
  • the nucleic acid sequence encoding HIV protease at codon 90 can be determined by direct nucleic acid sequencing using various primer extension-chain termination (Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam and Gilbert) methodologies or more recently developed sequencing methods such as matrix assisted laser desorption-ionization time of flight (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems) .
  • the nucleic acid sequence encoding amino acid position 90 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
  • evaluation of protease inhibitor susceptibility and of whether amino acid position 90 of HIV-1 protease was wild type or methionine was carried out using a phenotypic susceptibility assay or genotypic assay, respectively, using resistance test vector DNA prepared from the biological sample.
  • the plasma sample was collected, viral RNA was purified and an RT-PCR methodology was used to amplify a patient derived segment encoding the HIV-1 protease and reverse transcriptase regions .
  • the amplified patient derived segments were then incorporated, via DNA ligation and bacterial transformation, into an indicator gene viral vector thereby generating a resistance test vector.
  • Resistance test vector DNA was isolated from the bacterial culture and the phenotypic susceptibility assay was carried out and analyzed as described in Example 1.
  • the nucleic acid (DNA) sequence of the patient derived HIV-1 protease and reverse transcriptase regions was determined using a fluorescence detection chain termination cycle sequencing methodology (ABI/PE) .
  • the method was used to determine a consensus nucleic acid sequence representing the combination of sequences of the mixture of HIV-1 variants existing in the subject sample
  • Genotypes are analyzed as lists of amino acid differences between virus in the patient sample and a reference laboratory strain of HIV-1, NL4-3. Genotypes and corresponding phenotypes (fold-change in IC50 values) are entered in a relational database linking these two results with patient information. Large datasets can then be assembled from patient virus samples sharing particular characteristics, such as the presence of any given mutation, or combination of mutants, or reduced susceptibility to any drug or combination of drugs.
  • Phenotypic susceptibility profiles of 58 patient virus samples which contained a mutation at position 90 (L90M) , but no other primary mutations, were analyzed. According to most published guidelines, such viruses are expected to be resistant to ritonavir, nelfinavir, indinavir, and saquinavir. However, 28%, 9%, 31%, and 47% of these samples were phenotypically susceptible to these four protease inhibitors, respectively (see Table 6) . Thus, particularly for indinavir and saquinavir, there was poor correlation between the presence of mutations at position 90 and drug susceptibility.
  • Indinavir resistance in viruses containing mutations at position 90 was evaluated with respect to the presence of other specific mutations. Decreased indinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing L90M but no other primary mutations was correlated with the presence of mutations at secondary positions. Reduced indinavir susceptibility was observed in 17 of 19 samples containing mutations at both positions 73 and 90 (89%) and in 16 of 18 samples with both 71 and 90 (89%) (See Table 10) . The combination of mutations at position 90 with mutation at position 46 also significantly increased the proportion of samples that had reduced indinavir susceptibility (Table 10) .
  • Saquinavir resistance in viruses containing mutations at position 90 was evaluated with respect to the presence of other specific mutations. Decreased saquinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing L90M but no other primary mutations was correlated with the presence of mutations at secondary positions. Reduced saquinavir susceptibility was observed in 15 of 19 samples containing mutations at both positions 73 and 90 (79%) and in 14 of 18 samples with both 71 and 90 (78%) (See Table 11) . The combination of mutations at position 90 with mutations at other positions (e.g. 77 and 10) also significantly increased the proportion of samples that had reduced saquinavir susceptibility (Table 1) .
  • Indinavir resistance in viruses containing mutations at position 90 was evaluated with respect to the presence of a defined number of other mutations. Decreased indinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing L90M but no other primary mutations was correlated with the number of mutations at secondary positions. Reduced indinavir susceptibility was observed in 100% of samples with L90M and at least 5 other secondary mutations had (See Table 12). The proportion of samples that had reduced indinavir susceptibility increased significantly in samples with L90M combined with 3 or 4 other secondary mutations (Table 12) .
  • Saquinavir susceptibility of viruses containing combinations of mutations at amino acid 90 and many secondary mutations in HIV-1 Protease was evaluated with respect to the presence of a defined number of other mutations. Decreased saquinavir susceptibility (fold-change in IC 50 greater than 2.5) in viruses containing L90M but no other primary mutations was correlated with the number of mutations at secondary positions. Reduced saquinavir susceptibility was observed in 100% of samples with L90M and at least 5 other secondary mutations (See Table 12). The proportion of samples that had reduced saquinivir susceptibility increased significantly in samples with L90M combined with 3 or 4 other secondary mutations (Table 12) .
  • EXAMPLE 9 Predicting Response to Protease Inhibitors by Characterization of Amino Acids 82 and 90 of HIV-1
  • Protease changes in the amino acid at position 82 and 90 of the protease protein of
  • HIV-1 are evaluated using the following method comprising:
  • the biological sample comprises whole blood, blood components including peripheral mononuclear cells (PBMC) , serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin) , tissue biopsies, cerebral spinal fluid (CSF) , or other cell, tissue or body fluids.
  • PBMC peripheral mononuclear cells
  • plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
  • tissue biopsies tissue biopsies
  • cerebral spinal fluid (CSF) cerebral spinal fluid
  • the HIV-1 nucleic acid (genomic RNA) or reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample.
  • Evaluating whether the amino acid at position 82 of the HIV-1 protease is mutated to alanine, phenylalanine, serine, or threonine or at position 90 to methionine can be performed using various methods, such as direct characterization of the viral nucleic acid encoding protease or direct characterization of the protease protein itself. Defining the amino acid at positions 82 and 90 of protease can be performed by direct characterization of the protease protein by conventional or novel amino acid sequencing methodologies, epitope recognition by antibodies or other specific binding proteins or compounds.
  • the amino acid at positions 82 and 90 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
  • Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR) , NASBA, SDA, RCR, or 3SR.
  • the nucleic acid sequence encoding HIV protease at codons 82 and 90 can be determined by direct nucleic acid sequencing using various primer extension-chain termination (Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam and Gilbert) methodologies or more recently developed sequencing methods such as matrix assisted laser desorption-ionization time of flight (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems) .
  • the nucleic acid sequence encoding amino acid positions 82 and 90 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
  • evaluation of protease inhibitor susceptibility and of whether amino acid positions 82 and 90 of HIV-1 protease was wild type or alanine, phenylalanine, serine, or threonine in the case of position 82 and methionine at position 90 was carried out using a phenotypic susceptibility assay or genotypic assay, respectively, using resistance test vector DNA prepared from the biological sample.
  • plasma sample was collected, viral RNA was purified and an RT-PCR methodology was used to amplify a 5 patient derived segment encoding the HIV-1 protease and reverse transcriptase regions.
  • the amplified patient derived segments were then incorporated, via DNA ligation and bacterial transformation, into an indicator gene viral vector thereby generating a resistance test vector.
  • 10 Resistance test vector DNA was isolated from the bacterial culture and the phenotypic susceptibility assay was carried out and analyzed as described in Example 1.
  • HIV-1 protease and reverse transcriptase regions was determined using a fluorescence detection chain termination cycle sequencing methodology (ABI/PE) .
  • the method was used to determine a consensus nucleic acid sequence representing the combination of sequences of the
  • Genotypes are analyzed as lists of amino acid differences between virus in the patient sample and a reference laboratory
  • Genotypes and corresponding phenotypes are entered in a relational database linking these two results with patient information. Large datasets can then be assembled from patient virus samples sharing particular characteristics,
  • a means and method is provided for accurately measuring and reproducing the replication fitness of HIV-1.
  • This method for measuring replication fitness is applicable to other viruses, including, but not limited to hepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitis C virus) and herpesviruses (human cytomegalovirus) .
  • This example further provides a means and method for measuring the replication fitness of HIV-1 that exhibits reduced drug susceptibility to reverse transcriptase inhibitors and protease inhibitors.
  • This method can be used for measuring replication fitness for other classes of inhibitors of HIV-1 replication, including, but not limited to integration, virus assembly, and virus attachment and entry. Replication fitness tests are carried out using the means and methods for phenotypic drug susceptibility and resistance tests described in US Patent Number 5,837,464
  • patient-derived segment (s) corresponding to the HIV protease and reverse transcriptase coding regions were either patient-derived segments amplified by the reverse transcription-polymerase chain reaction method (RT-PCR) using viral RNA isolated from viral particles present in the serum of HIV-infected individuals or were mutants of wild type HIV-1 made by site directed mutagenesis of a parental clone of resistance test vector DNA. Resistance test vectors are also referred to as "fitness test vectors" when used to evaluate replication fitness. Isolation of viral RNA was performed using standard procedures (e.g. RNAgents Total RNA Isolation System, Promega, Madison WI or RNAzol, Tel-Test, Friendswood, TX) . The RT-PCR protocol was divided into two steps. A retroviral reverse transcriptase [e.g. Moloney MuLV reverse transcriptase
  • thermostable DNA polymerase e.g. Taq (Roche Molecular Systems, Inc., Branchburg, NJ) , Tth (Roche Molecular Systems, Inc., Branchburg, NJ) , PrimeZyme (isolated from Thermus brockianus, Biometra, Gottingen, Germany)
  • thermostable polymerases as described for the performance of "long PCR” (Barnes, W.M., (1994) Proc. Natl. Acad. Sci, USA 91, 2216-2220) [e.g.
  • PCR6 (Table 5, #1) is used for reverse transcription of viral RNA into cDNA.
  • the primers, Apal primer (PDSApa, Table 5, #2) and Agel primer (PDSAge, Table 5, #3) used to amplify the "test" patient-derived segments contained sequences resulting in Apal and Agel recognition sites being introduced into both ends of the PCR product, respectively.
  • WO 97/27319 Number WO 97/27319 (see Fig. 1) using an amplified DNA product of 1.5 kB prepared by RT-PCR using viral RNA as a template and oligonucleotides PCR6 (#1) , PDSApa (#2) and PDSAge (#3) as primers, followed by digestion with Apal and Agel or the isoschizomer PinAl .
  • PCR6 #1
  • PDSApa #2
  • PDSAge #3
  • the plasmid DNA corresponding to the resultant fitness test vector comprises a representative sample of the HIV viral quasi-species present in the serum of a given patient, many (>100) independent E. coli transformants obtained in the construction of a given fitness test vector were pooled and used for the preparation of plasmid DNA.
  • a packaging expression vector encoding an amphotrophic MuLV 4070A env gene product enables production in a fitness test vector host cell of fitness test vector viral particles which can efficiently infect human target cells.
  • Fitness test vectors encoding all HIV genes with the exception of env were used to transfect a packaging host cell (once transfected the host cell is referred to as a fitness test vector host cell) .
  • the packaging expression vector which encodes the amphotrophic MuLV 4070A env gene product is used with the resistance test vector to enable production in the fitness test vector host cell of infectious pseudotyped fitness test vector viral particles .
  • Fitness tests were carried out with fitness test vectors using two host cell types.
  • Fitness test vector viral particles were produced by a first host cell (the fitness test vector host cell) that was prepared by transfecting a packaging host cell with the fitness test vector and the packaging expression vector. The fitness test vector viral particles were then used to infect a second host cell (the target host cell) in which the expression of the indicator gene is measured (see Fig. A).
  • the fitness test vectors containing a functional luciferase gene cassette were constructed and host cells were transfected with the fitness test vector DNA.
  • the fitness test vectors contained patient-derived reverse transcriptase and protease DNA sequences that encode proteins which were either susceptible or resistant to the antiretroviral agents, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors.
  • the amount of luciferase activity detected in the infected cells is used as a direct measure of "infectivity", "replication capacity” or “fitness”, i.e. the ability of the virus to complete a single round of replication.
  • Relative fitness is assessed by comparing the amount of luciferase activity produced by patient derived viruses to the amount of luciferase activity produced by a well- characterized reference virus (wildtype) derived from a molecular clone of HIV-1, for example NL4-3 or HXB2.
  • Fitness measurements are expressed as a percent of the reference, for example 25%, 50%, 75%, 100% or 125% of reference ( Figure B, C) .
  • Host cells were seeded in 10-cm-diameter dishes and were transfected one day after plating with fitness test vector plasmid DNA and the envelope expression vector. Transfections were • performed using a calcium-phosphate co-precipitation procedure. The cell culture media containing the DNA precipitate was replaced with fresh medium, from one to 24 hours, after transfection. Cell culture media containing fitness test vector viral particles was harvested one to four days after transfection and was passed through a 0.45-mm filter before being stored at " 80°C. HIV capsid protein (p24) levels in the harvested cell culture media were determined by an EIA method as described by the manufacturer (SIAC; Frederick, MD) . Before infection, target cells (293 and 293/T) were plated in cell culture media.
  • Control infections were performed using cell culture media from mock transfections (no DNA) or transfections containing the fitness test vector plasmid DNA without the envelope expression plasmid.
  • One to three or more days after infection the media was removed and cell lysis buffer (Promega) was added to each well.
  • Cell lysates were assayed for luciferase activity.
  • cells were lysed and luciferase was measured by adding Steady- Glo (Promega) reagent directly to each well without aspirating the culture media from the well.
  • a means and method is provided for identifying mutations in reverse transcriptase that alter replication fitness.
  • a means and method is provided for identifying mutations that alter replication fitness and can be used to identify mutations associated with other aspects of HIV-1 replication, including, but not limited to integration, virus assembly, and virus attachment and entry.
  • This example also provides a means and method for quantifying the affect that specific mutations reverse transcriptase have on replication fitness .
  • a means and method for quantifying the affect that specfic protease and reverse transcriptase mutations have on replication fitness to mutations in other viral genes involved in HIV-1 replication, including, but not limited to the gag, pol, and envelope genes is also provided.
  • Fitness test vectors were constructed as described in example 10. Fitness test vectors derived from patient samples or clones derived from the fitness test vector pools, or fitness test vectors were engineered by site directed mutagenesis to contain specific mutations, and were tested in a fitness assay to determine accurately and quantitatively the relative fitness compared to a well- characterized reference standard. A patient sample was examined for increased or decreased reverse transcriptase activity and correlated with the relative fitness observed ( Figure C) . Reverse transcriptase activity of patient HIV samples
  • Reverse transcriptase activity can be measured by any number of widely used assay procedures, including but not limited to homopolymeric extension using (e.g. oligo dT:poly rC) or real time PCR based on molecular beacons (reference Kramer) or 5' exonuclease activity (Lie and Petropoulos, 1996) .
  • virion associated reverse transcriptase activity was measured using a quantitative PCR assay that detects the 5' exonuclease activity associated with thermo-stable DNA polymerases ( Figure C) .
  • the fitness of the patient virus was compared to a reference virus to determine the relative fitness compared to "wildtype" viruses that have not been exposed to reverse transcriptase inhibitor drugs.
  • the fitness of the patient virus was compared to viruses collected from the same patient at different timepoints, for example prior to initiating therapy, before or after changes in drug treatment, or before or after changes in virologic (RNA copy number) , immunologic (CD4 T-cells) , or clinical (opportunistic infection) markers of disease progression. Genotypic analysis of patient HIV samples
  • Fitness test vector DNAs are analyzed by any of the genotyping methods described in Example 1.
  • patient HIV sample sequences were determined using viral RNA purification, RT/PCR and ABI chain terminator automated sequencing. The sequence was determined and compared to reference sequences present in the database or compared to a sample from the patient prior to initiation of therapy. The genotype was examined for sequences that are different from the reference or pre-treatment sequence and correlated to the observed fitness.
  • Genotypic changes that are observed to correlate with changes in fitness were evaluated by construction of fitness vectors containing the specific mutation on a defined, wild-type (drug susceptible) genetic background. Mutations may be incorporated alone and/or in combination with other mutations that are thought to modulate the fitness of a virus. Mutations were introduced into the fitness test vector through any of the widely known methods for site-directed mutagenesis. In one embodiment of this invention the mega-primer PCR method for site-directed mutagenesis is used. A fitness test vector containing the specific mutation or group of mutations were then tested using the fitness assay described in Example 10 and the fitness was compared to that of a genetically defined wild-type (drug susceptible) fitness test vector which lacks the specific mutations.
  • a means and method for identifying mutations in protease that alter replication fitness is provided.
  • This example provides the means and methods for identifying mutations that alter replication fitness for various components of HIV-1 replication, including, but not limited to integration, virus assembly, and virus attachment and entry.
  • This example also provides a means and method for quantifying the affect that specific mutations in protease or reverse transcriptase have on replication fitness. This method can be used for quantifying the effect that specific protease mutations have on replication fitness and can be used to quantify the effect of other mutations in other viral genes involved in HIV-1 replication, including, but not limited to the gag, pol, and envelope genes.
  • Fitness test vectors were constructed as described in example 10.
  • Protease activity can be measured by any number of widely used assay procedures, including but not limited to in vitro reactions that measure protease cleavage activity
  • protease cleavage of the gag polyprotein (p55) was measured by
  • the fitness of the patient virus was compared to a reference virus to determine the relative fitness compared to "wildtype" viruses that have not been exposed to protease inhibitor drugs.
  • the fitness of the patient virus was compared to viruses collected from the same patient at different timepoints, for example prior to initiating therapy, before or after changes in drug treatment, or before or after changes in virologic (RNA copy number) , immunologic (CD4 T-cells) , or clinical (opportunistic infection) markers of disease progression.
  • Genotypic analysis of patient HIV samples Fitness test vector DNAs, either pools or clones, are analyzed by any of the genotyping methods described in Example 1.
  • patient HIV sample sequences were determined using viral RNA purification, RT/PCR and ABI chain terminator automated sequencing. The sequence was determined and compared to reference sequences present in the database or compared to a sample from the patient prior to initiation of therapy, if available. The genotype was examined for sequences that are different from the reference or pre-treatment sequence and correlated to the observed fitness.
  • Genotypic changes that are observed to correlate with changes in fitness are evaluated by construction of fitness vectors containing the specific mutation on a defined, wild-type (drug susceptible) genetic background. Mutations may be incorporated alone and/or in combination with other mutations that are thought to modulate the fitness of a virus. Mutations are introduced into the fitness test vector through any of the widely known methods for site-directed mutagenesis. In one embodiment of this invention the mega-primer PCR method for site-directed mutagenesis is used. A fitness test vector containing the specific mutation or group of mutations are then tested using the fitness assay described in Example 10 and the fitness is compared to that of a genetically defined wild-type (drug susceptible) fitness test vector which lacks the specific mutations.
  • fitness test vectors containing site directed mutations in reverse protease that result in amino acid substitutions at positions 30, 63, 77, 90 (list from Figure E) and that display • different amounts of protease activity are constructed and tested for fitness ( Figure E) .
  • the fitness results enable the correlation between specific protease amino acid substitutions and changes in viral fitness.
  • This example describes the high incidence of patient samples with reduced replication fitness.
  • This example also describes the general correlation between reduced drug susceptibility and reduced replication fitness.
  • This example further describes the occurrence of viruses with reduced fitness in patients receiving protease inhibitor and/or reverse transcriptase inhibitor treatment.
  • This example further describes the incidence of patient samples with reduced replication fitness in which the reduction in fitness is due to altered protease processing of the gag polyprotein (p55) .
  • This example further describes the incidence of protease mutations in patient samples that exhibit low, moderate or normal (wildtype) replication fitness.
  • protease mutations that are frequently observed, either alone or in combination, in viruses that exhibit reduced replication capacity.
  • This example also describes the incidence of patient samples with reduced replication fitness in which the reduction in fitness is due to altered reverse transcriptase activity.
  • This example describes the occurrence of viruses with reduced replication fitness in patients failing antiretroviral drug treatment..
  • Fitness/resistance test vectors were constructed as described in example 10. Fitness and drug susceptibility was measured in 134 random patient samples that were received for routing phenotypic testing by the ViroLogic Clinical Reference Laboratory. Fitness assays were performed as described in Example 10. Drug susceptibility testing and genotyping of the protease region was performed as described in Example 1. Reverse transcriptase activity was measured as described in Example 11. Protease processing was measured as described in Example 12.
  • Drug susceptibility of patient viruses Reduced drug susceptibility was observed for a majority of the patient virus samples (Table A) . 66 percent of the viruses exhibited large (define as >10X of the reference) reductions in susceptibility to one or more NRTI drugs. 52 percent of the viruses exhibited large reductions in susceptibility to one or more NNRTI drugs. 45 percent of the viruses exhibited large reductions in susceptibility to one or more PRI drugs .
  • Viruses with reduced drug susceptibility were much more likely to display reduced replication fitness ( Figures F, G, H, and I) .
  • protease mutations D30N, M46I/L, G48V, I54L/A/S/T/V, and I84V were observed at high incidences in viruses with reduced protease processing of the p55 gag polyprotein (Figure L) .
  • a means and method for using replication fitness measurements to guide the treatment of HIV-1 is provided.
  • This example further provides a means and method for using replication fitness measurements to guide the treatment of patients failing antiretroviral drug treatment.
  • This example further provides the means and methods for using replication fitness measurements to guide the treatment of patients newly infected with HIV-1.
  • physicians may choose to perform routine replication fitness assays for patients that have multi- drug resistant virus.
  • This assay could be used to monitor the replication fitness of patient viruses when complete suppression of virus replication is not possible due to multi-drug resistance.
  • the assay would be used to guide treatment decisions that prevent the drug resistant virus with low replication fitness from increasing its replication fitness. In this way, physicians may prolong the usefulness of antiretroviral drugs despite the presence of drug resistant virus in the patient.
  • This example provides a means and method for identifying mutations in protease that affect susceptibility (increased or decreased) to saquinavir.
  • the effects of combination of mutations at position 82 are evaluated using the following method comprising: (i) collecting a biological sample from an HIV-1 infected subject; (ii) evaluating whether the HIV-1 in the sample contains nucleic acid encoding protease having a valine to alanine (V82A) , phenylalanine (V82F) , serine (V82S) , or threonine (V82T) substitution at position 82 or a leucine to methionine substitution at position 90 (L90M) ; and (iii) determining susceptibility to protease inhibitors (PRIs) .
  • PRIs susceptibility to protease inhibitors
  • the biological sample comprises whole blood, blood components including peripheral mononuclear cells (PBMC) , serum, plasma (prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin) , tissue biopsies, cerebral spinal fluid (CSF) , or other cell, tissue or body fluids.
  • PBMC peripheral mononuclear cells
  • plasma prepared using various anticoagulants such as EDTA, acid citrate-dextrose, heparin
  • tissue biopsies tissue biopsies
  • cerebral spinal fluid (CSF) cerebral spinal fluid
  • the HIV-1 nucleic acid (genomic RNA) or reverse transcriptase protein can be isolated directly from the biological sample or after purification of virus particles from the biological sample.
  • Evaluating whether the amino acid at position 82 of the HIV-1 protease is mutated to alanine, phenylalanine, or threonine can be performed using various methods, such as direct characterization of the viral nucleic acid encoding protease or direct characterization of the protease protein itself. Defining the amino acid at position 82 of protease can be performed by direct characterization of the protease protein by conventional or novel amino acid sequencing methodologies, epitope recognition by antibodies or other specific binding proteins or compounds. Alternatively, the amino acid at position 82 of the HIV-1 protease protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the protease protein.
  • Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription-polymerase chain reaction (RT-PCR) , NASBA, SDA, RCR, or 3SR.
  • RT-PCR reverse transcription-polymerase chain reaction
  • the nucleic acid sequence encoding HIV protease at codon 82 can be determined by direct nucleic acid sequencing using various primer extension-chain termination (Sanger, ABI/PE and Visible Genetics) or chain cleavage (Maxam and Gilbert) methodologies or more recently developed sequencing methods such as matrix assisted laser desorption-ionization time of flight (MALDI-TOF) or mass spectrometry (Sequenom, Gene Trace Systems) .
  • MALDI-TOF matrix assisted laser desorption-ionization time of flight
  • MSequenom Gene Trace Systems
  • nucleic acid sequence encoding amino acid position 82 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
  • probe hybridization methodologies such as genechip hybridization sequencing (Affymetrix) , line probe assay (LiPA; Murex) , and differential hybridization (Chiron) .
  • evaluation of the effects of mutations at amino acid position 82 of HIV-1 protease on protease inhibitor susceptibility was carried out using a phenotypic susceptibility assay using resistance test vector DNA prepared from the biological sample.
  • plasma samples were collected, viral RNA was purified and an RT-PCR methodology was used to amplify a patient derived segment encoding the HIV-1 protease and reverse transcriptase regions .
  • the amplified patient derived segments were then incorporated, via DNA ligation and bacterial transformation, into an indicator gene viral vector thereby generating a resistance test vector.
  • Resistance test vector DNA was isolated from the bacterial culture and the phenotypic susceptibility assay was carried out as described in Example 1.
  • the genotype of the protease region was determined by dideoxy chain- termination sequencing of the resistance test vector DNA. The results are summarized for saquinavir (SQV) in Figure 6. Samples were categorized as having mutations in protease encoding alanine (A), phenylalanine (F), or threonine (T) at position 82, instead of the wild-type valine (V) , and the percentage of samples in each category displaying hyper-sensitivity to saquinavir (i.e., fold- change vs. reference of 0.4 or less) was determined.
  • SQV saquinavir
  • This example provides a means and method for identifying mutations in integrase that alter replication fitness.
  • This example provides the means and methods for identifying mutations that alter replication fitness for various components of HIV-1 replication, including, but not limited to integration, virus assembly, and virus attachment and entry.
  • This example also provides a means and method for quantifying the affect that specific mutations in protease, reverse transcriptase, or integrase have on replication fitness. This method can be used for quantifying the effect that specific integrase mutations have on replication fitness and can be used to quantify the effect of other mutations in other viral genes involved in HIV-1 replication, including, but not limited to the gag, pol, and envelope genes.
  • Fitness test vectors engineered by site directed mutagenesis to contain specific mutations in integrase were tested in a fitness assay to determine accurately and quantitatively the relative fitness compared to a well- characterized reference standard.
  • Genotypic changes that are observed to correlate with resistance to integrase inhibitors are evaluated by construction of fitness vectors containing the specific mutation on a defined, wild-type (drug susceptible) genetic background. Mutations may be incorporated alone and/or in combination with other mutations that are thought to modulate the fitness of a virus. Mutations are introduced into the fitness test vector through any of the widely known methods for site-directed mutagenesis. In one embodiment of this invention the mega-primer PCR method for site-directed mutagenesis is used (Sarkar, G. and Sommar, S.S., 1994, Biotechniques 8, 404-407).
  • a fitness test vector containing the specific mutation or group of mutations are then tested using the fitness assay described in Example 10 and the fitness is compared to that of a genetically defined wild-type (drug susceptible) fitness test vector which lacks the specific mutations. Observed changes in fitness are attributed to the specific mutations introduced into the fitness test vector.
  • fitness test vectors containing site directed mutations in integrase that result in amino acid substitutions at positions 66, 154, 66 and 153, and 66 and 154 are constructed and tested for fitness ( Figure 7) .
  • Figure 7 Relative luciferase activity of integrase inhibitor-resistant site-directed mutants .
  • Table 1 PRI susceptibility of selected patient samples. Viruses displaying increased susceptibility to amprenavir (5-fold or greater) were genotyped and found to contain the N88S mutation in PR. Samples were listed in order of decreasing amprenavir susceptibility.
  • Table 2 PRI susceptibility of site-directed mutants in PR. Mutations were introduced into the drug sensitive reference resistance test vector and the susceptibility to PRIs was determined.
  • Table 3 Relative luciferase activity levels for patient sample virus-derived resistance test vector pools.
  • the luciferase activity (relative light units, RLU) measured in the absence of drug for the patient sample was compared to that of the drug sensitive reference control from the same assay run, and expressed as a percentage of control. These values are from one assay each. All the samples that contain the N88S mutations in PR were found to have reduced luciferase activity compared to control.
  • Table 4 Relative luciferase activity levels for resistance test vectors containing site-directed mutations.
  • the luciferase activity (relative light units, RLU) measured in the absence of drug for the mutant was compared to that of the drug sensitive reference control from the same assay run, and expressed as a percentage of control. These values are from one to five assays each, and each alue was obtained using an independent clone for mutants which were tested multiple times. All the constructs that contain the N88S mutations in PR were found to have reduced luciferase activity compared to control. All the constructs with the K20T mutation were essentially inactive in the assay.

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Abstract

L'invention porte sur des essais de susceptibilité et se résistance à des médicaments antiviraux servant à identifier des posologies efficaces pour le traitement d'infections par le VIH, et du SIDA, dont en particulier des posologies incluant un inhibiteur de protéase. L'invention porte également sur des moyens et procédés de suivi de la progression de l'infection par le VIH, et de sa réponse à une thérapie antivirale dans le cadre d'essais de susceptibilité aux phénotypes et génotypes.
PCT/US2001/028754 2000-09-15 2001-09-14 Moyens et methodes de suivi d'une therapie antiretrovirale a base d'inhibiteurs de proteases et decisions d'orientation therapeutique dans le traitement du vih et du sida WO2002022076A2 (fr)

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EP01970982A EP1322779A4 (fr) 2000-09-15 2001-09-14 Moyens et methodes de suivi d'une therapie antiretrovirale a base d'inhibiteurs de proteases et decisions d'orientation therapeutique dans le traitement du vih et du sida
CA002422815A CA2422815A1 (fr) 2000-09-15 2001-09-14 Moyens et methodes de suivi d'une therapie antiretrovirale a base d'inhibiteurs de proteases et decisions d'orientation therapeutique dans le traitement du vih et du sida

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EP1373479A1 (fr) * 2001-01-19 2004-01-02 Virologic, Inc. Procedes pour controler une therapie antiretrovitale par inhibiteur de protease
WO2004003817A1 (fr) 2002-07-01 2004-01-08 Tibotec Pharmaceuticals Ltd. Nouveaux profils mutationnels dans une protease du vih-1 correles a une resistance phenotypique aux medicaments
US6869759B1 (en) 1999-06-22 2005-03-22 Virologic, Inc. Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS
EP1552023A2 (fr) * 2002-07-01 2005-07-13 Virologic, Inc. Compositions et methodes permettant de mesurer la sensibilite d'un virus pathogene a des inhibiteurs de la protease
US7186506B1 (en) 2000-06-12 2007-03-06 Monogram Biosciences, Inc. Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS
US7384734B2 (en) 2002-02-15 2008-06-10 Monogram Biosciences, Inc. Compositions and methods for determining the susceptibility of a pathogenic virus to protease inhibitors
WO2008145606A1 (fr) 2007-05-25 2008-12-04 Tibotec Pharmaceuticals Ltd. Nouveau profil mutationnel dans un site de clivage du gag du vih-1 corrélé à une résistance phénotypique aux médicaments
US7473524B2 (en) 2002-07-01 2009-01-06 Hilde Azijn Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance
US7993824B2 (en) 2002-07-01 2011-08-09 Monogram Biosciences, Inc. Compositions and methods for determining the susceptibility of a pathogenic virus to protease inhibitors
US8563236B2 (en) 1999-05-28 2013-10-22 Virco, N.V. Mutational profiles in HIV-1 reverse transcriptase correlated with phenotypic drug resistance
US8574831B2 (en) 2000-10-20 2013-11-05 Virco N.V. Mutational profiles in HIV-1 reverse transcriptase correlated with phenotypic drug resistance

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Publication number Priority date Publication date Assignee Title
AU5758200A (en) * 1999-06-22 2001-01-09 Virologic, Inc. Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of hiv/aids

Non-Patent Citations (2)

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Title
See also references of EP1322779A2 *
ZIERMANN ET AL.: 'A mutation in human immunodeficiency virus type 1 protease, N88S, that causes in vitro hypersensitivity to amprenavir' vol. 74, no. 9, May 2000, pages 4414 - 4419, XP002908352 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8563236B2 (en) 1999-05-28 2013-10-22 Virco, N.V. Mutational profiles in HIV-1 reverse transcriptase correlated with phenotypic drug resistance
US6869759B1 (en) 1999-06-22 2005-03-22 Virologic, Inc. Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS
US7186506B1 (en) 2000-06-12 2007-03-06 Monogram Biosciences, Inc. Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS
US7138231B2 (en) 2000-09-15 2006-11-21 Monogram Biosciences, Inc. Means and methods for monitoring protease inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS
US8574831B2 (en) 2000-10-20 2013-11-05 Virco N.V. Mutational profiles in HIV-1 reverse transcriptase correlated with phenotypic drug resistance
EP1373479A1 (fr) * 2001-01-19 2004-01-02 Virologic, Inc. Procedes pour controler une therapie antiretrovitale par inhibiteur de protease
EP1373479A4 (fr) * 2001-01-19 2005-12-07 Virologic Inc Procedes pour controler une therapie antiretrovitale par inhibiteur de protease
US7384734B2 (en) 2002-02-15 2008-06-10 Monogram Biosciences, Inc. Compositions and methods for determining the susceptibility of a pathogenic virus to protease inhibitors
US7217506B2 (en) 2002-07-01 2007-05-15 Tibotec Pharmaceuticals, Ltd. Mutational profiles in HIV-1 protease correlated with phenotypic protease inhibitor resistance
EP1552023A4 (fr) * 2002-07-01 2007-04-04 Virologic Inc Compositions et methodes permettant de mesurer la sensibilite d'un virus pathogene a des inhibiteurs de la protease
US7473524B2 (en) 2002-07-01 2009-01-06 Hilde Azijn Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance
US7553618B2 (en) 2002-07-01 2009-06-30 Monogram Biosciences, Inc. Method for determining human immunodeficiciency virus type 1 (HIV-1) hypersusceptibility to the protease inhibitor amprenavir
US7993824B2 (en) 2002-07-01 2011-08-09 Monogram Biosciences, Inc. Compositions and methods for determining the susceptibility of a pathogenic virus to protease inhibitors
US8076062B2 (en) 2002-07-01 2011-12-13 Tibotec Pharmaceuticals Ltd. Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance
EP1552023A2 (fr) * 2002-07-01 2005-07-13 Virologic, Inc. Compositions et methodes permettant de mesurer la sensibilite d'un virus pathogene a des inhibiteurs de la protease
WO2004003817A1 (fr) 2002-07-01 2004-01-08 Tibotec Pharmaceuticals Ltd. Nouveaux profils mutationnels dans une protease du vih-1 correles a une resistance phenotypique aux medicaments
US8592161B2 (en) 2002-07-01 2013-11-26 Janssen R&D Ireland Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance
WO2008145606A1 (fr) 2007-05-25 2008-12-04 Tibotec Pharmaceuticals Ltd. Nouveau profil mutationnel dans un site de clivage du gag du vih-1 corrélé à une résistance phénotypique aux médicaments
US8546076B2 (en) 2007-05-25 2013-10-01 Tibotec Pharmaceuticals Ltd. Mutational profile in HIV-1 GAG cleavage site correlated with phenotypic drug resistance

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